( C ) Immunolabelling shows subcellular localization of hClC - 4 ( top , green in merge ) , hClC - 3 ( middle , green in merge ) hClC - 3unc ( bottom , green in merge ) of transiently transfected COS - 7 , in comparison to either PDI or Lamp - 1 ( both red ) as marker for ER late endosomes / lysosomes , respectively .

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(C) Immunolabelling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge) and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison to either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively.


( D ) Co - localization of hClC - 3 hClC - 4 in vesicles of COS - 7 transfected with hClC - 4 cDNA ( hemagglutinin ( HA ) tagged ) together with either hClC - 3 or hClC - 3unc . Immunostaining used antibodies against ClC - 3 HA tag ( green red in merge , respectively ) .

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(D) Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA (hemagglutinin (HA) tagged) together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively).


( A ) Clcn3unc / unc / Clcn4 - / - Clcn3 - / - mice died within 3 - 4 after birth . Approximately 20 % of survived in either ( Clcn3unc / unc / Clcn4 - / - , n = 136 Clcn3 - / - , n = 187 ) . Clcn3 - / - / Clcn4 - / - mice ( n = 4 ) died within 1 - 2 after birth .

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(A) Clcn3unc/unc/Clcn4-/- and Clcn3-/- mice died within 3-4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4-/-, n = 136 and Clcn3-/-, n = 187). All Clcn3-/-/Clcn4-/- mice (n= 4) died within 1-2 weeks after birth.


( B ) Nissl - stained paraffin sections show progressive neuronal loss ( arrows ) that begins in hippocampal CA1 region of P14 Clcn3unc / unc / Clcn4 - / - mice results in a complete loss of hippocampus at P28 . Neurodegeneration progresses slower in Clcn3 - / - mice Stobrawa et al , 2001 ( ) ( scale bar : 200 µm ) .

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(B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm).


( C ) Semi - thin sections of P28 retinae revealed degeneration of photoreceptor in outer layer outer inner segment of Clcn3unc / unc / Clcn4 - / - , but not of Clcn3unc / unc or Clcn4 - / - mice ( scale bar : 50 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer layer ; OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , ganglion layer .

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(C) Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4-/-, but not of Clcn3unc/unc or Clcn4-/- mice (scale bar: 50 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


( A ) GFP antibodies immunolabel VenusClC - 3 ( green ) in somata of CA1 pyramidal neurons of Clcn3ven / ven , but not Clcn3 + / + brain sections . sp , stratum pyramidale ( scale bar : 20 µm ) .

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(A) GFP antibodies immunolabel VenusClC-3 (green) in the somata of CA1 pyramidal neurons of Clcn3ven/ven, but not Clcn3+/+ brain sections. sp, stratum pyramidale (scale bar: 20 µm).


( B ) Co - immunolabelling of VenusClC - 3 ( green ) with ClC - 4 ( red ) in CA3 region of Clcn3ven / ven mice ( scale bar : 20 µm ) . sl , stratum lucidum ; sp , stratum pyramidale .

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(B) Co-immunolabelling of VenusClC-3 (green) with ClC-4 (red) in the CA3 region of Clcn3ven/ven mice (scale bar: 20 µm). sl, stratum lucidum; sp, stratum pyramidale.


( C ) Co - immunostaining of VenusClC - 3 ( green ) with EEA1 ( red , left panel ) , Lamp - 1 ( red , middle panel ) or synaptophysin ( red , right panel ) in either CA1 or CA3 region of Clcn3ven / ven mice . Arrows indicate structures of overlap ( scale bar : 5 µm ) .

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(C) Co-immunostaining of VenusClC-3 (green) with EEA1 (red, left panel), Lamp-1 (red, middle panel) or synaptophysin (red, right panel) in either CA1 or CA3 region of Clcn3ven/ven mice. Arrows indicate structures of overlap (scale bar: 5 µm).


( D ) GFP antibody immunolabels VenusClC - 3 ( green ) in neurites of cultured neurons . EEA1 VGLUT1 are co - stained ( both in red ; left right panel , respectively ) . dendritic marker MAP2 is stained in blue ( right panel ) ( scale bars : 10 µm ) . stained with DAPI .

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(D) GFP antibody immunolabels VenusClC-3 (green) in neurites of cultured neurons. EEA1 and VGLUT1 are co-stained (both in red; left and right panel, respectively). The dendritic marker MAP2 is stained in blue (right panel) (scale bars: 10 µm). DNA stained with DAPI.


( E ) Immuno - blot of fractions collected during preparation of SVs from brain of Clcn3ven / ven mice . VenusClC - 3 ( detected with GFP antibody ) co - purified with SV marker synaptophysin .

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(E) Immuno-blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven/ven mice. VenusClC-3 (detected with GFP antibody) co-purified with SV marker protein synaptophysin.


( F ) of immobilized Venus - tagged SVs imaged with TIRF microscopy . Scale bar , 2 µm .

(F) Fluorescence of immobilized Venus-tagged SVs imaged with TIRF microscopy. Scale bar, 2 µm.


( G , H ) Co - localization between Venus - tagged vesicles with antibodies against VGLUT1 synaptophysin was analyzed . ( G ) 11 . 4 ± 0 . 6 ( SD ) of VGLUT1 - labeled SVs 8 . 2 ± 2 ( SD ) of synaptophysin - labeled SVs showed Venus ( n = 6 ) . ( H ) 65 ± 5 . 4 ( SD ) 79 . 4 ± 3 . 8 ( SD ) of Venus - tagged SVs showed co - localization with VGLUT1 synaptophysin , respectively ( n = 6 ) .

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(G, H) Co-localization between the Venus-tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed. (G) 11.4 ± 0.6 (SD) of VGLUT1-labeled SVs and 8.2 ± 2 (SD) of synaptophysin-labeled SVs showed Venus fluorescence (n=6). (H) 65 ± 5.4 (SD) and 79.4 ± 3.8 (SD) of Venus-tagged SVs showed co-localization with VGLUT1 and synaptophysin, respectively (n=6).


( I ) Co - immunolabelling of VenusClC - 3 ( green ) with synaptophysin ( red ) of immobilized SVs imaged with TIRF microscopy . Vesicle in quadrant delimited by dashed is shown in higher magnification in merge . Scale bar , 2 µm .

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(I) Co-immunolabelling of VenusClC-3 (green) with synaptophysin (red) of immobilized SVs imaged with TIRF microscopy. Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge. Scale bar, 2 µm.


( A ) Immunoblots for ClC - 3 , ClC - 4 , VGLUT1 , VGLUT2 , VGAT , GluR4 GABAA - R - α1 of whole lysates from brain of Clcn3 + / + Clcn3 - / - mice at 2 , 6 12 of .

(A) Immunoblots for ClC-3, ClC-4, VGLUT1, VGLUT2, VGAT, GluR4 and GABAA-R-α1 of whole lysates from brain of Clcn3+/+ and Clcn3-/- mice at 2, 6 and 12 weeks of age.


( B ) Acidification of synaptic vesicle LP2 fractions from Clcn3unc / unc Clcn3 + / + mice at one year of ( upper panel , n = 2 each ≥ 3 measurements per ) , 10 - - old Clcn4 - / - WT mice ( middle panel , n = 3 each ≥ 3 measurements per ) , 4 - 6 - - old Clcn3unc / unc / Clcn4 - / - control mice ( lower panel , 6 each with ≥ 2 measurements per ) . A decrease in reflects acidification . protonophore FCCP dissipated pH gradient . Mean values ± s . e . m .

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(B) Acidification of synaptic vesicle LP2 fractions from Clcn3unc/unc and Clcn3+/+ mice at one year of age (upper panel, n= 2 animals each and ≥3 measurements per animal), 10-weeks-old Clcn4-/- and WT mice (middle panel, n= 3 animals each and ≥3 measurements per animal), and 4-6-weeks-old Clcn3unc/unc/Clcn4-/- and control mice (lower panel, 6 animals each with ≥2 measurements per animal). A decrease in fluorescence reflects acidification. The protonophore FCCP dissipated the pH gradient. Mean values ± s.e.m.


( C ) of ATP - induced acidification of LP2 fractions derived from 2 , 6 , 10 - 12 - old Clcn3 - / - compared to wild - type mice . Acidification measured by acridine orange in presence of 60 mM KCl . At least 2 in at least 2 independent experiments were pooled . Each measurement was performed at least 3 times . Mean ± s . e . m . is shown . * p < 0 . 05 , * * * p < 0 . 0005 ( two - unpaired t ) .

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(C) Quantification of ATP-induced acidification of LP2 fractions derived from 2, 6, and 10-12 weeks-old Clcn3-/- compared to wild-type mice. Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl. At least 2 animals in at least 2 independent experiments were pooled. Each measurement was performed at least 3 times. Mean ± s.e.m. is shown.* p < 0.05, *** p < 0.0005 (two-tailed unpaired t test).


( D ) Immunoblot revealed strong reduction of VGLUT1 in homogenate ( H ) LP2 fraction of Clcn3unc / unc / Clcn4 - / - mice .

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(D) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate (H) and LP2 fraction of Clcn3unc/unc/Clcn4-/- mice.


( E ) Endosomal pH of Clcn3 + / + Clcn3 - / - hippocampal neurons determined with a pH - sensitive transferrin conjugate . N = 5 independent lines with at least 2 live dishes per with about 10 each ≥ 10 Tfn - positive compartments per image genotype were analysed . Mean ± s . e . m . is shown .

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(E) Endosomal pH of Clcn3+/+ and Clcn3-/- primary hippocampal neurons determined with a pH-sensitive transferrin conjugate. N= 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥10 Tfn-positive compartments per image and genotype were analysed. Mean ± s.e.m. is shown.


Miniature inhibitory postsynaptic currents ( mIPSCs ) measured in CA1 pyramidal neurons in different mouse models at P14 - P16 . ( A ) example mIPSCs of WT ( left ) Clcn3 - / - neurons .

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Miniature inhibitory postsynaptic currents (mIPSCs) measured in CA1 pyramidal neurons in different mouse models at P14-P16. (A) example mIPSCs of WT (left) and Clcn3-/- neurons.


Mean values ± s . e . m . cumulative histogram of amplitude ( top ) frequency / interevent - interval ( bottom ) of mIPSCs in different mouse - models as indicated . Several of 3 mice of each genotype were analyzed . Compared genotypes were siblings . No difference in amplitude or frequency / interevent - interval distribution of mIPSCs was found in ( B ) Clcn3 - / - Clcn3 + / + ( Clcn3 + / + : 16 , Clcn3 - / - : 15 ; mean amplitude : p > 0 . 8 , t ; mean frequency : p > 0 . 4 , MW - ) , in ( C ) Clcn3unc / unc Clcn3 + / + ( Clcn3 + / + : 21 , Clcn3unc / unc : 13 ; mean amplitude : p > 0 . 7 ; mean frequency : p = 0 . 3 , t ) , in ( D ) Clcn4 - / - Clcn3 + / + ( Clcn3 + / + : 18 , Clcn4 - / - : 18 ; mean amplitude : p > 0 . 3 , t - ; mean frequency : p = 0 . 6 , MW - ) .

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Mean values ± s.e.m. and cumulative histogram of amplitude (top) and frequency/interevent-interval (bottom) of mIPSCs in different mouse-models as indicated . Several cells of 3 mice of each genotype were analyzed. Compared genotypes were siblings. No difference in amplitude or frequency/ interevent-interval distribution of mIPSCs was found in (B) Clcn3-/- and Clcn3+/+ (Clcn3+/+: 16 cells, Clcn3-/-: 15 cells; mean amplitude: p > 0.8, t test; mean frequency: p > 0.4, MW-test), in (C) Clcn3unc/unc and Clcn3+/+ (Clcn3+/+: 21 cells, Clcn3unc/unc: 13 cells; mean amplitude: p > 0.7; mean frequency: p = 0.3, t test), and in (D) Clcn4-/- and Clcn3+/+ (Clcn3+/+: 18 cells, Clcn4-/-: 18 cells; mean amplitude: p > 0.3, t-test; mean frequency: p = 0.6, MW-test).


( E ) No difference in mean amplitude ( p > 0 . 5 , t ) , in cumulative amplitude mean frequency of mIPSCs ( p > 0 . 1 , t ) was found in Clcn3unc / unc / Clcn4 - / - Clcn3 + / + / Clcn4 - / - . interevent - interval distribution was shifted to right , indicating a reduced frequency of mIPSCs in Clcn3unc / unc / Clcn4 - / - ( KS - , p < 0 , 05 ) ( Clcn3 + / + / Clcn4 - / - : 16 , Clcn3unc / unc / Clcn4 - / - : 12 ) .

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(E) No difference in mean amplitude (p > 0.5, t test), in cumulative amplitude and mean frequency of mIPSCs (p > 0.1, t test) was found in Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/-. The interevent-interval distribution was shifted to the right, indicating a reduced frequency of mIPSCs in Clcn3unc/unc/Clcn4-/- (KS-test, p < 0,05) (Clcn3+/+/Clcn4-/-: 16 cells, Clcn3unc/unc/Clcn4-/-: 12 cells).


( F , G ) miniature excitatory postsynaptic currents ( mEPSCs ) . ( F ) example traces of mEPSCs in WT Clcn3 - / - neurons ( G ) No significant difference between Clcn3unc / unc / Clcn4 - / - Clcn3 + / + / Clcn4 - / - mEPSCs in mean amplitudes ( p > 0 . 9 , t ) or in distribution of amplitudes ( Clcn4 - / - : 12 , 2 mice ; Clcn3unc / unc / Clcn4 - / - : 8 , 2 mice ) .

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(F,G) miniature excitatory postsynaptic currents (mEPSCs). (F) example traces of mEPSCs in WT and Clcn3-/- neurons (G) No significant difference between Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/- mEPSCs in mean amplitudes (p > 0.9, t test) or in the distribution of amplitudes (Clcn4-/-: 12 cells, 2 mice; Clcn3unc/unc/Clcn4-/-: 8 cells, 2 mice).


( H ) hippocampal neurons co - transfected with ClC - 3 synaptobrevin ( syb2 ) , both luminally tagged with pHluorin or pH - sensitive mOrange Ramirez et al , 2012 ( ) , respectively , were stimulated by 200 action potentials ( APs ) at 20 Hz ( indicated by bar ) . Vesicle exocytosis was monitored by increase upon exposure to extracellular neutral pH . Note that only Syb2 not ClC - 3 shows signs of exocytosis . Traces were corrected for photobleaching . initial decay of pHluorin signal is probably due to high exposure , which forces fluorophores into a transient - state Dean et al , 2011 ( ) . Averaged traces ( n = 10 ) , error bars , s . e . m .

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(H) Primary hippocampal neurons co-transfected with ClC-3 and synaptobrevin (syb2), both luminally tagged with pHluorin or the pH-sensitive mOrange Ramirez et al, 2012(), respectively, were stimulated by 200 action potentials (APs) at 20 Hz (indicated by bar). Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH. Note that only Syb2 and not ClC-3 shows signs of exocytosis. Traces were corrected for photobleaching. The initial decay of the pHluorin signal is probably due to the high light exposure, which forces the fluorophores into a transient dark-state Dean et al, 2011(). Averaged fluorescence traces (n=10), error bars, s.e.m.


B Pim1 , 2 , or 3 GFP - EDC3 expression vectors were transfected into HEK - 293T . GFP - Trap Nano - beads ( GFP - Trap ) were used to immunoprecipitate ( IP ) GFP - EDC3 , associated were analyzed by Western blotting with indicated Abs .

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B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs.


B HEK - 293T were transfected with Pim1 each EDC3 domain ( Fig . 2A ) fused to C - terminus of GFP - tagged vector . fragments were immunoprecipitated with GFP - Trap beads analyzed by Western blotting with indicated Abs . EDC3 wild type ( W ) , F1 , F2 , F3 , F4 were EDC3 fragments vector fused to GFP . C1 lane was only transfected with GFP Flag vector while C2 contained EDC3 wild type fused to GFP no Pim1 but empty vector control ( pcDNA3 ) .

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B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).


F PC3 - LN4 CRISPR - Ctr Pim1 KO were treated with PIMi ( PIM447 , 3μM ) , AKTi ( GSK690693 , 5μM ) , or combination for 24 h . Extracts were subjected to Western blotting with indicated Abs .

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F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.


of EDC3 / DCP1a foci number from Control ( Ctrl ) , PIMi , AKTi AKTi + PIMi combination in ( A ) . Data information : data of ( B are presented as mean + / - ( n = 30 / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - , are considered significant . p values > 0 . 05 not significant ( n . s . ) . analyses done included multiple independent fields .

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Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A). Data information: The quantification data of (B are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


D of DCP1a foci number for Control ( Ctrl ) , PIMi , AKTi , AKTi + PIMi combination in ( C ) . Data information : data of D ) are presented as mean + / - ( n = 30 / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - , are considered significant . p values > 0 . 05 not significant ( n . s . ) . analyses done included multiple independent fields .

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D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C). Data information: The quantification data of D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


A , B Western blot of whole - ( WCL ) P - ( PB ) purified from PC3 - LN4 stably expressing GFP - DCP1a used as a P - tag . P - bodies ( PB ) were isolated with GFP - Trap beads analyzed by Western blotting with indicated Abs .

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A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs.


A PC3 - LN4 Naïve CRISPR / Cas9 genome - edited EDC3 S161A ( SA ) , S161D ( Fredriksson et al . ) , EDC3 knock - out ( KO ) lines were plated ( 4000 / well in 24 well plates ) imaged every 12 h using phase contrast in IncuCyte ZOOM platform . graph shown was generated with IncuCyte Basic Software following 5 days of growth . experiments were done in triplicates . Values are mean + / - S . E . M . p values < 0 . 05 , calculated using unpaired student t - . Data information : invasion migration assays were performed in triplicates 6 different fields at 10x magnification were counted in 3 separate inserts . data are presented as mean + / - S . D . p values < 0 . 05 , calculated using unpaired student ' s t - .

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A PC3-LN4 Naïve and CRISPR/Cas9 genome-edited EDC3 S161A (SA), S161D (Fredriksson et al.), and EDC3 knock-out (KO) cell lines were plated (4000 cells/well in 24 well plates) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform. The graph shown was generated with IncuCyte Basic Software following 5 days of growth. The experiments were done in triplicates. Values are mean +/- S.E.M. p values<0.05, calculated using unpaired student t-test. Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test.


H Relative mRNA expression of ITGA6 , ITGB1 , KLF4 in PC3 - LN4 EDC3 KO - C60 , containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information : Real - time PCR

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H Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 EDC3 KO-C60 cells, containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information: Real-time PCR data


J Western blot of extracted from PC3 - LN4 treated with DMSO or combination of Pim447 AZD5363 for 96 h using specified antibodies .

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J Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.


A Two independently derived CRISPR / Cas9 PC3 - LN4 EDC3 S161A mutants ( EDC3 SA # 1 , # 2 ) wild type were injected subcutaneously in flank ( 5X106 ) of SCID mice ( n = 8 / group ) , growth was monitored until control group reached 1000 mm3 . subcutaneous volume measurement ( mm3 ) was done as outlined in Materials Methods . Data are presented as mean ± , ( n = 8 / group ) .

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A Two independently derived CRISPR/Cas9 PC3-LN4 EDC3 S161A mutants (EDC3 SA#1, #2) and wild type cells were injected subcutaneously in the flank (5X106) of SCID mice (n=8/group), and tumor growth was monitored until the control group reached 1000 mm3. The subcutaneous tumor volume measurement (mm3) was done as outlined in Materials and Methods. Data are presented as mean ± SEM, (n = 8/group).


B Phosphorylated EDC3 were examined in normal immortalized breast lines , MCF10A MCF12A , human breast cancer lines HCC38 , HCC1954 , MDA - MB - 231 , MDA - MD - 468 , BT - 549 , MRKNU1 , mouse 4T1 by Western blotting with indicated Abs .

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B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines, MCF10A and MCF12A, human breast cancer cell lines HCC38, HCC1954, MDA-MB-231, MDA-MD-468, BT-549, MRKNU1, and mouse 4T1 by Western blotting with the indicated Abs.


C Dual immunohistochemistry of human breast with p - EDC3 Ab ( Fredriksson et al . ) total EDC3 ( brown ) . Scale bar : 50µm . D Dual immunohistochemistry of human normal resected breast tissue sections with p - EDC3 Ab ( Fredriksson et al . ) total EDC3 ( brown ) . Scale bar : 50µm .

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C Dual immunohistochemistry staining of human breast tumor with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm. D Dual immunohistochemistry staining of human normal resected breast tissue sections with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.


( A ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG , subjected to indicated treatments stained with antibodies against FLAG lysosomal marker LAMP2 . FLAG LAMP2 channels are in green red , respectively , in merge . DAPI ( blue ) is included in merge . Scale bars represent 10 μm .

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(A) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm.


( B ) of number of with TFEB - 3 × FLAG in four conditions in ( A ) . Each value represents mean ± s . d . from three independent fields with N = 300 .

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(B) Quantification of the number of cells with nuclear TFEB-3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300.


( C ) Immunofluorescence of HEK ‐ 293T treated as indicated stained with antibodies against endogenous TFEB lysosomal RagC ( green red , respectively , in merge ) . DAPI is included in merge . Scale bars represent 10 μm .

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(C) Immunofluorescence of HEK‐293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm.


( D ) Immunoblotting of extracted from HeLa that express TFEB - 3 × FLAG treated with DMSO , chloroquine ( CQ ) or SalA , subjected to / fractionation blotted with antibody against FLAG to detect TFEB . H3 tubulin were used as markers , respectively . Blots are representative of triplicate experiments .

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(D) Immunoblotting of proteins extracted from HeLa cells that express TFEB-3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.


( A ) Lysosomal stress inhibits mTOR signalling . Immunoblotting of extracts isolated from HeLa treated overnight as indicated . Membranes were probed with antibodies against p ‐ T202 / Y204 ‐ ERK1 / 2 , ERK1 / 2 , p ‐ T389 ‐ S6K , S6K to measure ERK mTORC1 activities .

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(A) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p‐T202/Y204‐ERK1/2, ERK1/2, p‐T389‐S6K, and S6K to measure ERK and mTORC1 activities.


( B ) Torin 1 induces TFEB dephosphorylation translocation . FLAG immunoblotting of fractions isolated from TFEB - 3 × FLAG HeLa starved in acid ‐ free media subsequently stimulated as indicated for at least 3 h . Correct subcellular fractionation was verified with H3 tubulin antibodies .

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(B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB-3 × FLAG HeLa cells starved in amino acid‐free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies.


( C ) Effects of ERK mTOR inhibitors on TFEB translocation . TFEB - GFP HeLa were seeded in 96 ‐ well plates , cultured for 12 h , then treated with indicated concentrations of ERK inhibitor U0126 , or mTOR inhibitors Rapamycin , Torin 1 , Torin 2 . After 3 h at 37 ° C , were processed were acquired using OPERA automated confocal microscope ( Perkin ‐ Elmer ) . Scale bars represent 30 μm .

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(C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB-GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin‐Elmer). Scale bars represent 30 μm.


( D ) Dose - response curves of effects of ERK mTOR inhibitors on TFEBnuclear translocation . TFEB - GFPHeLa were seeded in 384 ‐ well plates , cultured for 12 h , treated with 10 different concentrations of ERK inhibitor U0126 , or mTOR inhibitors Rapamycin , Torin 1 , Torin 2 ranging from 2 . 54 nM to 50 μM . graph shows of translocation at different concentrations of each compound ( in log of ) . EC50 for each compound was calculated using Prism software ( see Materials methods for details ) .

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(D) Dose-response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation. TFEB-GFPHeLa cells were seeded in 384‐well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details).


( E ) Immunofluorescence of HEK ‐ 293T treated with DMSO or Torin 1 stained with antibodies against endogenous TFEB lysosomal RagC ( green red , respectively , in merge ) . DAPI is included in merge . Scale bars represent 10 μM .

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(E) Immunofluorescence of HEK‐293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM.


( F ) Rag GTPase knockdown induces TFEB translocation . HeLa stably expressing TFEB - 3 × FLAG were infected with lentiviruses encoding Short hairpin ( Sh ‐ ) RNAs targeting luciferase ( control ) or RagC RagD mRNAs . In samples , 96 h post infection , were left untreated ( N = normal media ) , starved ( S = starved media ) or treated with Torin 1 ( T = Torin 1 ) for 4 h then subjected to / fractionation . TFEB localization was detected with a FLAG antibody , whereas tubulin H3 were used as controls for fraction , respectively ; of S6K phosphorylation were used to RagC RagD knockdown efficiency .

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(F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB-3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh‐) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency.


( G ) Loss of mTORC2 does not affect TFEB phosphorylation . Mouse embryonic fibroblasts ( MEFs ) isolated from Sin1 − / − or control ( E14 . 5 ) were infected with a retrovirus encoding TFEB - 3 × FLAG ; 48 h post infection , were treated with Torin 1 ( T ) for 4 h where indicated , subjected to / fractionation immunoblotted for FLAG , tubulin , H3 .

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(G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB-3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3.


( H ) Binding of TFEB to mTORC1 . HEK ‐ 293T that express TFEB - 3 × FLAG were lysed subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR , mTORC1 subunit raptor mTORC2 components rictor Sin1 . FLAG - Rap2A served as negative control .

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(H) Binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG-Rap2A served as negative control.


( A ) Torin 1 induces S142 dephosphorylation . HeLa were treated as indicated total extracts were probed with a TFEB p ‐ S142 phosphoantibody with anti ‐ FLAG antibody . Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in fraction .

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(A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction.


( B ) mTORC1 in ‐ vitro kinase assays . Highly purified FLAG - S6K1 , TFEB - 3 × FLAG , or TFEBS142A - 3 × FLAG were incubated with radiolabelled ATP without kinase , with purified mTORC1 or with mTORC1 + Torin 1 , analysed by autoradiography . lower panel shows a FLAG immunoblot of substrates .

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(B) mTORC1 in‐vitro kinase assays. Highly purified FLAG-S6K1, TFEB-3 × FLAG, or TFEBS142A-3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates.


( D ) conservation scores of phosphosites quantitative agreement between mTOR consensus motif around phosphosites of TFEB .

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(D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB.


( E ) S142 S211 regulate TFEB localization . FLAGimmunostaining ( red ) of HeLa expressing serine ‐ to ‐ alanine mutated versions of TFEB - 3 × FLAG . Nuclei were stained with DAPI ( blue ) . Values are means of five fields containing at least 50 transfected . Student ' s t ‐ ( unpaired ) * * * P0 . 001 . Scale bars represent 30 μm .

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(E) S142 and S211 regulate TFEB localization. FLAGimmunostaining (red) of HeLa cells expressing serine‐to‐alanine mutated versions of TFEB-3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t‐test (unpaired) ***P0.001. Scale bars represent 30 μm.


( A ) Spinning disk confocal image of a MEF that co ‐ expresses TFEB - GFP mRFP - Rab7 ( green red in merge , respectively ) .

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(A) Spinning disk confocal image of a MEF cell that co‐expresses TFEB-GFP and mRFP-Rab7 (green and red in the merge, respectively).


( B ) Time ‐ lapse of TFEB ‐ Rab7 ‐ positive lysosomes from boxed region in ( A ) . Time intervals are in seconds .

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(B) Time‐lapse of TFEB‐ and Rab7‐positive lysosomes from the boxed region in (A). Time intervals are in seconds.


( C ) Time ‐ lapse of Torin 1 treatment in a MEF expressing TFEB - GFP . Arrow indicates time of Torin 1 addition . Yellow arrowheads indicate Torin 1 ‐ induced lysosomal accumulation of TFEB - GFP . Time intervals are in minutes .

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(C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB-GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB-GFP. Time intervals are in minutes.


( D ) Immunofluorescence of HEK ‐ 293T expressing TFEB - 3 × FLAG , treated with DMSO ( top ) or Torin1 ( bottom ) stained with antibodies against FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(D) Immunofluorescence of HEK‐293T cells expressing TFEB-3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( E ) FRAP of TFEB - GFP ‐ positive lysosomes from control MEFs ( blue ) or MEFs treated with Torin 1 ( red ) . Each data point represents mean ± s . d . from five independent spots .

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(E) FRAP analysis of TFEB-GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots.


( F ) Time ‐ lapse of photobleaching recovery of TFEB - GFP ‐ positive lysosomes from control ‐ treated MEFs ( top ) or MEFs treated with Torin 1 ( bottom ) . Red arrowheads indicate time of photobleaching . Time intervals are in seconds .

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(F) Time‐lapse of photobleaching and fluorescence recovery of TFEB-GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds.


( G ) Torin 1 increases binding of TFEB to mTORC1 . HEK ‐ 293T that express TFEB - 3 × FLAG along with HAGST ‐ Rap2A or HAGST ‐ RagsDN were treated with vehicle or with Torin1 , lysed subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR raptor . FLAG - Metap2 served as negative control .

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(G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG-Metap2 served as negative control.


( H ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with Rap2A ( top ) or RagsDN mutants ( bottom ) , treated with Torin 1 stained with antibodies against FLAG LAMP2 ( green red in merge , respectively ; DAPI is in blue ) . In , scale bars represent 10 μm .

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(H) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.


( A - C ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with a control GTPase or indicated Rag mutants . were deprived of ( top ) or deprived then stimulated ( bottom ) for indicated times stained for FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(A-C) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( D ) of number of with TFEB from each condition in ( A - C ) .

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(D) Quantification of the number of cells with nuclear TFEB from each condition in (A-C).


( E , F ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with Rap2A ( E ) or RagsCA mutants ( F ) , subjected to indicated treatments stained with antibodies against FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(E, F) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (E) or the RagsCA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( G ) of number of with TFEB from DMSO ‐ CQ ‐ treated fields in ( E ) ( F ) . In fields , scale bars represent 10 μm . In histograms , each value represents mean ± s . d . from three independent fields with N = 300 .

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(G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.


( A ) Chloroquine treatment inhibits mTORC1 activity in hepatocytes . hepatocytes isolated from 2 ‐ month ‐ old Tcfebflox / flox ( control ) Tcfebflox / flox ; Alb ‐ Cre ( Tcfeb − / − ) mice were left untreated , or treated overnight with Torin 1 , U0126 , or Chloroquine . Subsequently , were lysed extracts were immunoblotted with indicated antibodies .

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(A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies.


( B , C ) TFEB mediates transcriptional response to chloroquine Torin 1 . Quantitative PCR ( qPCR ) of TFEB target genes in hepatocytes from control ( flox / flox ) Tcfeb − / − mice . were treated with Chloroquine ( left ) or Torin 1 ( right ) . graphs show relative increased expression in treated versus corresponding untreated samples . Values represent means ± s . d . of three independent hepatocyte preparations ( three mice / genotype ) . Student ' s t ‐ ( two ) * P ‐ value ⩽ 0 . 05 .

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(B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.


( A - B ) of miR - 122 in Huh7 released EVs either untreated ( Fed ) or subjected to starvation for metabolites including for 16h ( Starved ) . miR - 122 signals were detected by Northern blotting position of 32P - labelled Oligos that served as markers are shown in lane ( A ) . U6 snRNA was used as loading control .

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(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control.


( A - B ) of miR - 122 in Huh7 released EVs either untreated ( Fed ) or subjected to starvation for metabolites including for 16h ( Starved ) . CAT - 1 were measured by qRT - PCR using GAPDH mRNA as control ( B ) . A scheme of experiment is shown in top panel in B .

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(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.


( C - D ) extracellular of different miRNAs EV associated in Fed Starved Huh7 . Relative changes in extracellular ( EV associated ) of miR - 122 miR - 24 in Fed or 8h Starved Huh7 were quantified by qRT - PCR plotted ( mean + / - s . e . m . , n = 3 ) . of miRNAs in Fed condition were taken as unit . Fold change ( with SD ) in level of miRNAs pre - miR - 122 measured were shown in bottom panel ( C ) . miRNA were normalized against U6 snRNA .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV associated) of miR-122 and miR-24 in Fed or 8h Starved Huh7 cells were quantified by qRT-PCR and plotted (mean+/- s.e.m., n=3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of all miRNAs and pre-miR-122 measured were shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA.


( C - D ) extracellular of different miRNAs EV associated in Fed Starved Huh7 . Western blot of different exosomal Ago2 in EVs isolated under Fed or Starved conditions . CD63 is an exosomal marker of HuR in EVs were quantified against respective CD63 . Mean of three independent measurements are plotted ( middle panel in D ) . Calnexin GAPDH are not detected in EVs but visible in extract . EV associated HA - HuR for Fed Starved Huh7 expressing HA - HuR were also detected in bottom panel in D .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements are plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV associated HA-HuR levels for Fed and Starved Huh7 cells expressing HA-HuR were also detected in the bottom panel in D.


( E ) Effect of GW4869 treatment on miRNA content in Fed Starved . of miRNAs were measured by real - time normalized against U6 snRNA . Mean data are from three independent experiments . DMSO treatment was used as control for GW4869 treated .

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(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.


( F ) CAT - 1 Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 . qRT - PCR techniques was adopted for using 18S rRNA values for normalization ( mean + / - s . e . m . , n = 3 ) .

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(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).


( G ) Effect of siSMPD2 treatment on miR - 122 content of EVs from Fed Starved Huh7 . EVs from Fed Starved Huh7 , depleted for SMPD2 , were isolated miR - 122 content were measured by qRT - PCR normalized against content of EVs . Fold change in EV associated miR - 122 upon starvation both for control siSMPD2 treated were shown above respective bars .

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(G) Effect of siSMPD2 treatment on miR-122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 content were measured by qRT-PCR and normalized against protein content of EVs. Fold change in EV associated miR-122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars.


( H ) Effect of siSMPD2 treatment on CAT - 1 mRNA content ( left panel ) miR - 122 ( right panel ) in Huh7 . RNAs , from Fed Starved Huh7 , depleted for SMPD2 , were isolated miR - 122 CAT - 1 mRNA contents were measured by qRT - PCR . miR - 122 contents were normalized against U6 snRNA . GAPDH mRNA was used for normalization of CAT - 1 mRNA ( mean + / - s . e . m . , n = 3 ) . miR - 122 for siCon treated ( Fed Starved ) were considered as units .

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(H) Effect of siSMPD2 treatment on CAT-1 mRNA content (left panel) and miR-122 levels (right panel) in Huh7 cells. RNAs, from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 and CAT-1 mRNA contents were measured by qRT-PCR. miR-122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT-1 mRNA levels (mean +/- s.e.m., n=3). miR-122 levels for siCon treated cells (Fed and Starved) were considered as units.


( I - J ) A schematic representation of experiment to separate Extracellular Vesicles ( EVs ) on OptiPrep @ density gradient ( left panel ) . Densities of fractions 1 - 10 are plotted a best - fit curve is drawn ( right upper panel ) . CD63 in fractions of Fed Starved were detected by western blots to confirm presence of exosomes of Fed Straved ( right lower panel ) . Mean Ct values of miR - 122 in RNAs isolated from Exosome enriched ( 8 - 9 ) vs Non - exosomal ( 1 - 3 ) fractions were analyzed plotted ( J ) ( mean + / - s . e . m . , n = 3 ) .

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(I-J) A schematic representation of the experiment to separate Extracellular Vesicles (EVs) on OptiPrep@ density gradient (left panel). Densities of fractions 1-10 are plotted and a best-fit curve is drawn (right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells (right lower panel). Mean Ct values of miR-122 in RNAs isolated from Exosome enriched (8-9) vs Non-exosomal (1-3) fractions were analyzed and plotted (J) (mean +/- s.e.m., n=3).


( K ) Effect of Starvation on apoptic numbers in Huh7 . Western Blot detection of apoptosis marker Cytochrome C in in EV associated fractions of Fed Starved Huh 7 . β - Actin CD63 are used as loading control for EV associated fraction respectively ( left panel ) . TUNEL positive in Fed Starved Huh7 . TUNEL assays of Fed Starved Huh 7 were performed TUNEL positive were quantified . Fed , but DNase treated , were used as positive control ( + ve control ) ( right panel ) .

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(K) Effect of Starvation on apoptic cell numbers in Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells. β-Actin and CD63 are used as loading control for cellular and EV associated fraction respectively (left panel). TUNEL positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified. Fed, but DNase treated, cells were used as positive control (+ve control) (right panel).


( L ) Effect of thapsigargain ( TG ) treatment of Huh7 on EV associated miR - 122 level . A schematic representation of experiment ( upper panel ) . miR - 122 were measured by qRT - PCR in total in EVs released by treated . Values were normalized either against U6 snRNA or content of EVs ( mean + / - s . e . m . , n = 3 ) ( lower panels ) for EV associated respectively .

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(L) Effect of thapsigargain (TG) treatment of Huh7 cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.


( M ) Effect of TG on level of eIF2 - a its phosphorylated form , Ago2 CD63 in EVs . b - Actin was used as loading control for samples .

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(M) Effect of TG on cellular level of eIF2-a and its phosphorylated form, and Ago2 and CD63 in EVs. b-Actin was used as loading control for cellular samples.


( A ) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 . Immunoprecipitated materials were separated into two equal halves to do western blot of HuR relative of associated miR - 122 miR - 21 were done by quantitative RT - PCR ( mean + / - s . e . m . , n = 3 ) . GFP used as negative control to confer specificity of anti - HuR3A2 antibody in immunoprecipitating HuR . miR - 122 content in GFP - antibody immunoprecipitated materials was too low to get detected reliably using qRT - PCR .

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(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR.


( B - C ) Effect of HuR depletion on EV content of miRNAs in Huh7 . Schemes of HuR depletion experiments in Huh7 ( B ) . In siHuR treated , depletion of HuR was monitored by western blotting ( mean + / - s . e . m . , n = 4 )

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4)


( B - C ) Effect of HuR depletion on EV content of miRNAs in Huh7 . Relative of miRNAs were measured in EVs released from siCon or siHuR treated by qRT - PCR . miRNA contents were normalized against U6 snRNA . of each miRNA in control siRNA ( siCon ) treated set were taken as unit . ( C ) . CAT - 1 Aldolase mRNA of siHuR siCon treated Fed Huh7 were quantified by qRT - PCR normalized against GAPDH mRNA .

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA.


( D ) Effect of HuR knockdown on level of different miRNAs in starved Huh7 . Relative were estimated by real time qPCR , normalized with respect to U6 snRNA plotted in left panel . of miR - 122 in EVs of starved siHuR siCon treated Huh7 were also measured by RT - PCR plotted in right panel ( mean + / - s . e . m . , n = 3 ) .

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(D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3).


( E ) Level of eIF - 2a its phosphorylated form , estimated by western blot using specific antibodies , in siCon or siHuR treated starved Huh7 extracts . Western blot data of HuR confirm reduction of this upon siRNA treatment . CAT - 1 Aldolase mRNA were estimated by real time normalized against GAPDH level . Values of siCon treated samples were taken as unit ( mean + / - s . e . m . , n = 3 ) . In right panel relative quantity of phosphorylated eIF - 2a measured by densitometry were plotted . of p38 phosphor - p38 in siCon siHuR treated Starved Huh7 were detected by western blot .

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(E) Level of eIF-2a and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2a measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot.


( F ) Western blot to detect level of phosphorylated eIF - 2a eIF - 4E - BP1 in lysates of Starved HuR depleted Huh7 either transfected with ant - let - 7a or anti - miR - 122 oligos . miR - 122 inactivation was monitored by measuring cat - 1 level by qRT - PCR using GAPDH as control ( mean + / - s . e . m . , n = 3 ) . * denotes phosphorylated eIF - 4E - BP1 . b - Actin was used as loading control . Relative of phosphorylated eIF - 2a were measured by densitometric estimation of three independent measurements .

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(F) Western blot analysis to detect the level of phosphorylated eIF-2a and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. b-Actin was used as loading control. Relative levels of phosphorylated eIF-2a were measured by densitometric estimation of three independent measurements.


( A ) Northern blot of miR - 122 in Huh7 expressing Control HA - HuR expression plasmids . U6 was used for loading control . Position of 32P - labelled Oligos , blotted to used for Northern blotting , served as markers are shown in M lane .

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(A) Northern blot of miR-122 in Huh7 cells expressing the Control and HA-HuR expression plasmids. The U6 was used for loading control. Position of the 32P-labelled Oligos, blotted to the membrane used for Northern blotting, served as size markers are shown in the M lane.


( B ) Schematic representation of experiments done in Huh7 expressing HA - HuR ( bottom ) . For control , pCIneo transfected Huh7 were used . Relative expression of HuR in transfected were analysed by western blot ( top ) . b - actin blots were used as loading controls .

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(B) Schematic representation of experiments done in Huh7 cells expressing HA-HuR (bottom). For control, pCIneo transfected Huh7 cells were used. Relative expression of HuR in transfected cells were analysed by western blot (top). b-actin blots were used as loading controls.


( C ) EV - associated miRNA were measured in Huh7 either expressing pCIneo or HA - HuR expressing vectors by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . miRNA were normalized against U6 snRNA .

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(C) Cellular and EV-associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA-HuR expressing vectors by qRT-PCR (mean+/- s.e.m., n=5). Cellular miRNA levels were normalized against U6 snRNA.


( E ) Effect of HA - HuR expression on miRNA on EV non - EV fractions of culture supernatant collected from control HA - HuR encoding plasmid transfected Huh7 . For isolation of non - EV fraction , supernatant obtained after removal of EVs by ultracentrifugation was used for preparation ( left panel ) ( mean + / - s . e . m . , n = 3 ) . Effect of HuR expression on CD63 , Alix HuR in EVs . Extracts from EVs isolated from control or HA - HuR expressing Huh7 were western blotted for respective ( right panel ) .

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(E) Effect of HA-HuR expression on miRNA levels on EV and non-EV fractions of the culture supernatant collected from control and HA-HuR encoding plasmid transfected Huh7 cells. For isolation of non-EV fraction, supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation (left panel) (mean+/- s.e.m., n=3). Effect of HuR expression on CD63, Alix and HuR levels in EVs. Extracts from EVs isolated from control or HA-HuR expressing Huh7 cells were western blotted for respective proteins (right panel).


( F ) Relative miR - 122 in CD63 positive affinity purified EVs isolated from HA - HuR pCIneo transfected Huh7 were quantified by qRT - PCR ( mean + / - s . e . m . , n = 3 ) . of miR - 122 were normalized against CD63 content of EVs .

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(F) Relative miR-122 levels in CD63 positive affinity purified EVs isolated from HA-HuR and pCIneo transfected Huh7 cells were quantified by qRT-PCR (mean+/- s.e.m., n=3). The levels of miR-122 were normalized against CD63 content of EVs.


( G ) Effect of GW4869 on EV associated content of three different miRNAs in HA - HuR expressing Huh7 quantified by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . EVs from DMSO treated were used as control .

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(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.


( H ) A schematic representation of experiment done to characterize EVs isolated from Huh7 expressing HA - HuR or pCIneo respectively by running OptiPrepTM density gradient ( left panel ) western blotting for CD63 ( right upper panel ) . Relative miR - 122 in fractions 1 - 2 8 - 9 were compared by qRT - PCR ( right lower panel ) .

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(H) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA-HuR or pCIneo respectively by running OptiPrepTM density gradient (left panel) and western blotting for CD63 (right upper panel). Relative miR-122 levels in fractions 1-2 and 8-9 were compared by qRT-PCR (right lower panel).


( I ) of apoptic in HA - HuR expressing Huh7 . Western Blot detection of apoptosis marker Cytochrome C was done ( top ) for EV associated fractions of HA - HuR pCIneo ( control ) expressing Huh 7 respectively . Western blot for HA - HuR was done to analyse expression of HA - HuR in EVs isolated from HA - HuR pCIneo expressing Huh7 . CD63 β - actin are used as loading control for EV associated fractions fractions respectively ( upper panels ) . TUNEL of HA - HuR pCIneo expressing non - transfected Huh 7 was performed representative pictures of HA - HuR pCIneo expressing along with picture of non - transfected but DNase treated ( positive control ) were compared ( lower panels ) . Scale bar 50 µM .

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(I) Levels of apoptic cells in HA-HuR expressing Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C was done (top) for EV associated and cellular fractions of HA-HuR and pCIneo (control) expressing Huh 7 cells respectively. Western blot for HA-HuR was done to analyse the expression of HA-HuR in EVs isolated from HA-HuR and pCIneo expressing Huh7 cells. CD63 and β-actin are used as loading control for EV associated fractions and cellular fractions respectively (upper panels). TUNEL assay of HA-HuR and pCIneo expressing and non-transfected Huh 7 cells was performed and representative pictures of HA-HuR and pCIneo expressing cells along with picture of non-transfected but DNase treated (positive control ) cells were compared (lower panels). Scale bar 50 µM.


( B ) Change in miR - 122 level in starved mouse liver . Endogenous miR - 122 ( left panel ) CAT - 1 mRNA ( middle panel ) in mouse liver starved for 12h were quantified by qRT - PCR . miR - 122 in blood serum of corresponding were also measured ( right panel ) . Ct values of miR - 122 CAT - 1 mRNA obtained for each liver blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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(B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( C ) Relative level of HuR in both liver lung of Fed Starved mice were analysed by western blots , β - actin was used as loading control .

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(C)Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control .


( E ) Western Blot to check expression of HA - HuR in liver samples of injected mice . β - Actin was used as loading control . HA - HuR expressing Huh7 lysate was used as positive control .

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(E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control.


( F ) . Change in miR - 122 level upon expression of HA - HuR in mouse liver . Endogenous miR - 122 level in HA - HuR expression plasmid injected mouse liver were quantified by RT - PCR compared against liver samples collected from control ( pCIneo injected ) group of ( mean + / - s . e . m . , n = 5 ) .

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(F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5).


( G ) Endogenous miR - 122 target CAT - 1 ( left panel ) Aldolase ( right panel ) mRNA in HA - HuR or pCIneo ( control ) injected mouse liver were quantified by RT - PCR ( mean + / - s . e . m . , n = 4 ) .

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(G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4).


( H ) miR - 122 in blood serum of pCIneo ( control ) or HA - HuR expression plasmid injected mice were measured . Ct values of miR - 122 CAT - 1 Aldolase mRNAs obtained for each liver blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( A ) Time course experiment of HuR binding of miR - 122 replaced from Ago2miRNPs . A schematic representation of in vitro miRNA binding of HuR ( left panel ) . Equal amounts of recombinant HuR ( rHuR ) , after indicated time of miRNP interaction on FLAG - beads , were immunoprecipitated HuR associated miR - 122 were detected by qRT - PCR ( right top panel ) . Ago2western blot detects its presence in FLAG beads used in its absence in HuRimmunoprecipitated materials . HuRwestern blot was used to confirm its presence of its in immunoprecipitated materials , at different time points ( right bottom panel ) .

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(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).


( C ) Coomassie stained gel picture denotes purity of recombinant HuR used in . same were western blotted for HuR to show specificity of antibody used for immunoprecipitation . recombinant P N of Chandipura was used as negative control to confirm specificity of 3A2 anti - HuR antibody .

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(C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody.


( D ) HuR western blot in middle right panel confirmed presence of HuR truncated version of it in immunoprecipitated materials . A representative domain picture of HuR HuRΔIII are shown in upper panel . Relative of HuR bound miR - 122 were determined by qRT - PCR ( mean + / - s . e . m . , n = 3 ) ( middle left panel ) . Amount of miR - 122 recovered from immunoprecipitated materials were normalized against amount of HuR or HuRΔIII present in Immunoprecipitated materials . Ago2 western blot confirmed its presence in FLAG beads used in while Ago2 was not detected in HuR immunoprecipitated materials ( bottom panel ) .

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(D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel).


( E ) electrophoretic mobility shift done with 32P - labeled synthetic miR - 122 recombinant HuR . Radiolabelled miR - 122 ( 100 fmol ) was incubated with increasing of FL - HuR ( 5 - 100nM ) or HuRΔIII ( 100 nM ) gel shifting of miR - 122 were marked by arrowhead . Position of free probes is marked by an arrow . In right panel , 1pmol of unlabelled TNF - alpha AU - rich element encoding synthetic was used to compete with miR - 122 binding of full HuR . Amount of HuR or HuRΔIII used for gel shift were premeasured by densitometric estimation of coomassie stained gel bands from SDS page .

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(E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page.


( F ) Association of miRNAs with recombinant HuR in vitro . Small pool of Huh7 isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction HuR associated , recovered from immunoprecipitated HuR after binding reaction , were quantified along with input used for binding . mean Ct values of from samples from three independent binding reactions along with input were measured plotted ( mean + / - s . e . m . , n = 3 ) . Input recombinant HuR used in binding in immunoprecipitated materials were detected by western blot .

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(F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.


( A ) Subcellular distribution of HuR Ago2 in Huh7 . Isotonic lysates of Huh7 transfected with HA - HuR expression or pCIneo control vector were analyzed on 3 - 30 % OptiprepTM gradients for separation of organelles localization of Ago2 HuR were determined by western blotting . Alix Calnexin were used as markers of late endosome / MVB ER respectively . Positions of specific organelle containing fractions were marked above panels . * denotes position of high molecular HuR bands detected in MVB fractions .

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(A) Subcellular distribution of HuR and Ago2 in Huh7 cells. Isotonic lysates of Huh7 cells transfected with HA-HuR expression or pCIneo control vector were analyzed on 3-30% OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis. Alix and Calnexin were used as markers of late endosome/MVB and ER respectively. Positions of specific organelle containing fractions were marked above the panels. * denotes the position of the high molecular weight HuR bands detected in the MVB fractions.


( B ) Detection of ubiquitinated HuR in Huh7 expressing HA - Ub . HuR was immunoprecipitated with anti - HuR antibody from lysate of Huh7 transfected with HA - Ub expression or pCIneo ( control ) plasmids western blotted for HA or HuR ( left panel ) . In vitro ubiquitination was done with recombinant His - tagged HuR extract of Huh7 expressing HA - Ub . scheme of experiment western blot data for input extract along with immunoprecipitated materials are shown . Specific bands of Ub - HuR are marked by arrows HuR is marked by arrowhead .

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(B) Detection of ubiquitinated HuR in Huh7 cells expressing HA-Ub. HuR was immunoprecipitated with anti-HuR antibody from cell lysate of Huh7 cells transfected with HA-Ub expression or pCIneo (control) plasmids and western blotted for HA or HuR (left panel). In vitro ubiquitination assay was done with recombinant His-tagged HuR and cell extract of Huh7 cells expressing HA-Ub. The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown. Specific bands of Ub-HuR are marked by arrows and HuR is marked by arrowhead.


( C ) Changes in relative level of miR - 122 in subcellular ER MVB associated fractions of OptiprepTM gradients done with lysates from pCIneo or HA - HuR expression vector transfected were measured by qRT - PCR ( mean + / - s . e . m . , n = 5 ) .

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(C) Changes in relative level of miR-122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA-HuR expression vector transfected cells were measured by qRT-PCR (mean+/- s.e.m., n=5).


( D ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER MVB enriched fractions of Huh7 . Organelles were separated on OptiprepTM gradients , HuR in organeller lysates were immunoprecipitated with HuR specific antibody western blotted for ubiquitin to detect ubiquitinated form of HuR . Whole lysates used for optiprep were western blotted for ubiquitinated . Positions of high molecular bands of ubiquitinated HuR are marked by arrowheads . of HuR in immunoprecipitated materials were detected using anti - HuR antibody . Anti - GFP antibody immnoprecipitated materials were used as control .

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(D) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells. Organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR. Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins. Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads. Levels of HuR in the immunoprecipitated materials were detected using anti-HuR antibody. Anti-GFP antibody immnoprecipitated materials were used as control.


( E ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER MVB enriched fractions of co - expressing HA - Ubiquitin FLAG - HuR . were lysed organelles were separated on OptiprepTM gradients , HuR in organeller lysates were immunoprecipitated with HuR specific antibody western blotted for HA to detect ubiquitinated form of HuR . HuR in lysate of FLAG - HuR HA - Ub co expressing were detected by HuR specific antibody .

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(E) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co-expressing HA-Ubiquitin and FLAG-HuR. Cells were lysed and organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR. HuR in the lysate of the FLAG-HuR and HA-Ub co expressing cells were detected by HuR specific antibody.


( F ) Loss of miRNA association of HuR on MVB . HA - HuR expressing Huh7 lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA - HuR associated miRNA were measured by qRT - PCR normalized against immunoprecipitated . of immunoprecipitated HA - HuR were detected by western blotting quantified by densitometry . Data represented as mean + / - s . e . m . , n = 3 .

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(F) Loss of miRNA association of HuR on MVB. HA-HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA-HuR and associated miRNA levels were measured by qRT-PCR and normalized against immunoprecipitated proteins. Levels of immunoprecipitated HA-HuR were detected by western blotting and quantified by densitometry. Data represented as mean+/- s.e.m., n=3.


( G ) Loss of miRNA from ubiquitinated form of HuR . Scheme of double immunoprecipitation experiment is shown in left panel . Western blot for HuR in anti - HA anti - HuRimmunoprecipitatedFLAG - HuR along with input of FLAG - HuR used . Lysates of expressing FLAG - HuR used for FLAGHuR purification is also shown ( right upper panels ) . qRT - PCR estimation of miRNA per unit of HuRimmunoprecipitated were estimated for both HA HuRimmunoprecipitated materials normalized against respective quantified by densitometric of western blots ( right lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . Position of ubiquitinated band of FLAG - HuR is marked by arrow .

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(G) Loss of miRNA from ubiquitinated form of HuR. Scheme of the double immunoprecipitation experiment is shown in the left panel. Western blot analysis for HuR in the anti-HA and anti-HuRimmunoprecipitatedFLAG-HuR along with the input of FLAG-HuR used. Lysates of cell expressing the FLAG-HuR and used for FLAGHuR purification is also shown (right upper panels). qRT-PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots (right lower panel). Data represented as mean+/- s.e.m., n=3. Position of ubiquitinated band of FLAG-HuR is marked by arrow.