( C ) 3D reconstruction of multi - channel confocal stacks acquired from a RFP + apical dendrite of a granule showing co - localization with pre - post - synaptic markers VIAAT ( granule neuron ) gephyrin ( mitral ) , respectively ( magnification shown in insets ) .

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(C) 3D reconstruction of multi-channel confocal stacks acquired from a RFP+ apical dendrite of a granule cell showing co-localization with the pre- and post-synaptic markers VIAAT (granule neuron) and gephyrin (mitral cell), respectively (magnification shown in insets).


( D ) Anti - RFP immunogold labeling of a in superficial granule layer of a 4D + mouse . Inset shows a representative RFP + synapse ( out of > 10 analyzed from 4D + mice ) .

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(D) Anti-RFP immunogold labeling of a cell in the superficial granule cell layer of a 4D+ mouse. Inset shows a representative RFP+ synapse (out of >10 analyzed from 4D+ mice).


Current clamp recordings showing examples of spontaneous barrages of synaptic potentials ( E ) , Inset in E ( 4D + ) is magnified ( bottom ) .

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Current clamp recordings showing examples of spontaneous barrages of synaptic potentials (E), Inset in E (4D+ cell) is magnified (bottom).


Current clamp recordings showing examples of repetitive spiking in response to depolarizing current steps ( F - G ) of 4D - ( top ) 4D + ( bottom ) mice . Note in G that lag preventing spike initiation is longer at lower currents ( green ) shorter at higher ( black ) , suggesting presence of an A - type K current typical of granule in 4D - vs . 4D + .

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Current clamp recordings showing examples of repetitive spiking in response to depolarizing current steps (F-G) of 4D- (top) and 4D+ (bottom) mice. Note in G that the lag preventing spike initiation is longer at lower currents (green) and shorter at higher (black), suggesting the presence of an A-type K current typical of granule cells in 4D- vs. 4D+.


( H ) Box whiskers plots representing electrophysiological properties of neurons derived from 4D - 4D + NSC ( black red , respectively ) including from left to right : spike number , resting potential ( Vrest ) , input resistance ( Rin ) lag to spiking of recorded superficial granule ( see Fig . EV3 for additional parameters ) .

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(H) Box and whiskers plots representing electrophysiological properties of neurons derived from 4D- and 4D+ NSC (black and red, respectively) including from left to right: spike number, resting membrane potential (Vrest), input resistance (Rin) and lag to spiking of the recorded superficial granule cells (see Fig. EV3 for additional parameters).


( C ) graph indicating proportion of correct responses ( performance ) for bins of 100 trials each during testing with binary mixtures of concentrated octanols . Discontinuous indicates similar 95 % plateau performance of 4D - ( black ) 4D + ( red ) mice . ( D ) Whiskers box plots of performance ( left ) lick frequency ( right ) during a probe of 200 trials with 1 : 10 diluted octanols after testing as in ( C ) .

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(C) Line graph indicating the proportion of correct responses (performance) for bins of 100 trials each during testing with binary mixtures of concentrated octanols. Discontinuous line indicates the similar 95% plateau performance of 4D- (black) and 4D+ (red) mice. (D) Whiskers box plots of performance (left) and lick frequency (right) during a probe of 200 trials with 1:10 diluted octanols after testing as in (C).


( A ) ATX serum were measured in blood serum samples from IBD healthy controls using ELISA : active UC ( n = 26 ) , active CD ( n = 34 ) , control ( Con ) ( n = 26 ) .

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(A) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA: active UC (n=26), active CD (n=34), control (Con) (n=26).


( B ) - sex - matched Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - mice ( n = 9 ) Atx + / + ; Il10 - / - ( n = 9 ) were examined for development of spontaneous colitis . changes were monitored every other day , starting at of 37 days ending at of 6 months . Results are means ± SD . Data were compared by Two - way ANOVA ( with treatment times ) , followed by multiple - comparison Bonferroni t to assess differences between groups .

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(B) Age- and sex-matched Atx∆ΜΕ/∆ΜΕ;Il10-/- mice (n=9) and Atx+/+;Il10-/- littermates (n=9) were examined for the development of spontaneous colitis. Body weight changes were monitored every other day, starting at the age of 37 days and ending at the age of 6 months. Results are means ± SD. Data were compared by Two-way ANOVA (with treatment and times), followed by the multiple-comparison Bonferroni t test to assess differences between groups.


( C ) Gross appearance of full - mouse colon . Each on a ruler represents a millimeter ( mm ) .

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(C) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).


( D E ) H & E stained section of mouse colon . Insets are enlarged in lower panel . Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope ( n = 14 / group ) ( E ) .

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(D and E) H&E stained section of the mouse colon. Insets are enlarged in the lower panel. Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope (n=14/group) (E).


( F ) Histological parameters were quantified with H & E - colon sections ( n = 10 / group ) ( D ) shown as mean ± .

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(F) Histological parameters were quantified with H&E-colon sections (n=10/group) (D) and shown as mean ± SEM.


( G ) Survival of Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - ( n = 17 ) Atx + / + ; Il10 - / - ( n = 18 ) was analyzed by Kaplan - Meier method ( Log - rank P = 0 . 0046 ) .

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(G) Survival of Atx∆ΜΕ/∆ΜΕ;Il10-/- (n=17) and Atx+/+;Il10-/- littermates (n=18) was analyzed by the Kaplan-Meier method (Log-rank P=0.0046).


( H ) Presented are H & E - stained sections of small intestine in a ' Swiss - Roll ' form . Inset areas are enlarged in right panel .

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(H) Presented are H&E-stained sections of the small intestine in a 'Swiss-Roll' form. Inset areas are enlarged in the right panel.


( A ) Total lysates of peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were subjected to immunoblotting to confirm Atx deletion .

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(A) Total cell lysates of peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were subjected to immunoblotting analysis to confirm Atx deletion.


( B ) Peritoneal macrophages from mice were subjected to ESI - MS / MS method to quantify endogenous LPA . molecular species of LPA total LPA ( inset graph ) were compared . data are shown as mean ± ( n = 3 / group ) * P < 0 . 05 ( one - unpaired t - ) .

(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).


( C D ) Thioglycolate - elicited peritoneal immune harvested from mice were immediately fixed for transmission electron microscopy to visualize appearance of macrophages ( C ) DCs ( D ) . Scale bar indicates 1 μm .

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(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.


Lipid rafts from peritoneal macrophages ( E , F ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . was purified concentrated with 3K cut - off filter , followed by Western blot with antibody recognizing lipid raft marker Caveolin or Flotillin - 1 .

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Lipid rafts from peritoneal macrophages (E, F) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.


Lipid rafts from mouse macrophage Raw264 . 7 treated with Atx inhibitor ( Atx - i ) PF8380 ( 50 μM , 30 min ) or vehicle ( Veh . , DMSO 0 . 1 % ) ( G ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . was purified concentrated with 3K cut - off filter , followed by Western blot with antibody recognizing lipid raft marker Flotillin - 1 .

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Lipid rafts from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin-1.


( H ) Peritoneal macrophages were stained with Alexa Fluor 594 - cholera toxin subunit B ( Red ) . plasma was examined with FV10i confocal scanning microscopy . Inset areas were magnified at right panel . Each scale bar indicates 10 μm .

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(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.


Peritoneal macrophages ( A ) were stimulated with LPS ( 20 ng / mL , 20 min ) lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


Raw264 . 7 ( B ) were stimulated with LPS ( 20 ng / mL , 20 min ) in presence / absence of ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) . lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


physical interaction of TLR4 - CD14 was examined by FRET . HEK293 were transfected with TLR4 - EYFP ( yellow ) CD14 - ECFP ( blue ) ( C ) followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . were washed with PBS fixed , then visualized with filter sets for CFP YFP . FRET were visualized with FRET filter set , expressed as corrected FRET efficiency displayed in quantitative pseudocolor indicating between ( arbitrary linear units of ) . Presented are representative from at least three independent experiments . Scale bar indicates 5 μm .

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The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


physical interaction of TLR4 - TLR4 was examined by FRET . HEK293 were transfected with TLR4 - EYFP TLR4 - ECFP ( D ) , followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . were washed with PBS fixed , then visualized with filter sets for CFP YFP . FRET were visualized with FRET filter set , expressed as corrected FRET efficiency displayed in quantitative pseudocolor indicating between ( arbitrary linear units of ) . Presented are representative from at least three independent experiments . Scale bar indicates 5 μm .

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The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


( A B ) Peritoneal macrophages from Atx + / + ( A ) Atx ∆ ΜΕ / ∆ ΜΕ ( B ) mice were stimulated with LPS ( 20 ng / mL , 20 min ) , followed by fixation in 4 % paraformaldehyde for 15 min at room with TLR4 - anti rabbit FITC ( Green ) , early endosomal marker EEA1 - anti mouse Dylight405 ( Blue ) , Alexa Fluor 594 - CTXB ( Red ) . TLR4 internalization was examined with confocal microscopy . Presented are representative from 3 independent experiments in which more than 95 % of exhibited similar results . Scale bar , 10 μm .

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(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with antibodies indicated . Presented is representative from three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


Raw264 . 7 were stimulated by LPS with ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with antibodies indicated . Presented is representative from three independent experiments .

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Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min : lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 , were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min lysates were subjected to immunoblotting with antibodies as indicated . regular Akt were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. regular Akt were used for a loading control. Presented are the representative image from at least three independent experiments.


Atx - heterozygous ( Atx - Het ) mice wild type ( C ) were stimulated by LPS indicated time point lysates were subjected to immunoblotting with antibodies as indicated . RasGAP were used for a loading control . Presented are representative image from at least three independent experiments .

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Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.


( D ) Raw264 . 7 were stimulated by LPS with ATX inhibitor . lysates were subjected to immunoblotting with antibodies as indicated . , β - Actin were used for a loading control . Presented are representative image from at least three independent experiments .

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(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. , β-Actin were used for a loading control. Presented are the representative image from at least three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with TLR2 ligand Peptidoglycan ( PGN , 30 ng / mL ) ( E ) for indicated time points . lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with Pam3CSK4 ( 1 μg / mL ) ( F ) for indicated time points . lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with Pam3CSK4 (1 μg/mL) (F) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with IL - 1β ( 100 ng / mL ) for indicated time points . : lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with IL-1β (100 ng/mL) for the indicated time points. : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with TNFα ( 25 ng / mL ) ( H ) for indicated time points . lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TNFα (25 ng/mL) (H) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


Peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + ( A ) were stimulated with LPS ( 50 ng / mL , 8 h ) culture medium was collected for ELISA to measure level of secreted cytokines . assays were performed in triplicate , data are shown as mean ± .

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Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) were stimulated with LPS (50 ng/mL, 8 h) The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Raw264 . 7 ( B ) were stimulated with LPS ( 50 ng / mL , 8 h ) in presence / absence of ATX inhibitor ( 50 μM , 30 min ) . culture medium was collected for ELISA to measure level of secreted cytokines . assays were performed in triplicate , data are shown as mean ± .

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Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Peritoneal macrophages were stimulated with LPS or vehicle ( endotoxin free water ) , after which intracellular cytokine production was measured through FACS . With scatter dot plot , gate was set so that viable were selected to analyze of signal by flow cytometry . overlay plot represents cytometry data .

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Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.


( F ) To induce sepsis via LPS injection ( Shirey et al . , 2013 ; Voss et al . , 2016 ) , ( 9 - 10 ) - sex - matched Atx ∆ ΜΕ / ∆ ΜΕ mice ( n = 9 ) Atx + / + ( n = 21 ) were injected with LPS ( i . p . 25 mg / kg ) . Survival was monitored for up to 16 h , analyzed by Kaplan - Meier method ( Log - rank P = 0 . 0004 ) .

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(F) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).


( G ) Blood samples were collected from Atx ∆ ΜΕ / ∆ ΜΕ ( n = 7 ) Atx + / + ( n = 6 ) mice 10 h after LPS injection , after which serum TNFα level was measured . data are shown as mean ± .

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(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.


Confocal scanning of macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + . were incubated with IgG - opsonized latex beads in absence / presence of LPS ( 20 ng / mL , 2 h ) stained with Phalloidin ( F - actin ) DAPI . Internalized beads were examined under a higher ( A )

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Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A)


Confocal scanning of macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + . were incubated with IgG - opsonized latex beads in absence / presence of LPS ( 20 ng / mL , 2 h ) stained with Phalloidin ( F - actin ) DAPI . Internalized beads were examined under a lower magnification ( B ) to quantify % phagocytosis by dividing latex bead - positive numbers by total number of DAPI - positive ( n = 11 - 16 per group ) . data are analyzed with results accumulated from three independent experiments shown as mean ± ( B ) . * * P < 0 . 01 ( Mann - Whitney U ) .

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Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).


( C ) LPS - stimulated macrophages were co - incubated with IgG - opsonized latex beads for 40 min . Phagocytic cups were visualized by F - actin with Phalloidin - iFluor 647 . arrow indicates phagocytic cup formed in plasma . Scale bar is 10 μm .

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(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.


After LPS ( 20 ng / mL , 4 h ) treatment , LPS - induced iNos , Arginase - 1 ( Arg - 1 ) , Ym - 1 mRNA expression were evaluated by semi - quantitative PCR ( D ) .

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After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D).


With LPS stimulation ( 20 ng / mL , 8 h ) , iNOS production was examined by immunoblotting ( E ) .

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With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).


( A ) Time - dependent killing of a panel of isolates in presence of meropenem ( MEM10 , 10 µg / ml ) . were grown overnight sub - cultured for 30 minutes at 37 ° C before being exposed to meropenem at 10 µg / mL moved to a 37 ° C stationary incubator . Samples were taken at each indicated time point spot - titered onto LB medium to quantify colony forming units ( CFU / mL ) throughout meropenem treatment . ( B ) of corresponding to time points in ( A ) were taken after labeling with stain FM4 - 64 . E . cloacae , Enterobacter cloacae ; K . aerogenes , Klebsiella aerogenes ; K . pneumoniae , Klebsiella pneumoniae ; E . coli , Escherichia coli

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(A) Time-dependent killing of a panel of bacterial isolates in the presence of meropenem (MEM10, 10 µg/ml). Cells were grown overnight and sub-cultured for 30 minutes at 37°C before being exposed to meropenem at 10 µg/mL and moved to a 37°C stationary incubator. Samples were taken at each indicated time point and spot-titered onto LB medium to quantify colony forming units (CFU/mL) throughout meropenem treatment. (B) images of cells corresponding to the time points in (A) were taken after labeling with membrane stain FM4-64. E. cloacae, Enterobacter cloacae; K. aerogenes, Klebsiella aerogenes; K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli


( D ) Regulatory modes of representative VxrB - controlled genes in response to PenG . Upper plot shows VxrB binding patterns ( assessed via ChIP - Seq ) , lower panel shows transcriptional response upon overexpression of VxrB D78E ( assessed via - Seq ) . expression was confirmed by S1 nuclease mapping in wild type ΔvxrAB mutant strains after exposure to PenG ( 100 µg / ml for 3 hours ) . demarcation between time points indicates that these data points were not adjacent in original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


Reduction of wall synthesis gene expression causes a tolerance defect . ( C ) Survival of a ∆ pbp1b ∆ pbp1a Piptg : pbp1a after 6 hours of PenG exposure . Pup indicates conditions of high IPTG concentrations ( 100 µM ) , Pdown low IPTG ( 2 µM ) . Survival fraction is cfu / ml of PenG - treated cultures normalized to untreated cultures grown in plated on medium containing same IPTG .

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Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.


( D ) Time - dependent killing experiment in presence of PenG ( 100 µg / ml , 10 x MIC ) . data are means ( + / - standard error ) of 3 independent biological replicates .

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(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.


( C ) Time - course of iron accumulation in PenG - treated WT ∆ vxrAB as measured by ICP - MS . Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ P = 0 . 001 ; ∗ ∗ ∗ P < 0 . 0003 ( paired t - ) .

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(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).


( B ) . To PenG - mediated induction of Fur - regulated genes , S1 nuclease mapping assays were performed on small ryhB , RyhB target sodB , hutA / B , coding for heme transport systems vc2212 , which encodes a component of a ferric citrate uptake system ) . were grown to mid - exponential phase followed by exposure to H2O2 or PenG for indicated duration . Numbers represent expression normalized to untreated control ( averages from three independent replicates ) . demarcation between time points indicates that these data points were not adjacent in original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


( B ) ( C ) Iron uptake mutants restore ∆ vxrAB growth on 10 % sucrose . indicated strains were grown to exponential phase ( OD600 ~ 0 . 5 ) in LB medium spot - plated on 10 % Sucrose plates with or without additional iron sulfate . An example is shown in ( B ) , of cfu is shown in ( C ) . Data information : Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ ∗ P < 0 . 0003 ; ∗ ∗ ∗ ∗ P < 0 . 0001 ( paired t - ) .

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(B) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).


( B ) Excretion of V . cholerae . Fecal samples were collected every two hours assessed for their viable content ( Colony Forming Units , CFU ) to monitor level of V . cholerae excretion . Each symbol represents fecal samples from an mouse ( n = 4 ) . number of CFU was normalized per milligram of feces ( CFU / mg ) . Error bars represent standard deviation . ∗ ∗ P < 0 . 005 ; ∗ ∗ ∗ P < 0 . 001 ( two - way ANOVA ) .

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(B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).


( E ) Time - dependent killing of respiratory chain mutants ( ∆ nqrA , encoding NADH dehydrogenase ∆ ubiA , encoding an early step of ubiquinone synthesis ) in presence of PenG ( 100 µg / ml , 10xMIC ) . strains were grown in LB 0 . 2 % glucose . Data information : Data are mean of three independent biological replicates error bars represent standard deviation .

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(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


( F ) Growth - phase dependent killing of ROS detoxification systems ( ∆ fur , ∆ sodB , ∆ oxyR1 ∆ katG ∆ katB ) . At designated time - points , a 5 ml aliquot was withdrawn exposed to 100 µg / ml PenG for 3 h . CFU / mL for each time point was assessed before ( solid lines ) after addition of PenG ( dotted lines ) . Data information : Data are mean of three independent biological replicates error bars represent standard deviation .

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(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


IIA1 . 6 B lymphoma were stimulated with BCR - ligand - ( IgM ) or BCR - ligand + ( IgG ) beads for 60 min , fixed stained for F - actin ( top ) α - tubulin ( bottom ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm.


Histograms show quantifications of polymerized tubulin filamentous F - actin at centrosome ( dashed outline on image , values correspond to fraction of in a 2 - micron - wide around centrosome relative to total in ) total amount of polymerized tubulin ( bottom right , values were normalized with respect to mean of control condition ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney . Scale bar : 2 µm .

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Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm.


differences of centrosomal F - actin centrosomal microtubule intensities in stimulated with BCR - ligand + beads with respect to stimulated with BCR - ligand - beads . data set is identical to panel ( B ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with one - sample t - ( i . e . comparison to a theoretical mean of " 0 " ) .

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Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


graph shows variations of total amount of tubulin per with respect to content of actin at centrosome in an XY representation of measurements . two lines correspond to linear regressions of two sets of data relative to stimulated with BCR - ligand + ( activated ) or BCR - ligand - ( resting ) beads .

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The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads.


IIA1 . 6 B lymphoma were fixed immuno - stained for F - actin ( red ) microtubules ( green ) . show projection of maximal of three confocal slices spaced by 0 . 5 µm apart from centrosome . Scale bars : 2 µm ( 0 . 5 µm in zoomed insets ) .

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IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).


IIA1 . 6 B lymphoma were treated 45 min with indicated inhibitors ( CK666 at 25 µM , SMIFH2 at 25µM ) or DMSO as control prior to being fixed stained for α - tubulin ( left column ) F - actin ( right column ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were treated 45 min with indicated inhibitors (CK666 at 25 µM, SMIFH2 at 25µM) or DMSO as control prior to being fixed and stained for α-tubulin (left column) and F-actin (right column). Scale bar: 3 µm.


Histograms show quantifications of amount of polymerized tubulin ( right , values were normalized with respect to mean of control condition ) filamentous actin at centrosome ( left , values correspond to fraction of in a 2 - micron - wide around centrosome relative to total in ) . Measurements were pooled from 3 independent experiments ; DMSO : n = 91 , CK666 : n = 82 , SMIFH2 : n = 74 , latrunculinA : n = 96 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney .

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Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right, values were normalized with respect to the mean of control condition) and filamentous actin at the centrosome (left, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell). Measurements were pooled from 3 independent experiments; DMSO: n= 91, CK666: n= 82, SMIFH2: n= 74, latrunculinA: n= 96. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test.


differences of centrosomal F - actin microtubule intensities in treated with cytoskeleton inhibitors in comparison with respective densities in treated with DMSO . Errors bars represent standard deviations . P values were calculated with one - sample t - ( i . e . comparison to a theoretical mean of " 0 " ) .

Percentage differences of centrosomal F-actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


graph shows same measurements as in panel ( B ) in an XY representation of measurements . three lines correspond to linear regressions of three sets of data relative to stimulated with each actin drug .

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The graph shows the same measurements as in panel (B) in an XY representation of individual measurements. The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug.


IIA1 . 6 B lymphoma were transfected to transiently express centrin1 - GFP ( red ) EB3 - mCherry ( green ) video - recorded at contact site with glass coverslip ( left ) at centrosome ( right ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were transfected to transiently express centrin1-GFP (red) and EB3-mCherry (green) and video-recorded at the contact site with the glass coverslip (left) and at the centrosome (right). Scale bar: 3 µm.


duration of EB3 - positive comets presence in bottom plane was measured in DMSO CK666 treated ( left ) . Error bars correspond to standard deviations . number of EB3 - positive comets exiting a 2 - µm wide centrosomal was also compared between two conditions ( right ) . In both cases P values were calculated with Mann - Whitney .

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The duration of EB3-positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells (left). Error bars correspond to standard deviations. The number of EB3-positive comets exiting the a 2-µm wide centrosomal area was also compared between the two conditions (right). In both cases P values were calculated with Mann-Whitney test.


IIA1 . 6 B lymphoma were transfected to transiently express centrin1 - VCA - GFP ( bottom ) or centrin1 - GFP ( top ) as control prior to be fixed stained for α - tubulin ( left column ) F - actin ( middle column ) . GFP signal of centrin1 or centrin1 - VCA is shown in right column to illustrate proper centrosome targeting . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were transfected to transiently express centrin1-VCA-GFP (bottom) or centrin1-GFP (top) as control prior to be fixed and stained for α-tubulin (left column) and F-actin (middle column). The GFP signal of centrin1 or centrin1-VCA is shown in the right column to illustrate the proper centrosome targeting. Scale bar: 3 µm.


Histograms show quantifications of amount of polymerized tubulin ( right ) filamentous actin at centrosome ( left ) . Values correspond to fraction of in a 2 - micron - wide around centrosome relative to total in . Measurements were pooled from 3 independent experiments ; centrin1 - GFP : n = 88 , centrin1 - VCA - GFP : n = 87 . Errors bars represent standard deviations . P values were calculated with Mann - Whitney .

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Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right) and filamentous actin at the centrosome (left). Values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell. Measurements were pooled from 3 independent experiments; centrin1-GFP: n= 88, centrin1-VCA-GFP: n= 87. Errors bars represent standard deviations. P values were calculated with Mann-Whitney test.


differences of F - actin microtubule intensities at centrosome were compared in transfected either with centrin1 - VCA - GFP or with centrin1 - GFP . Errors bars represent standard deviations . P values were calculated with one - sample t - ( i . e . comparison to a theoretical mean of " 0 " ) .

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Percentage differences of F-actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1-VCA-GFP or with centrin1-GFP. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


graph shows variations of total amount of tubulin per with respect to content of actin at centrosome . two lines correspond to linear regressions of two sets of data relative to transfected with centrin1 - VCA - GFP or centrin1 - GFP .

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The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome. The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1-VCA-GFP or centrin1-GFP.


Two sets of representative showing microtubules actin filaments assembled from isolated centrosomes . Centrosomes were isolated from Jurkat expressing centrin1 - GFP . Upper lower lines show actin filaments microtubules radiating from two distinct centrosomes with low ( top ) high ( bottom ) densities of actin filaments . Scale bars : 10 μm .

Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm.


graph shows number of microtubules per centrosome relative to density of actin filaments . Inset shows actin filaments at centrosome with a FIRE look - up table a 20 µm - wide circle in which actin is measured . Measurements were pooled from 5 independent experiments ; n = 50 .

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The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50.


Microtubules ( top ) actin filaments ( bottom ) assembly from isolated centrosomes in presence of increasing of monomeric actin ( from left to right ) . Scale bar : 20 μm .

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Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.


graph shows number of microtubules per centrosome in response to increasing concentrations of monomeric actin . Data were pooled from 2 independent experiments ; 0 µM : n = 21 ; 0 . 3 µM : n = 17 ; 0 . 5 µM : n = 17 ; 1 . 0 µM : n = 17 . * * * * : P < 0 . 001 Mann - Whitney .

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The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test.


image shows density of actin filaments ( in presence of 1µM of actin monomers ) at centrosome color - coded with FIRE look - up table definition of central , peripheral distal regions corresponding to decreasing concentrations of actin filaments . Scale bar : 20 μm . graph shows measurements of microtubule growth rate in each region . Data were pooled from 3 independent experiments : central zone : n = 58 , peripheral : n = 104 , outside : n = 61 ; n . s . means no statistical difference between data set according to Mann - Whitney .

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The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test.


graphs show various intensities of centrosome immuno - with antibodies against gamma - tubulin on same coverslip depending on presence / absence of actin filaments ( left ) or on amount of actin filaments ( right ) . Data were pooled from 3 independent experiments . Left graph : without actin n = 69 , with actin n = 26 , right graph n = 26 . N . s . means no statistical difference between data set according to Mann - Whitney .

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The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.


Schematic illustration of first dynamic : sequential addition of tubulin followed by tubulin actin on isolated centrosomes . Representative showing microtubules ( top ) merged of actin microtubules ( bottom ) for two steps of ; in presence of tubulin only ( left column ) in presence of tubulin actin ( right column ) . Scale bar : 10 µm . of differences in number of microtubules per centrosome between two stages of experiment described above on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were collected from a single experiment ; asters without actin filaments : n = 29 ; asters with actin filaments : n = 13 . Data were analyzed using Mann - Whitney .

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Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm. Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test.


Schematic illustration of second dynamic : tubulin is added to measure centrosome nucleation capacity washed out . Then actin is added followed by actin tubulin . Representative showing microtubules ( top ) merged of actin microtubule ( bottom ) during three steps of ; in presence of tubulin only ( left column ) , in absence of tubulin presence of actin ( middle column ) in presence of tubulin actin ( right column ) . Scale bar : 10 µm . Arrowheads indicate microtubules unable to re - grow after assembly of actin filaments . of differences in number of microtubules per centrosome between first last steps of experiment described above ( panels D E ) on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were pooled from 2 independent experiments ; asters without actin filaments : n = 24 ; asters with actin filaments : n = 13 . * * * * : P < 0 . 001 Student t - .

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Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin. Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments. Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.


Representative of microtubules ( top ) actin filaments ( bottom ) growth from micropatterns . Scale bars : 20 μm .

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Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm.


Schematic illustration of on micropatterned substrate . Tubulin was first added alone to measure nucleation capacity of each micropattern , then washed out . Later on , actin was added followed by actin tubulin . Finally , actin was rinsed out gelsolin was added to fully disassemble actin filaments . Representative showing microtubules ( top ) merged of actin microtubules ( bottom ) during four steps of ; in presence of tubulin only , in absence of tubulin presence of actin , in presence of tubulin actin , finally in presence of tubulin gelsolin but in absence of actin filaments ( from left to right ) . Scale bar : 10 μm .

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Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm.


of number of microtubules per micropattern in presence of tubulin only ( left ) , actin tubulin ( middle ) tubulin only after actin filaments disassembly ( right ) . Data were pooled from 2 independent experiments ; n = 133 . * * * * : P < 0 . 001 Student t - . graph shows same measurements as in panel ( E ) in an XY representation of measurements . It illustrates differences in number of microtubules per micropattern between first to second step ( tubulin only versus actin tubulin together ) with respect to density of actin filaments per micropattern .

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Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.


RPE1 stably expressing centrin1 - GFP were plated for 3 h on coverslips coated with different ratios ( 100 : 0 ; 50 : 50 or 1 : 99 ) of fibronectin PLL - PEG prior to fixation for F - actin ( top magnified views around centrosome below . Scale bars : 10 µm 2 µm , respectively ) α - tubulin ( bottom . Scale bar : 10 µm ) .

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RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).


IIA1 . 6 B lymphoma were plated for 60 min on poly - L - lysine , fibronectin or ICAM - 1 coated cover slides prior to be fixed stained for F - actin ( top ) α - tubulin ( bottom ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.


of occupied by RPE1 on substrate ( top left ) , F - actin content at centrosome ( top right ) polymerized tubulin at centrosome ( bottom left ) in entire ( bottom right ) for three conditions of adhesion described in ( B ) . Measurements came from 3 independent experiments with more than 60 analyzed in each . Errors bars represent standard deviations . F - actin microtubule contents were compared using Mann - Whitney , variations of were compared using unpaired t - .

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Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


of occupied by B lymphoma on substrate ( top left ) , F - actin content at centrosome ( top right ) polymerized tubulin at centrosome ( bottom left ) in entire ( bottom right ) for three conditions of adhesion described in ( D ) . Measurements came from 3 independent experiments with more than 80 analyzed in each . Errors bars represent standard deviations . F - actin microtubule contents were compared using Mann - Whitney , variations of were compared using unpaired t - .

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Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


( G ) GTT or ITT in 17 - week - old wild - type or JFKTG mice fed on HFD . Error bars represent mean ± ( n = 6 , * p < 0 . 05 , paired two - Student ' s t - ) .

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(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( H ) H & E Oil Red O of liver sections from wild - type or JFKTG mice fed on ND or HFD . Scale bar , 100 μm .

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(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.


( D ) of wild - type or JfkKO mice fed on HFD . Error bars represent mean ± ( n = 6 , * p < 0 . 05 , paired two - Student ' s t - ) .

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(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( G ) H & E Oil Red O of liver sections from wild - type or JfkKO mice fed on HFD . Scale bar , 100 μm .

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(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.


( H ) Wild - type or JfkKO mice were fed with HFD for 11 prior to injection of adenovirally - delivered vector or JFK via tail vein . Seven days after injection , total prepared from liver tissues were subjected to western blotting for expression of Jfk or measurement for hepatic TG NEFA content . Error bars represent mean ± ( n = 6 , * p < 0 . 05 , one - way ANOVA with Tukey ' s HSD ) . H & E Oil Red O of liver sections are shown . Scale bar , 100 μm .

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(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.


( A ) HepG2 were stably transfected with vector or FLAG - JFK . extracts were immunopurified with anti - FLAG immunoafﬁnity resin eluted with FLAG peptides . eluates were resolved on SDS - PAGE silver - stained . bands were retrieved analyzed by mass spectrometry .

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(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.


( B ) extracts from HepG2 were immunoprecipitated with antibodies against ING5 followed by immunoblotting with antibodies against indicated .

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(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.


( E ) HepG2 were transfected with FLAG - JFK treated with 50 μg / ml CHX for indicated times in presence or absence of MG132 for western blotting . Quantitation was done by densitometry expressed as signals of ING5 / β - actin .

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(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.


( H ) Bacterially - expressed GST - JFK or JFK mutants was incubated with in vitro transcribed / translated ING5 for in vitro ubiquitination assays .

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(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.


( D ) ChIP - seq track at representative promoter of target genes . red rectangles indicate peak regions / binding sites of ING5 on target genes promoter .

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(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.


( C ) HepG2 were treated with control siRNAs or siRNAs against JFK or / ING5 , or transfected with vector , JFK , or / ING5 . extracts were prepared for western blotting with indicated antibodies .

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(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.


( D ) HepG2 were transfected with vector or JFK , AMPKα1 , or / ING5 , or treated with control siRNAs or siRNAs against JFK , AMPKα1 , or / ING5 , extracts were prepared for AMPK activity assays by ELISA . Error bars represent mean ± SD for triplicate experiments ( * p < 0 . 05 , paired two - Student ' s t - ) .

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(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).


( D ) measurement of AMPK activity rate of fatty acid β - oxidation of wild - type or JfkKO mice . extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA . Mitochondria were isolated from MEFs or liver tissues , rate of fatty acid β - oxidation was quantified by spectrophotometric detection for ferricyanide - trapped reduced products . Error bars represent mean ± ( n = 6 , * p < 0 . 05 , paired two - Student ' s t - ) .

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(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


Wild - type or JfkKO mice were fed with HFD for 11 prior to injection of adenovirally - delivered vector control , shIng5 , or shAmpkα1 via tail vein . Seven days after injection , extracts prepared from liver tissues were subjected to western blotting or measurement for hepatic TG NEFA content ( E ) . Asterisk ( * ) represents significant comparison to wild - type mice , octothorpe ( # ) represents significant comparison to JfkKO mice . Error bars represent mean ± ( n = 6 , * p < 0 . 05 , # p < 0 . 05 , one - way ANOVA with Tukey ' s HSD ) .

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Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).


D . Expression distribution of lncORF in both groups by Ribo - seq .

D. Expression distribution of lncORF in both groups by Ribo-seq.


H . Scatter diagram of lncRNA - seq in HCT116 / L - OHP HCT116 .

H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.


A . Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation , PCR verification of 12 screened lncRNA in both RNC - total from two groups of CRC , among which translation level of lnc - AP ( marked with a green box ) was most notably dysregulated in both groups of drug - resistant ( n = 3 , biological replicates , mean ± , t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance .

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A. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance.


B . PCR of lnc - AP in serum samples from oxaliplatin - resistant ( PD , n = 15 ) - sensitive ( PR , n = 9 ) CRC ( mean ± , t ) .

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B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).


C . In public database lncCAR , high expression level of lnc - AP was correlated with long overall survival in CRC .

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C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients.


E . PCR WB of HEK293T transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± , Dunnett - t ) . F . PCR WB of HCT116 / L - OHP SW480 / L - OHP transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± , Dunnett - t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector .

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E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


J . IF detection of pep - AP in HCT116 / L - OHP SW480 / L - OHP transfected with either HA - lnc - AP OE plasmid . Scale bar , 10 µm .

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J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.


B . CCK - 8 of inhibition by L - OHP in two groups of CRC with down - or up - regulation of lnc - AP ( n = 3 , biological replicates , mean ± ) . Data information : * si - NC was abbreviation of commercial normal control of siRNA .

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B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


C . IF detection of H2AFX for damage by L - OHP in two groups of CRC with down - or up - regulation of lnc - AP , nuclei were stained with DAPI ( n = 3 , biological replicates , mean ± , Dunnett - t ) .

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C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).


G . Verification of interaction between CEBPA upstream region of AP002387 via ChIP followed by PCR ( n = 3 , biological replicates , mean ± , t ) . Data information : * for P < 0 . 05 ; * * for P < 0 .

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G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01


H . PCR of lnc - AP attenuation restoration in HCT116 SW480 ( n = 3 , biological replicates , mean ± , Dunnett - t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector si - NC was abbreviation of commercial normal control of siRNA .

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H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


J . CCK - 8 of inhibition by L - OHP in HCT116 SW480 with lnc - AP / pep - AP attenuation restoration ( n = 3 , biological replicates , mean ± ) . Data information : * si - NC was abbreviation of commercial normal control of siRNA .

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J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


D . Survival correlated with TALDO1 using TCGA data . Upper : Kaplan curve of overall survival probability for with high or low level of TALDO1 . Lower : expression graph of TALDO1 in who were dead or not .

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D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not.


F . IF detection of colocalization of pep - AP - Flag TALDO1 in HCT116 / L - OHP SW480 / L - OHP . Scale bar , 10 µm .

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F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.


G . WB confirmation of interaction between HA - pep - AP TALDO1 in HCT116 / L - OHP SW480 / L - OHP .

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G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.


A . WB of TALDO1 overexpression or knock down in two groups of CRC , which was quantified by gray on right ( n = 3 , biological replicates , mean ± , t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to corresponding plasmid vector si - NC was abbreviation of commercial normal control of siRNA .

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A. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


D . ROS was measured by flow cytometry shown in both curve bar charts ( n = 3 , biological replicates , mean ± , t ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to corresponding plasmid vector si - NC was abbreviation of commercial normal control of siRNA .

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D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . MMP detection . Scale bar , 10 µm . F . IF detection of H2AFX for damage by L - OHP . Scale bar , 10 µm .

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E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.


E . ROS was measured by flow cytometry shown in both curve ( left ) bar ( right ) charts ( n = 3 , biological replicates , mean ± , Dunnett - t ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector si - NC was abbreviation of commercial normal control of siRNA .

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E. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


F . MMP detection with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC . Scale bar , 10 µm .

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F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.


C . Flow cytometry of apoptosis under L - OHP with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC , which was quantified in bar charts on right ( n = 3 , biological replicates , mean ± , Dunnett - t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector si - NC was abbreviation of commercial normal control of siRNA .

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C. Flow cytometry of the cell apoptosis ratio under L-OHP with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.


E . CCK - 8 of inhibition by L - OHP with inhibitors of apoptosis Z - VAD - FMK or ferroptosis Fer - 1 in HCT116 / L - OHP SW480 / L - OHP ( n = 3 , biological replicates , mean ± ) . Data information : * Control referred to corresponding plasmid

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E. CCK-8 assay of the inhibition ratio by L-OHP with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector


B . Alterations of volume in different groups with or without pep - AP OE ( n = 6 , biological replicates , mean ± ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector .

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B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


F WB of pep - AP , TALDO1 , PARP , - PARP - caspase - 3 within tumors in C ( a ) .

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F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a).


G . Relative NADPH / NADP + within tumors in C ( a ) ( n = 6 , mean ± , Dunnett - t ) . H . GSH contents within tumors in C ( a ) ( n = 6 , mean ± , Dunnett - t ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to corresponding plasmid vector .

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G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.


I . IF detection of pep - AP , TALDO1 H2AFX , TUNEL of fragmentation of Ki67 within tumors in C ( a ) .

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I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).


( a , b ) HeLa were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( a ) Endogenous LC3 was detected by immunoblot in total lysates , quantified by densitometric normalized to actin . Relative were expressed in with control set to 100 shown in graph .

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(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph.


( a , b ) HeLa were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( b ) mRNA was obtained of Ptch1 or Gli1 actin mRNA were amplified by standard PCR visualized in agarose gels . Graph shows mRNA quantified by densitometric normalized to actin .

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(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin.


( c ) 8xGli - luciferase construct was transfected in HeLa for 24 h , followed by treatment with 10 μM purmorphamine , or DMSO as a control , for another 24 h . Graph represents mean value of firefly luciferase activity relative to Renilla transfection control .

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(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.


( d ) Endogenous LC3 - II was detected in lysates from treated with 10 μM purmorphamine for 24 h , either in absence or presence of trehalose ( 100 mM ) . Where indicated , bafA1 ( 400 nM ) was added for last 4 h . by densitometric relative to actin is shown in graph .

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(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.


( e ) were treated with 10 μM purmorphamine for 24 h under starvation conditions , either in presence of bafA1 or DMSO as a control , LC3 - II actin were detected .

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(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.


( f ) HA - HttQ74 construct was transfected into HeLa followed by treatment with 10 μM purmorphamine for 24 h . of transfected with detected by HA immunofluorescence is shown in graph . P - values were calculated by odds .

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(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.