D ) IGV analyses of H2A . Z localisation ( from ENCODE ) on Aurora B ( green ) c - MYC ( blue ) genes showing H2A . Z enrichment at TSS at upstream region respectively ( bottom panels ) . IGV analyses of ATAC seq peaks for Control si H2A . Z . 1 si for Aurora B MYC genes . number in red represents log2Ratio H2AZ . 1 si / Control si .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si.


E ) Representative of Hela stained for Ki67 . arrows indicate different patterns quantified : strong signal ( grey arrow ) , weak signal ( brown arrow ) no signal ( brown arrow ( scale bar 10μm ) . F ) of experiment in ( E ) . Error bars indicate standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square . * * * = p < 0 . 001 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Representative images of Hela cell stained for Ki67. The arrows indicate the different staining patterns quantified: strong signal (grey arrow), weak signal (light brown arrow) and no signal (dark brown arrow (scale bar 10μm). F) Quantification of the experiment in (E). Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test. ***=p<0.001.


G ) Representative of HeLa transfected with control ( top ) or H2A . Z . 1 ( bottom ) siRNA stained for Lamin A / C ( green ) . Scale bar = 10μm .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

G) Representative images of HeLa cells transfected with control (top) or H2A.Z.1 (bottom) siRNA and stained for Lamin A/C (green). Scale bar = 10μm.


C ) Kaplan Meier survival curve for neuroblastoma with low ( red ) or high ( blue ) expression of H2AFZ ( H2A . Z . 1 ) obtained from R2 genomics .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Kaplan Meier survival curve for neuroblastoma patients with low (red) or high (blue) expression of H2AFZ (H2A.Z.1) obtained from R2 genomics.


A - G Hematoxylin eosin ( H & E ) of frontal sections of miniature pig mandible slices showing morphological changes during permanent canine ( PC ) initiation stage from embryonic day 50 ( E50 ) to postnatal day 10 ( PN10 ) ; ( A ' - F ' ) are magnifications of boxed regions in their corresponding figure panel . DC , deciduous canine ; PC , permanent canine ; SDL , successional dental lamina ; n = 3 . Scale bars = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-G Hematoxylin and eosin (H&E) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine (PC) initiation stage from embryonic day 50 (E50) to postnatal day 10 (PN10); (A'-F') are magnifications of boxed regions in their corresponding figure panel. DC, deciduous canine; PC, permanent canine; SDL, successional dental lamina; n = 3. Scale bars = 100 µm.


H In situ hybridization ( ISH ) showing expression pattern of Sox2 in initiation stage from E50 to E90 ; right figure panels are magnifications of boxed regions in left panels . Dashed lines mark position of successional dental lamina ( SDL ) ; green arrowhead indicates positive of Sox2 at tip of SDL . n = 3 . Scale bars = 100 µm .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

H In situ hybridization (ISH) showing the expression pattern of Sox2 in the initiation stage from E50 to E90; right figure panels are magnifications of boxed regions in left panels. Dashed lines mark the position of the successional dental lamina (SDL); green arrowhead indicates positive staining of Sox2 at the tip of the SDL. n = 3. Scale bars = 100 µm.


I Immunofluorescence ( IF ) of Ki67 at SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Immunofluorescence (IF) of Ki67 at the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


J Semi - comparison of Ki67 expression during E60 , E70 , E90 stages . n = 3 . Data information : Data represent means ± . * P < 0 . 05 ( one - way ANOVA Newman - Keuls post - hoc ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Semi-quantification and comparison of Ki67 expression levels during E60, E70, and E90 stages. n = 3. Data information: Data represent the means ± SEM. *P < 0.05 (one-way ANOVA and Newman-Keuls post-hoc tests).


K TUNEL showing apoptosis status of SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K TUNEL assay showing the apoptosis status of the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


A Three - dimensional reconstruction of serial H & E frontal sections of miniature pig mandibles at embryonic day 60 ( E60 ) E90 ; deciduous canine ( DC ) in purple , permanent canine ( PC ) in yellow , alveolar socket in green . red , blue , green arrows indicate width of labial alveolar socket , lingual alveolar socket , DC , respectively . n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Three-dimensional reconstruction of serial H&E frontal sections of miniature pig mandibles at embryonic day 60 (E60) and E90; deciduous canine (DC) in purple, permanent canine (PC) in yellow, and alveolar socket in green. The red, blue, and green arrows indicate the width of the labial alveolar socket, lingual alveolar socket, and DC, respectively. n = 3.


B DC labial lingual alveolar socket widths in horizontal plane at bottom of PC during E60 E90 . n = 3 . Data information : Data represent means ± . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


C proportion of DC width relative to total alveolar socket width during E60 E90 . n = 3 . Data information : Data represent means ± . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The proportion of DC width relative to the total alveolar socket width during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


D Micro - CT imaging of whole mandible at E60 ; boxed region is magnified in D ' ( top panel ) . ( D ' ) Mandible slice isolated with Geomagic software . dashed lines mark DC . E 3 - D color map ( left ) after alignment of mandible slices before after ( sham ) surgery showing a comparison of surface points . Coronal sections through cusp tips ( transparent squares , left ) were selected for 2 - D comparisons ( middle ) ; right panels are magnifications of yellow - boxed regions . solid purple contour dotted black contour indicate pre - post - surgery shapes , respectively . between two contours are colored segments showing direction of movement . colored ball in 2 - D comparison marks position of maximum displacement . PC position is indicated in pink . n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Micro-CT imaging of the whole mandible at E60; boxed region is magnified in D' (top panel). (D') Mandible slice isolated with Geomagic software. White dashed lines mark DC. E 3-D color map (left) after alignment of mandible slices before and after (sham) surgery showing a comparison of the surface points. Coronal sections through cusp tips (transparent squares, left) were selected for 2-D comparisons (middle); right panels are magnifications of yellow-boxed regions. The solid purple contour and dotted black contour indicate the pre- and post-surgery shapes, respectively. The distance between the two contours are the colored line segments showing the distance and direction of the movement. The colored ball in the 2-D comparison marks the position of the maximum displacement. The PC position is indicated in pink. n = 3.


F Dissected mandible slice with a " U " shape .

F Dissected mandible slice with a "U" shape.


A Diagram of dissected miniature pig mandible slice illustrating deciduous ( DC ) permanent ( PC ) canine application of compression in vitro using Flexcell FX - 5000 Compression System .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Diagram of the dissected miniature pig mandible slice illustrating the deciduous (DC) and permanent (PC) canine and application of compression in vitro using Flexcell FX-5000 Compression System.


B - D H & E of canine frontal sections from embryonic day 60 ( E60 ) ( B ) , after culturing for two days without stress ( 0 kPa ) ( C ) , or with stress ( 3 kPa ) ( D ) . ( B ' - D ' ) are magnifications of boxed regions in their corresponding figure panels . Scale bars = 50 µm . n = 6 / 6 in E60 group ; n = 12 / 12 in 0 - kPa group ; n = 4 / 8 in 3 - kPa group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-D H&E staining of canine frontal sections from embryonic day 60 (E60) (B), after culturing for two days without stress (0 kPa) (C), or with stress (3 kPa) (D). (B'-D') are magnifications of boxed regions in their corresponding figure panels. Scale bars = 50 µm. n = 6/6 in E60 group; n = 12/12 in 0-kPa group; n = 4/8 in 3-kPa group.


F RT - qPCR of several mechanosensitive ( TGFB1 , NFKB1 , EGR1 , RUNX2 ) of four mechanoreceptors ( ITGB1 , ITGB3 , ITGAV ITGA2 ) in PC surrounding soft tissue at E60 after culturing for two days with ( 3 kPa ) or without extra mechanical stress ( 0 kPa ) . n = 3 for each group . Data represent means ± . One - way ANOVA ( Newman - Keuls for post - hoc comparisons between two groups ) , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . =

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F RT-qPCR of several mechanosensitive proteins (TGFB1, NFKB1, EGR1, and RUNX2) and of four mechanoreceptors (ITGB1, ITGB3, ITGAV and ITGA2) in PC and surrounding soft tissue at E60 and after culturing for two days with (3 kPa) or without extra mechanical stress (0 kPa). n = 3 for each group. Data represent the means ± SEM. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups), *P < .05, **P < .01, ***P < .001; NS, not significant. =


A - L IF of integrin β1 , ERK1 , RUNX2 in miniature pig canine frontal sections at embryonic day 60 ( E60 ) , E90 , E60 cultured under 3 kPa stress for two days E60 cultured under 0 kPa stress for two days ; ( A ' - L ' ) are magnifications of boxed regions in corresponding figure panels . M Relative IF expression of integrin β1 , ERK1 , RUNX2 during E60 , E90 , E60 cultured under 3 kPa stress for two days E60 cultured under 0 kPa stress for two days . Data information : Data represent means ± . n = 3 . Scale bars = 25 µm ( A ' , G ' ) 50 µm ( other panels ) . Unpaired t - for M , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant.


N of human PC at 18 - 19 ( H & E ) ; ( N ' ) is magnification of boxed region in ( N ) . DC , deciduous canine ; PC , permanent canine ; IEE , inner enamel epithelium ; OEE , outer enamel epithelium . Mesenchyme between DC PC is indicated with a green arrow .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.


O - Q ISH expression patterns of ITGB1 , ERK1 , RUNX2 in human PCs . Green arrows indicate mesenchyme between DC PC .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.


R IF of RUNX2 in human PC . Mesenchyme between DC PC is indicated with a red arrow . Red arrow indicates mesenchyme between DC PC .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.


S PC epithelium areas in E60 , E90 , E60 cultured under 3 kPa stress for two days E60 cultured under 0 kPa stress for two days . Data information : Data represent means ± . n = 3 . , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant.


IF of RUNX2 phospho - ERK1 / 2 ( p - ERK1 / 2 ) upon force loading with 0 or 1 . 0 g / cm2 for 2 h or after force was removed cultured for an additional 1 , 2 , 4 hours .

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.


V Relative IF expression of RUNX2 p - ERK1 / 2 between groups Data information : Data represent means ± . n = 3 . One - way ANOVA ( Newman - Keuls for post - hoc comparisons between two groups ) for V , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant.


W Western blots of p - ERK1 / 2 RUNX2 after 1 . 0 g / cm2 was applied for 2 h , with or without anti - integrin β1 antibody ERK1 inhibitor ( U0126 ) . X Relative expression of p - ERK1 / 2 , RUNX2 , Actin in ( W ) . Data information : Data represent means ± . n = 3 . Unpaired t - for M ; One - way ANOVA ( Newman - Keuls for post - hoc comparisons between two groups ) for X , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.


A - C H & E of miniature pig canine frontal sections from embryonic day 60 ( E60 group ) ( A ) after mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag ( overexpression group ) ( B ) or overexpression control lentiviral vector with a Myc tag ( O - control group ) ( C ) ; ( A ' - C ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


D - F Immunohistochemical ( ) of Myc expression in mandible slices from E60 , overexpression , O - control groups . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 100 µm ( D - F )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F)


G - I IF of RUNX2 expression ( yellow arrows ) in mandible slices of E60 , overexpression , O - control groups . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 50

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


J , K Relative expression of Myc RUNX2 in E60 , O - control , overexpression groups . Data information : Data represent means ± . n = 3 for experiments . One - way ANOVA ( Newman - Keuls for post - hoc comparisons between two groups ) for J , K , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001.


L , M IF of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector ( knockdown control , K - control ) ( L ) or RUNX2 shRNA lentiviral vector ( knockdown ) ( M ) ; ( L ' - M ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 50 µm ( other panels ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels).


N Relative RUNX2 expression between K - control knockdown groups . Data information : Data represent means ± . n = 3 for experiments . Unpaired t - for N , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001.


O - Q H & E of mandible slice sections from K - control , knockdown + force ( 3 kPa ) , K - control + force ( 3 kPa ) groups . ( O ' - Q ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


R - T IF of RUNX2 expression ( yellow arrows ) of mandible slice sections from K - control , knockdown + force ( 3 kPa ) , K - control + force ( 3 kPa ) groups . ( R ' - T ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Data represent means ± . n = 3 for experiments . Scale bars = 25 µm ( R ' - T ' )

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

R-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T')


U Relative RUNX2 expression among three groups Data information : Data represent means ± . n = 3 for experiments . One - way ANOVA ( Newman - Keuls for post - hoc comparisons between two groups ) for U , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001.


V PC epithelium areas in overexpression , O - control , knockdown , knockdown + force , K - control , K - control + force groups . Data information : Data represent means ± . n = 3 for experiments . Unpaired t - for V ; , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.


A , B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 ( E60 ) E90 . ( A ' , B ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Scale bars = 50 µm .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


C Relative ISH expression of RUNX2 in epithelium ( Epi . ) mesenchyme ( Mes . ) at E60 E90 . Data information Data represent means ± . n = 3 for experiments . Unpaired t - for C , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001.


D , E ISH of miniature pig canine frontal sections showing expression patterns of β - catenin at E60 E90 . ( D ' , E ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Scale bars = 50 µm .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


F Relative ISH expression of β - catenin in epithelium ( Epi . ) mesenchyme ( Mes . ) at E60 E90 . Data information : Data represent means ± . n = 3 for experiments . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


G , H IF of Lef1 at E60 E90 . ( G , H ' ) are magnifications of boxed regions in corresponding figure panels . Data information : Scale bars = 50 µm .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


I Relative IF expression of Lef1 in epithelium ( Epi . ) mesenchyme ( Mes . ) at E60 E90 . Data represent means ± . n = 3 for experiments . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


J FISH of β - catenin in E60 E90 mandible slices E60 mandible slices after 12 24 h in culture via RNAscope . Boxed regions of mesenchyme are magnified in right panels . Epithelium is indicated with a yellow arrow . Data information : Scale bars = 50 µm .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow. Data information: Scale bars = 50 µm.


K Relative FISH expression of β - catenin in nucleus cytoplasm of epithelium mesenchyme at E60 , E90 , E60 cultured for 12 24 h . Data information : Data represent means ± . n = 3 for experiments . One - way ANOVA for K , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA for K, *P < .05, **P < .01, ***P < .001.


L , M IF of β - catenin at E60 E90 ; yellow arrows indicate localization of β - catenin in nucleus . Data information : Scale bars = 50 µm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus. Data information: Scale bars = 50 µm.


N Relative IF expression of β - catenin in nucleus of epithelium mesenchyme at E60 E90 . Data information : Data represent means ± . n = 3 for experiments . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


O , P FISH of β - catenin in mandible slices infected with RUNX2 shRNA lentivirus ( knockdown ) or scrambled shRNA lentivirus ( scramsh ) via RNAscope . Data information : Scale bars = 50 µm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm.


Q Relative FISH expression of β - catenin in nucleus cytoplasm of epithelium mesenchyme in knockdown scramsh groups . Data information : Data represent means ± . n = 3 for experiments . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


A , B IF of Lef1 in mandible slices at embryonic day 60 ( E60 ) infected with RUNX2 overexpression lentiviral vector ( overexpression ) or overexpression control lentiviral vector ( O - control ) . ( A ' - B ' ) are magnifications of boxed regions in corresponding figure panels . Dashed lines mark epithelium of PC . C Relative IF expression of Lef1 in epithelium ( Epi . ) mesenchyme ( Mes . ) of overexpression O - control groups . Data information : Data represent means ± . Scale bars = 100 µm . n = 3 for experiments . Unpaired t - for C , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C , **P < .01, ***P < .001; NS. not significant.


D , E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days . ( D ' , E ' ) are magnifications of boxed regions in corresponding figure panels . Dashed lines mark epithelium of PC . F Relative IF expression of Lef1 in epithelium mesenchyme after applying 3 0 kPa pressure . Data information : Data represent means ± . Scale bars = 100 µm . n = 3 for experiments . Unpaired t - for , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for , *P < .05, **P < .01, ***P < .001; NS. not significant.


G Western blots of Lef1 , non - phospho - β - catenin , RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector . ( G ' ) Relative expression between control vector RUNX2 overexpression groups . Data information : Data represent means ± . Unpaired t - , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups. Data information: Data represent the means ± SEM. Unpaired t-tests , *P < .05, **P < .01, ***P < .001; NS. not significant.


H Western blots of Lef1 , non - phospho - β - catenin , RUNX2 after DFCs were infected with scrambled shRNA ( scramsh ) or RUNX2 knockdown shRNA ( knockdown ) lentiviral vectors . ( H ' ) Relative expression between scramsh knockdown groups .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups.


I Western blots of non - phospho - β - catenin Lamin B in DFCs of RUNX2 overexpression ( O ) , control vector ( V ) , RUNX2 knockdown ( K ) , scramsh ( S ) , compressed force ( F ; 1 . 0 g / cm2 ) , control ( C ; 0 g / cm2 ) groups . Sample were loaded in a gradient ( 7 . 5 , 15 , 30 , 60 μg ) showing a linear relationship for each group .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group.


J Western blots of non - phospho - β - catenin Lamin B in DFCs treated with IWR - 1 - endo . Relative expression were compared between force no force groups between overexpression control groups with or without IWR - 1 - endo treatment .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.


( C ) Survival curves after injection of EL4 lymphoma ( n = 6 / group ) .

(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).


( H ) Ifng Ifng mRNA ( n = 4 / group ) were determined by ELISA quantitative PCR .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.


( A ) m6A dot blot using total from NK92 - siCTRL or NK92 - siFTO .

(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.


( D ) Western blotting for detection of GZMB in NK92 - siCTRL NK92 - siFTO after treatment with IL - 2 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.


( E ) of IFNγ ( n = 5 biological replicates ) TNFα ( n = 3 biological replicates ) in culture media 24 hours after IL - 2 treatment ( left ) or co - culture media with K562 for 4 hours ( right ) were determined by ELISA .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.


( B ) Western blotting . NK92 - siCTRL or NK92 - siFTO were incubated with 20 ng / ml IL - 2 for 24 hours , washed , cultured without IL - 2 ( IL - 2 depletion ) for indicated times ( n = 3 ) . ( C ) A western blotting of NK92 + MOCK or NK92 + FTO incubated with 20 ng / ml IL - 2 for indicated times was performed ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3). (C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3). D


( C ) m6A methylated immunoprecipitation & quantitative PCR ( MeRIP - qPCR ) using a m6A antibody in NK92 - siFTO ( upper ) or NK92 + FTO ( lower ) compared to NK92 - siCTRL or NK92 + MOCK .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.


( D ) SOCS3 3 ' - UTR reporter using AANAT reporter system . Plasmid information for AANAT reporter system with or without SOCS3 3 ' - UTR ( upper ) . of Aanat ( left ) Aanat mRNA ( right ) expression in HEK - 293T co - transfected with plasmid - expressing AANAT reporter containing SOCS3 3 ' - UTR siCTRL or siFTO .

O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O

(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.


( B ) Relative mRNA expression of JAK / STAT target genes , IFNγ ( n = 10 biological replicates ) , GZMB ( n = 6 biological replicates ) PRF1 ( n = 8 biological replicates ) were determined by qPCR in CD56 + - siCTRL or CD56 + - siFTO .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.


( C ) Western blotting of CD56 + - siCTRL or CD56 + - siFTO incubated with 20 ng / ml IL - 15 20 ng / ml IL - 21 for indicated times was performed . were lysed analyzed by immunoblotting with antibodies to indicated phosphorylated ( p - ) total ( n = 2 ~ 3 ) . Data are representative of three times independent experiments with similar results . were expressed as relative band density of corresponding ( lower ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).


( E ) of ventral bioluminescence ( BLI ) following injection of NK92 - TRC or NK92 - shFTO .

(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.


( G ) survival curves after injection of K562 - Luc .

(G) The survival curves after the injection of K562-Luc cells.


Negative stain image of B . cereus NVH 0075 - 95 endospore , showing spore ( SB ) , exosporium ( E ) , endospore appendages ( Ena ) , which emerge from endospore individually or as fiber bundles . At distal end , Enas terminate in a single or multiple thin ruffles ( R ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Negative stain TEM image of B. cereus NVH 0075-95 endospore, showing spore body (SB), exosporium (E), and endospore appendages (Ena), which emerge from the endospore individually or as fiber bundles. At the distal end, Enas terminate in a single or multiple thin ruffles (R).


cryoEM ( left ) negative stain 2D class averages of single S - Ena ( E ) L - Ena ( F ) . S - Ena show ~ 100 Å diameter a helical or staggered appearance with a ~ 37 Å rise , whereas L - Ena have a ~ 80 Å diameter appear as stacked disks of ~ 40 Å height ~ 60 Å translation along fiber . L - S - Ena show , respectively , a single or multiple ruffle ( s ) ( black arrow , labeled ' R ' ) at distal end of fiber .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cryoEM images (left) and negative stain 2D class averages of single S-Ena (E) and L-Ena (F) fibers. S-Ena show ~100 Å diameter and a helical or staggered appearance with a ~37 Å rise, whereas L-Ena have a ~80 Å diameter and appear as stacked disks of ~40 Å height and ~60 Å translation along the fiber. L- and S-Ena show, respectively, a single or multiple ruffle(s) (black arrow, labeled 'R') at the distal end of fiber.


SDS - PAGE of recEna1B , treated with β - mercaptoethanol or TEV protease ( to remove N - terminal 6xHis tag ) as indicated . Bands with apparent MW of ~ 13 ~ 15 kDa correspond recEna1B monomer with ( Ena1B6His ) without ( Ena1BC ) 6xHis tag TEV recognition site , respectively . In uncleaved sample , a band running at ~ 30 kDa ( labeled * ) corresponds to a non - physiological disulfide bound S - Ena1B dimer . Lower in recEna1B results from a loss of monomers to SDS - resistant high molecular complexes stuck in stacking gel .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.


Negative stain of rec1Ena1B oligomers formed after refolding , but prior to TEV removal of N - terminal 6xHis tag . Close - up view that shows recEna1B oligomers form open crescents similar in dimensions shape to single helical turns or arcs found in S - Ena fiber ( model - right ) . Steric hindrance by 6xHis is thought to arrest recEna1B polymerization into single helical arcs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.


Transcription level of ena1A ( x ) , ena1B ( ▲ ) , ena1C ( ○ ) dedA ( ♦ ) relative to rpoB determined by RT - qPCR during 16 hours of growth of B . cereus NVH 0075 - 95 . dotted represents growth measured by increase in OD600 . Of note , transcription of ena1C was surprisingly higher than ena1A ena1B , major components of isolated S - Ena ( Fig EV2 ) .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

Transcription level of ena1A (x), ena1B (▲), ena1C (○) and dedA (♦) relative to rpoB determined by RT-qPCR during 16 hours of growth of B. cereus strain NVH 0075-95. The dotted line represents the bacterial growth measured by increase in OD600. Of note, the transcription of ena1C was surprisingly higher than ena1A and ena1B, the major components of the isolated S-Ena (Fig EV2).


Representative negative stain of endospores of NVH 0075 - 95 mutants lacking ena1A , ena1B , or ena1C , as well as endospores of strains complemented with respective ena subunit expressed from plasmid ( i . e . pA , pAB pC ) . ena1B mutant was complemented with a plasmid carrying both ena1A 1B ( pAB ) due to repeated failure to transform with a plasmid holding ena1B only . Inset are 2D class averages of Enas observed on respective mutants . Knockout of ena1A , ena1B , or ena1C results in loss of S - Ena , a phenotype that is restored by plasmid - complementation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative negative stain images of endospores of NVH 0075-95 mutants lacking ena1A, ena1B, or ena1C, as well as endospores of strains complemented with the respective ena subunit expressed from plasmid (i.e. pA, pAB and pC). The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B (pAB) due to repeated failure to transform with a plasmid holding ena1B only. Inset are 2D class averages of Enas observed on the respective mutants. Knockout of ena1A, ena1B, or ena1C results in the loss of S-Ena, a phenotype that is restored by plasmid-based complementation.


Number ( top ) ( bottom ) of Enas found on WT , mutant ( ∆ ena1A , ∆ ena1B , or ∆ ena1C ) plasmid complemented ( ∆ ena1A : pena1A , ∆ ena1B : pena1AB , or ∆ ena1C : pena1C ) NVH 0075 - 95 endospores . Statistics : pair - wise Mann - Whitney U against WT ( n : ≥ 18 spores ; n : ≥ 50 Enas ; ns : not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 . - - - : mean ± s . d . )

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Number (top) and length (bottom) of Enas found on WT, mutant (∆ena1A, ∆ena1B, or ∆ena1C) and plasmid complemented (∆ena1A:pena1A, ∆ena1B:pena1AB, or ∆ena1C:pena1C) NVH 0075-95 endospores. Statistics: pair-wise Mann-Whitney U tests against WT (n: ≥18 spores; n: ≥50 Enas; ns: not significant, * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. ---: mean ± s.d.)


A , B MAGE - A3 / 6 decrease upon EBSS treatment of HeLa for indicated times . Note both bands are MAGE - A3 / 6 . Quantitation represents average of n = 3 with standard deviation shown .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

A, B MAGE-A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE-A3/6. Quantitation represents average of n=3 with standard deviation shown.


C Quantitative TMT proteomics of A375 grown in complete media ( CM ) or EBSS for 3 hrs . Number of affected are shown . Autophagy related are indicated in grey MAGE - A in blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 hrs. Number of proteins affected are shown. Autophagy related proteins are indicated in grey and MAGE-A proteins in blue.


D , E MAGE - A3 / 6 are actively degraded upon nutrient deprivation . HeLa were treated for indicated times with complete media ( DMEM + 10 % FBS ) or EBSS containing 100 µg / mL translation inhibitor cycloheximide ( CHX ) . degradation rates are shown ( E ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.


FBS ( 10 % ) , but not non - essential ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 upon EBSS treatment . HeLa were treated with EBSS indicated supplements for indicated times . Quantitation represents average of n = 3 with standard deviation shown .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.


FBS ( 10 % ) , but not non - essential ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 upon EBSS treatment . HeLa were treated with EBSS indicated supplements for indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


C Removal of FBS from normal growth conditions ( DMEM + 10 % FBS ) results in MAGE - A3 / 6 degradation in HeLa .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

C Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells.


D , E MAGE - A3 / 6 stability is controlled by a < 3 kDa , heat stable , lipophilic molecule present in FBS . HeLa were treated with EBSS alone , or EBSS containing 10 % complete FBS , 10 % charcoal - dextran stripped FBS , 10 % boiled FBS , 10 % dialyzed > 3 kDa FBS , or 10 % dialyzed < 3 kDa FBS for indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


F MAGE - A3 / 6 instability upon nutrient deprivation of HeLa is not altered by EGF 0 . 2 ng / mL , testosterone , or bovine pituitary extract 25 µg / mL .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.


Insulin partially rescues MAGE - A3 / 6 upon nutrient deprivation . HeLa were treated for indicated times with EBSS alone or EBSS containing NEAA ( 10 mM ) or insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown.


, H Insulin partially rescues MAGE - A3 / 6 upon nutrient deprivation . HeLa were treated for indicated times with EBSS alone or EBSS containing insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

, H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


A MAGE - A3 / 6 mRNA do not change upon nutrient deprivation . was isolated from HeLa treated with EBSS for indicated times . RT - QPCR was performed to detect both MAGE - A3 / 6 with a common primer set . Data was normalized to 18S rRNA . Quantitation represents average of n = 3 with standard deviation shown .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.


B , Proteasome inhibition rescues MAGE - A3 / 6 upon nutrient deprivation . HeLa were treated for indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 µM ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM).


C Proteasome inhibition rescues MAGE - A3 / 6 upon nutrient deprivation . HeLa were treated for indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 µM ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


D Nutrient deprivation increases ubiquitination of endogenous MAGE - A3 / 6 . HeLa were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated . Input pulldown samples were probed for indicated .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.


E MAGE - A3 / 6 instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase , TRIM28 . HeLa were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for indicated times .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

E MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.


F , MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa were treated with EBSS alone or EBSS containing NAE1 inhibitor MLN4924 ( 1 µM ) that blocks activity of Cullin E3 ubiquitin ligases for indicated times .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times.


G MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa were treated with EBSS alone or EBSS containing NAE1 inhibitor MLN4924 ( 1 µM ) that blocks activity of Cullin E3 ubiquitin ligases for indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


H Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase . HeLa were transfected with vector control or indicated dominant negative ( D / N ) Cullin constructs for 48 hrs before harvesting after 0 or 4 hrs EBSS treatment .

H Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment.


I Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on Cul4A / B . HeLa were transfected with control siRNAs or siRNAs targeting both Cul4A Cul4B . 72 hrs after treatment , were incubated for indicated times with EBSS before harvesting immunoblotting . Note , time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.


A Tandem affinity purification of MAGE - A3 from HEK293 identified DDB1 , DCAF5 , DCAF12 as binding partners . Mascot score , number of peptides identified , coverage is shown .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown.


B MAGE - A3 interacts with DCAF12 , but not DCAF5 . HeLa were transfected with indicated constructs ( Myc - MAGE - A3 HA - DCAF5 or HA - DCAF12 ) . Two days later were treated with complete media or EBSS for 4 hrs . were treated in presence of 1 µM MLN4924 to stabilize MAGE - A3 . extracts were subjected to anti - Myc IP .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.


C Nutrient deprivation increases endogenous DCAF12 binding to MAGE - A3 . HeLa were transfect with Myc - vector or Myc - MAGE - A3 for 48 hrs . were then treated with 10 µM MG132 in complete media , EBSS , or EBSS containing 20 µg / mL insulin for 6 hrs . extracts were subjected to anti - Myc IP .

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.


Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 partially rescued its instability upon nutrient deprivation . HeLa were treated with control , DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for indicated times with or without MLN4924 ( 1 µM ) .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).


E Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 partially rescued its instability upon nutrient deprivation . HeLa were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for indicated times with or without MLN4924 ( 1 µM ) .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).


F Knockdown of DCAF12 with siRNAs blocks MAGE - A3 / 6 degradation upon nutrient deprivation . HeLa were treated with indicated siRNAs for 72 hrs before incubation in EBSS for indicated times . Note , time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation .

O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.


G Ubiquitination of MAGE - A3 / 6 upon nutrient deprivation is dependent on DCAF12 . A375 parental or DCAF12 KO were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated . Input pulldown samples were probed for indicated .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.


A Identification of DCAF12 substrates . Control ( WT ) or DCAF12 knockout ( KO ) A375 were subjected to quantitative TMT proteomics to identify potential DCAF12 targets . MAGE - A ( shown in blue ) are stabilized in DCAF12 knockout .

O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O

A Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE-A proteins (shown in blue) are stabilized in DCAF12 knockout cells.


B Knockout of DCAF12 rescues degradation of MAGE - A in A375 . DCAF12 KO A375 were treated with complete media or EBSS before quantitative TMT proteomics . Note MAGE - A genes are not significantly altered by EBSS in DCAF12 KO .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

B Knockout of DCAF12 rescues degradation of MAGE-A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE-A genes are not significantly altered by EBSS in DCAF12 KO cells.


C DCAF12 target are differentially affected by EBSS compared to remainder of proteome . DCAF12 targets ( n = 33 ) identified show significantly greater decrease upon EBSS treatment compared to non - DCAF12 altered genes ( n = 8243 ) . Average with standard deviation is shown with statistical by student t - . Asterisks * * * indicate p < 0 . 001 ( student t - ) .

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C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n=33) identified show significantly greater decrease upon EBSS treatment compared to non-DCAF12 altered genes (n=8243). Average with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).


E Overexpression of DCAF12 decreases wild - type MAGE - A3 , but not MAGE - A3 degron mutant . indicated constructs were expressed for 48 hrs in HeLa before immunoblotting . MAGE - A3 degron mutant was created ] , in which DNYNEPKANQ * were added to extreme C - term to obscure DCAF12 degron motif .

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E Overexpression of DCAF12 decreases wild-type MAGE-A3, but not MAGE-A3 degron mutant. The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting. The MAGE-A3 degron mutant was created ], in which DNYNEPKANQ* were added to the extreme C-term to obscure the DCAF12 degron motif.


F of proteomics indicates that MAGE - A1 , MAGE - A4 , MAGE - A10 , MAGE - A11 are not regulated by nutrient deprivation or DCAF12 . Average ( n = 3 ) with standard deviation is shown with statistical by student t - . Asterisks * * * indicate p < 0 . 001 ( student t - ) .

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F Analysis of proteomics indicates that MAGE-A1, MAGE-A4, MAGE-A10, and MAGE-A11 are not regulated by nutrient deprivation or DCAF12. Average (n=3) with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).


G MAGE - A10 MAGE - A11 are not altered upon nutrient deprivation or DCAF12 deletion . A375 parental or DCAF12 KO were treated with EBSS for indicated times . lysates were probed for indicated .

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G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.


A - B U2OS GFP - LC3 were transfected with indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . were then fixed , imaged , number of GFP - LC3 were quantitated per . Representative ( A ) quantitation of > 50 from three independent experiments ( B ) are shown . Scale bar indicates 50 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - .

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A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


C - D A375 parental or DCAF12 KO were transfected with indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . were then fixed , endogenous LC3 was immunostained , number of LC3 were quantitated per . Representative ( C ) quantitation of > 50 from three independent experiments ( D ) are shown . Scale bar indicates 20 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - .

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C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


E A375 parental or DCAF12 KO were treated lysates were collected immunoblotted for indicated antibodies to confirm MAGE - A3 / 6 depletion monitor autophagy adaptor / substrate p62 / SQSTM1 .

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E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.


a , Expression of ISX , TWIST1 , Snail1 , Fibronectin ( FN1 ) , VEGF , E - cadherin ( CDH1 ) mRNA were examined in a ISX - GFP inducible Tet - ON transformants at 8 h after addition of DOX . Data are presented as mean ± SD in graph ( p < 0 . 001 compared to time point 0h ; Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

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a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.


b , Western blotting of of ISX , HIF1α , TWIST1 , Snail1 , Slug , ZEB1 , Bmi1 , E - cadherin ( E - Cad . ) , Fibronectin , N - cadherin ( N - Cad . ) , Vimentin , VEGF in A549 H1299 with DOX - inducible ISX expression system 8hs after DOX induction .

b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.


ISX transcriptionally activates luciferase activity driven by TWIST1 ( c ) promoter regions in A549 . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

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ISX transcriptionally activates luciferase activity driven by TWIST1 (c) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


d , ISX transcriptionally activates luciferase activity driven by Snail1 ( d ) promoter regions in A549 . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

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d, ISX transcriptionally activates luciferase activity driven by Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


e , ChIP of ISX binding to TWIST1 promoter region in A549 H1299 ( 10 % input of each groups were pull down applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

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e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


f , ISX transactivation activity analyzed by luciferase activity driven by TWIST1 promoter . Data are presented as mean ± SD in graph ( p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


g , ChIP of ISX binding to endogenous promoters of TWIST1 ( - 180 / + 35 ) Snail1 ( - 160 / + 40 ) in A549 ( 10 % input of each groups were pull down applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate .

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g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


h , Effect of forced expression knockdown of ISX on migration ( wound healing ) measured in A549 . ISXi , ISX - specific shRNAi Data are presented as mean ± SD in graph ( * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm .

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h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.


i , Effect of forced expression knockdown of ISX on invasion ( transwell ) activity measured in A549 . Data are presented as mean ± SD in bar graph ( * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm .

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i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.


a , BRD4 peptide ( 291 - 314 a . a ) obtained from anti - ISX immunoprecipitates of A549 lysates identified through liquid chromatography ‒ tandem mass spectrometry ( LC - tandem - MS ) . Data information : Each experiment was repeated at least three times .

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a, BRD4 peptide (291-314 a.a) obtained from anti-ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography‒tandem mass spectrometry (LC-tandem -MS). Data information: Each experiment was repeated at least three times.


b , ISX association ( PCAF , BRD4 , CBP , CREB , Pol II , Histone H3 ) determined by Western blot in immunoprecipitation of A549 . Data information : Each experiment was repeated at least three times .

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b, ISX association proteins (PCAF, BRD4, CBP, CREB, RNA Pol II, and Histone H3) determined by Western blot in immunoprecipitation of A549 cells. Data information: Each experiment was repeated at least three times.


c , ISX association ( PCAF , BRD4 , CBP , CREB , Pol II ) determined by Western blot in anti - ISX immunoprecipitates of tissues from with lung cancer . Data information : Each experiment was repeated at least three times .

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c, ISX association proteins (PCAF, BRD4, CBP, CREB, and RNA Pol II) determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with lung cancer. Data information: Each experiment was repeated at least three times.


d , Proximity ligation ( PLA ) of ISX BRD4 ( PCAF ) interaction in A549 . Red foci indicate close proximity of two . NC : negative control . Blank arrows indicate interactions of ISX with BRD4 ( PCAF ) in . Data information : Each experiment was repeated at least three times .

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d, Proximity ligation assay (PLA) of ISX and BRD4(PCAF) interaction in A549 cells. Red foci indicate close proximity of the two proteins. NC: negative control. Blank arrows indicate interactions of ISX with BRD4 (PCAF) in the cells. Data information: Each experiment was repeated at least three times.


e , Western blotting of of ISX , PCAF , EMT markers in DOX - inducible ISX expression system A549 treated with TH1834 , Garcinol , C646 , MB - 3 for 8 h after DOX induction . Data information : Each experiment was repeated at least three times .

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e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction. Data information: Each experiment was repeated at least three times.


f , Proximity ligation of ISX BRD4 interaction in A549 treated with Garcinol . Red foci indicate close proximity of two . Blank arrows indicate interactions of ISX with BRD4 in . NC : negative control . Data information : Each experiment was repeated at least three times .

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f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control. Data information: Each experiment was repeated at least three times.


g , Western blotting of of ISX , PCAF , EMT markers in A549 with PCAF knockdown DOX - inducible ISX expression system at 8 h time point after DOX induction . Data information : Each experiment was repeated at least three times .

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g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction. Data information: Each experiment was repeated at least three times.


migration ( wound healing , h ) ssay of A549 lung cancer treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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Cell migration (wound healing, h) ssay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


i , invasion ( transwell , i ) of A549 lung cancer treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

i, Cell invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


c , Recombinant PCAF acetylates His6 - ISX at lysine residue 69 by in vitro acetylation . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


d e , of GFP - tagged WT or mutant ISX , PCAF BRD4 were determined in cytosol , nuclei , anti - GFP immunoprecipites of A549 by Western blotting . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


f , of total acetylated histone H3 were determined in anti - histone H3 immunoprecipites of A549 by Western blotting . Data information : Each experiment was repeated at least three times .

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f, The protein levels of total and acetylated histone H3 were determined in anti-histone H3 immunoprecipites of A549 cells by Western blotting. Data information: Each experiment was repeated at least three times.


migration ( wound healing , g ) activity were determined in A549 with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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The cell migration (wound healing, g) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


h , invasion ( transwell , h ) activity were determined in A549 with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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h, The cell invasion (transwell, h) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


j . xenografts metastasis activity of constitutively expressing RFP A549 transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth Data information : Each experiment was repeated at least three times .

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j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information: Each experiment was repeated at least three times.


k , Kaplan - Meier survival curve of nude mice xenograft injected with A549 carrying GFP , ISX - GFP ISX Ac - GFP ( AC3 ) ( n = 10 ) . p = 0 . 0003 . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) comparing two Kaplan - Meier curves . Data information : Each experiment was repeated at least three times .

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k, Kaplan-Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP, ISX-GFP and ISX Ac-GFP (AC3) (n = 10). p = 0.0003. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves. Data information: Each experiment was repeated at least three times.


c , Recombinant PCAF acetylates His6 - BRD4 at lysine residue 332 . Acetylated BRD4 was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


d , mCherry - tagged WT BRD4 mutants were detected in anti - GFP immunoprecipitates by Western blotting in A549 . Data information : Each experiment was repeated at least three times .

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d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells. Data information: Each experiment was repeated at least three times.