Drug response curves upon treatment with inhibitors for ( C ) B ‑ RAF ( LY3009120 , TAK ‑ 632 , vemurafenib , dabrafenib ) , ( biological replicates n = 3 , data are shown as mean ± SD ) .

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

Drug response curves upon treatment with inhibitors for (C) B‑RAF (LY3009120, TAK‑632, vemurafenib, dabrafenib), (biological replicates n=3, data are shown as mean ±SD).


Drug response curves upon treatment with inhibitors for ( D ) MEK1 / 2 ( selumetinib , binimetinib ) ( biological replicates n = 3 , data are shown as mean ± SD ) .

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

Drug response curves upon treatment with inhibitors for (D) MEK1/2 (selumetinib, binimetinib) (biological replicates n=3, data are shown as mean ±SD).


( A ) Brigthfield of human derived organoids ( PDOs ) from gastric cancer with without RTK / MAPK alterations ( scale bar 100µm , zoom in 40µm ) .

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(A) Brigthfield images of human patient derived organoids (PDOs) from gastric cancer with and without RTK/MAPK alterations (scale bar 100µm, zoom in 40µm).


( B ) Drug response curves of PDOs upon treatment with MEK1 / 2 inhibitor trametinib ( biological replicates n = 3 , data are presented as mean , SD values are shown in Table EV2 ) . ( C ) Drug response curves upon HDAC inhibition with panobinostat ( PAN ) entinostat ( ENT ) ( biological replicates n = 3 , data are presented as mean , SD values are shown in Table EV2 ) . ( D ) Comparison of relative under curve ( AUCrel ) upon trametinib treatment in RTK / MAPK altered ( mean AUCrel : 0 . 5308 ; biological replicate n = 7 ) vs . non ‑ altered ( mean AUCrel : 0 . 7101 ; biological replicates n = 6 ) human PDOs ( two ‑ Student ' s t ‑ ; * p = 0 . 0356 ) .

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

(B) Drug response curves of PDOs upon treatment with the MEK1/2 inhibitor trametinib (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (C) Drug response curves upon HDAC inhibition with panobinostat (PAN) and entinostat (ENT) (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (D) Comparison of the relative area under the curve (AUCrel) upon trametinib treatment in RTK/MAPK altered (mean AUCrel: 0.5308; biological replicate n=7) vs. non‑altered (mean AUCrel: 0.7101; biological replicates n=6) human PDOs (two‑tailed Student's t‑Test; *p= 0.0356).


A microscopy showing UBQLN2 ( Alexa Fluor 647 ) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48 - K63 - linked chains of different lengths in 20 mM NaPhosphate 200 mM NaCl ( pH 6 . 8 ) at 30 ºC . [ Ub ] : [ UBQLN2 ] reflects between Ub monomers UBQLN2 molecules . same image is used for 0 : 1 condition . Scale bar , 5 µm . Yellow cartoons illustrate one major solution conformation of each chain .

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

A Fluorescence microscopy showing UBQLN2 (Alexa Fluor 647) in solutions contained 50 µM FL UBQLN2 with increasing amounts of K48- and K63-linked chains of different lengths in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. The same image is used for the 0:1 condition. Scale bar, 5 µm. Yellow cartoons illustrate one major solution conformation of each chain.


B Representative results from spectrophotometric turbidity comparing effects of varying amounts of K48 - K63 - linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2 .

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

B Representative results from spectrophotometric turbidity assay comparing the effects of varying amounts of K48- and K63-linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2.


A CSPs for UBQLN2 450 - 624 residues at 1 : 1 with Ub4 . highlighted regions in domain map are important for homotypic interactions that drive UBQLN2 LLPS ( Dao et al , 2018 ) . B Amide peak intensities between unbound Ub4 - bound forms of UBQLN2 450 - 624 with 555 - 570 region highlighted in gray . C CSPs for residues in UBQLN2 487 - 624 at 1 : 1 with Ub4 . D CSPs for residues in UBQLN2 450 - 624 lacking residues 551 - 569 ( red / gray region ) at 1 : 1 with Ub4 . E Normalized titration curves of 11 amide resonances for UBQLN2 450 - 624 .

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

A CSPs for UBQLN2 450-624 residues at 1:1 ratio with Ub4. The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS (Dao et al, 2018). B Amide peak intensities between the unbound and Ub4-bound forms of UBQLN2 450-624 with the 555-570 region highlighted in gray. C CSPs for residues in UBQLN2 487-624 at 1:1 ratio with Ub4. D CSPs for residues in UBQLN2 450-624 lacking residues 551-569 (red/gray region) at 1:1 ratio with Ub4. E Normalized titration curves of 11 amide resonances for UBQLN2 450-624.


A CSPs for UBQLN2 450 - 624 residues at 1 : 1 with Ub4 . highlighted regions in domain map are important for homotypic interactions that drive UBQLN2 LLPS ( Dao et al , 2018 ) .

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

A CSPs for UBQLN2 450-624 residues at 1:1 ratio with Ub4. The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS (Dao et al, 2018).


F Comparison of binding affinity ( Kd ) measurements obtained from NMR anisotropy . Error bars for FA numbers represent standard deviation over three biological trials for TITAN numbers are from jackknife . For visual comparison , gray dashed is Kd of Ub binding to UBQLN2 450 - 624 as determined from NMR FA experiments ( green bars ) .

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

F Comparison of binding affinity (Kd) measurements obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).


G anisotropy values for fluorescently - labeled UBQLN2 450 - 624 in presence of increasing K48 - or K63 - Ub4 . Error bars represent standard deviation over three biological trials .

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

G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing K48- or K63-Ub4. Error bars represent the standard deviation over three biological trials.


H anisotropy values for fluorescently - labeled K48 - or K63 - Ub4 in presence of increasing UBQLN2 450 - 624 . Error bars represent standard deviation over three biological trials .

H Fluorescence anisotropy values for fluorescently-labeled K48- or K63-Ub4 in the presence of increasing UBQLN2 450-624. Error bars represent the standard deviation over three biological trials.


B Proximal Ub resonances in Ub4 were compared to monoUb , as noted in schematic . CSPs > 0 . 05 ppm ( orange dashed ) are mapped onto proximal Ub unit of Ub4 in panel A .

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

B Proximal Ub resonances in Ub4 were compared to monoUb, as noted in schematic. CSPs > 0.05 ppm (orange dashed line) are mapped onto the proximal Ub unit of Ub4 in panel A.


C 15N - 1H SOFAST - HMQC NMR spectra of proximal Ub of K48 - Ub4 or K63 - Ub4 at 50 µM in absence presence of UBQLN2 450 - 624 at a 2 : 1 UBQLN2 : Ub4 . Unbound monoUb spectrum ( black ) is overlaid for comparison . NMR spectra collected processed under identical conditions .

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

C 15N-1H SOFAST-HMQC NMR spectra of proximal Ub of K48-Ub4 or K63-Ub4 at 50 µM in the absence and presence of UBQLN2 450-624 at a 2:1 UBQLN2:Ub4 ratio. Unbound monoUb spectrum (black) is overlaid for comparison. NMR spectra collected and processed under identical conditions.


D CSPs of same proximal Ub resonances in K63 - Ub4 vs K48 - Ub4 . Gray bars indicate K48 - Ub4 resonances that were too weak to be detected .

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

D CSPs of the same proximal Ub resonances in K63-Ub4 vs K48-Ub4. Gray bars indicate K48-Ub4 resonances that were too weak to be detected.


B Normalized pair distribution function P ( r ) from SAXS data for four Ub4 chain types .

B Normalized pair distance distribution function P(r) from SAXS data for four Ub4 chain types.


C CSPs at 1 : 1 Ub4 : UBQLN2 for chain types , but clustered into two categories for clarity .

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

C CSPs at 1:1 Ub4:UBQLN2 for all chain types, but clustered into two categories for clarity.


F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48 - Ub4 into UBQLN2 450 - 624 than upon titrations of K11 - Ub4 , K63 - Ub4 , M1 - Ub4 HOTag6 - Ub .

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

F Normalized titration curves of UBA resonances showing more pronounced sigmoidal behaviors upon titration of K48-Ub4 into UBQLN2 450-624 than upon titrations of K11-Ub4, K63-Ub4, M1-Ub4 and HOTag6-Ub.


G anisotropy values for fluorescently - labeled UBQLN2 450 - 624 in presence of increasing amounts of different Ub4 chains . Error bars represent standard deviation over three biological trials .

G Fluorescence anisotropy values for fluorescently-labeled UBQLN2 450-624 in the presence of increasing amounts of different Ub4 chains. Error bars represent the standard deviation over three biological trials.


H Comparison of Kd values obtained from NMR anisotropy . Error bars for FA numbers represent standard deviation over three biological trials for TITAN numbers are from jackknife . For visual comparison , gray dashed is Kd of Ub binding to UBQLN2 450 - 624 as determined from NMR FA experiments ( green bars ) .

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

H Comparison of Kd values obtained from NMR and fluorescence anisotropy. Error bars for the FA numbers represent the standard deviation over three biological trials and for the TITAN numbers are from jackknife analysis. For visual comparison, gray dashed line is Kd of Ub binding to UBQLN2 450-624 as determined from NMR and FA experiments (green bars).


A microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate 200 mM NaCl ( pH 6 . 8 ) at 30 ºC . [ Ub ] : [ UBQLN2 ] reflects between Ub monomers UBQLN2 molecules . Solution is spiked with Alexa Fluor 647 - labeled UBQLN2 . same image is used for 0 : 1 condition . For visual comparison , for K48 - Ub4 K63 - Ub4 are identical to those shown in Figure 1A . Scale bar , 5 µm .

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

A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet formation at increasing amounts of chains at 50 µM UBQLN2 in 20 mM NaPhosphate and 200 mM NaCl (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub monomers and UBQLN2 molecules. Solution is spiked with Alexa Fluor 647-labeled UBQLN2. The same image is used for the 0:1 condition. For visual comparison, images for K48-Ub4 and K63-Ub4 are identical to those shown in Figure 1A. Scale bar, 5 µm.


B Representative results from spectrophotometric turbidity as a function of at 30 μM UBQLN2 .

B Representative results from spectrophotometric turbidity assay as a function of temperature at 30 μM UBQLN2.


E microscopy showing distribution of Ub4 ( spiked with Dylight 488 - labeled chains ) inside outside droplets containing 50 µM UBQLN2 ( spiked with Alexa Fluor 647 - labeled UBQLN2 ) . Scale bar , 5 µm .

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

E Fluorescence microscopy showing the distribution of Ub4 (spiked with Dylight 488-labeled chains) inside and outside droplets containing 50 µM UBQLN2 (spiked with Alexa Fluor 647-labeled UBQLN2). Scale bar, 5 µm.


A SDS - PAGE gels monitoring formation of K63 - linked polyUb chains possibly ubiquitinated GST - Ubc13 over time , as indicated by asterisks curly brackets . K63 - Ub3 migrates on gel similarly as His - Mms2 , right below 20 kDa . Experimental conditions : ± 50 µM Ub UBQLN2 , 30 nM Dylight 650 - labeled UBQLN2 , 1 µM mE1 , 2 µM His - Mms2 , 4 µM GST - Ubc13 , 10 mM ATP MgCl2 , 3 mM TCEP , 50 mM Tris pH8 , 37 ºC . K63 - linked polyUb chain reactions were done in absence ( top ) presence ( bottom ) of UBQLN2 . Since UBQLN2 competes with E1 E2 to bind to Ub , presence of UBQLN2 slowed down chain formation ( indicated by comparing disappearance rates of Ub ) . band for GST - Ubc13 decreased with time . time - dependent appearance of species between 50 - 85 kDa were obstructed in presence of UBQLN2 . These species are most likely ubiquitinated GST - Ubc13 , not free polyUb chains , since these bands were observed with BioRad Stain - free gels ( right ) that detect with tryptophan residues unlike Ub or polyUb chains .

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

A SDS-PAGE gels monitoring the formation of K63-linked polyUb chains and possibly ubiquitinated GST-Ubc13 over time, as indicated by asterisks and curly brackets. K63-Ub3 migrates on the gel similarly as His-Mms2, right below 20 kDa. Experimental conditions: ± 50 µM Ub and UBQLN2, 30 nM Dylight 650-labeled UBQLN2, 1 µM mE1, 2 µM His-Mms2, 4 µM GST-Ubc13, 10 mM ATP and MgCl2, 3 mM TCEP, 50 mM Tris pH8, 37 ºC. The K63-linked polyUb chain reactions were done in the absence (top) and presence (bottom) of UBQLN2. Since UBQLN2 competes with E1 and E2 to bind to Ub, the presence of UBQLN2 slowed down chain formation (indicated by comparing disappearance rates of Ub). The band intensity for GST-Ubc13 decreased with time. The time-dependent appearance of species between 50-85 kDa were obstructed in the presence of UBQLN2. These species are most likely ubiquitinated GST-Ubc13, and not free polyUb chains, since these bands were observed with BioRad Stain-free gels (right) that detect proteins with tryptophan residues unlike Ub or polyUb chains.


A Generation of myeloid genetically inactivated for Elp3 . Elp3 mRNA were assessed by Real - Time PCR with extracts isolated from peritoneal macrophages of Elp3Control Elp3 ∆ Mye mice . mRNA in Elp3Control mice were set to 1 in other experimental conditions were relative to that after normalization with Gapdh mRNA ( n = 3 mice ; mean + / - SD , Student T , * * * = p < 0 . 001 ) . An anti - Elp3 western blot ( WB ) is also illustrated . anti - Gapdh blot is shown for normalization purposes .

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

A Generation of myeloid cells genetically inactivated for Elp3. Elp3 mRNA and protein levels were assessed by Real-Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3∆Mye mice. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, ***= p< 0.001). An anti-Elp3 western blot (WB) is also illustrated. The anti-Gapdh blot is shown for normalization purposes.


C D Defective mTORC2 signaling upon Elp3 deficiency in myeloid . Peritoneal macrophages from Elp3Control Elp3 ∆ Mye mice were isolated cultured ex - vivo . They were untreated or stimulated with a combination of LPS ( 100 ng / ml ) IFNγ ( 50 ng / ml ) for indicated periods of time resulting extracts were subjected to WB analyses .

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

C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS (100 ng/ml) and IFNγ (50 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.


F mTORC2 activation by M1 polarization signals relies on Ctu2 . Control Ctu2 - depleted bone marrow - derived macrophages ( BMDMs ) ( SiRNA - mediated depletions ) were treated or not with IFNγ ( 50 ng / ml ) LPS ( 100 ng / ml ) resulting extracts were subjected to WB analyses .

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

F mTORC2 activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) and the resulting cell extracts were subjected to WB analyses.


C Elp3 deficiency in myeloid exacerbates decrease of colon upon DSS administration ( n = 7 mice ; mean + / - SD , * * = p < 0 . 01 ) .

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

C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration (n = 7 mice; mean +/- SD, **= p< 0.01).


E More severe histological score in mice lacking Elp3 in myeloid upon DSS administration . Representative intestinal crypts from indicated genotypes are illustrated . histological score was established as described in methods ( n = 5 mice per genotype ; mean + / - SD , Student T , * = p < 0 . 05 ) .

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

E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological score was established as described in methods (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).


F Increased infiltration of iNOS + / CD68 + upon DSS administration in Elp3 ∆ Mye mice . of iNOS + in CD68 + was established in both genotypes treated with DSS ( n = 7 mice per genotype ; mean + / - SD , Student T , * * = p < 0 . 01 ) . Anti - iNOS - CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated .

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

F Increased infiltration of iNOS+/CD68+ cells upon DSS administration in Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS (n = 7 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS and -CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated.


G suffering from ulcerative colitis ( UC ) show reduced of both Elp1 Elp3 . central band represents mean value of relative expression in investigated cohort . Boxes represent 75th 25th percentile . Whiskers represent maximum minimum values before upper lower fence , respectively ( * * = p < 0 . 01 , * * * = p < 0 . 001 ) ( GSE9452 - GEO DataSets , Definition of an ulcerative colitis pre - inflammatory state ) .

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G Patients suffering from ulcerative colitis (UC) show reduced levels of both Elp1 and Elp3. The central band represents the mean value of relative expression in the investigated cohort. Boxes represent 75th and 25th percentile. Whiskers represent maximum and minimum values before the upper and lower fence, respectively (**= p< 0.01, ***= p<0.001) (GSE9452-GEO DataSets, Definition of an ulcerative colitis pre-inflammatory state).


A IL - 4 IL - 13 induce expression of tRNA - modifying enzymes in macrophages . Peritoneal macrophages were untreated ( « Control » ) or stimulated with IL - 4 ( 10 ng / ml ) , IL - 13 ( 10 ng / ml ) or with both cytokines for 24 hours extracts from resulting were subjected to WB analyses .

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A IL-4 and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses.


B pool of thiolated tRNAs in macrophages is enhanced by IL - 4 / IL - 13 but decreased by LPS / IFNγ . On left , Northern blot assessing t : Q ( UUG ) tRNA thiolation in Thioglycollate - elicited peritoneal macrophages subjected to indicated treatments for 24 hours . On right , of t : Q ( UUG ) tRNA thiolation , calculated as ration of thiolated over nonthiolated t : Q ( UUG ) , in indicated experimental conditions .

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

B The pool of thiolated tRNAs in macrophages is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot analysis assessing t:Q(UUG) tRNA thiolation in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:Q(UUG), in the indicated experimental conditions.


C Elp3 induction by IL - 4 / IL - 13 occurs through a PI3K - dependent pathway . Peritoneal macrophages were pretreated with DMSO ( control vehicle ) or with Wortmannin ( 200 nM ) for one hour subsequently left untreated or stimulated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for indicated periods of time . extracts from resulting were subjected to WB analyses .

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C Elp3 induction by IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.


E Rictor but not Raptor deficiency impairs Elp3 expression . BMDMs were transfected with indicated siRNAs resulting were treated or not with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for 24 hours . extracts were subjected to WB analyses .

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

E Rictor but not Raptor deficiency impairs Elp3 expression. BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for 24 hours. Cell extracts were subjected to WB analyses.


F IL - 4 / IL - 13 rely on Stat6 to induce Elp3 expression . Peritoneal macrophages were pretreated with control vehicle ( DMSO ) or with Stat6 pharmacological inhibitor AS1517499 ( 5 μM ) for one hour subsequently left unstimulated or treated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for indicated periods of time . extracts from resulting were subjected to WB analyses .

F IL-4/IL-13 rely on Stat6 to induce Elp3 expression. Peritoneal macrophages were pretreated with the control vehicle (DMSO) or with the Stat6 pharmacological inhibitor AS1517499 (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.


Elp3 deficiency in myeloid impairs multiple signaling pathways triggered by IL - 4 ( H , , IL - 13 ( H ) or by both cytokines ( H ) . Peritoneal macrophages isolated from indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) ( H , IL - 13 ( 10 ng / ml ) ( H ) or with both cytokines ( H ) for 8 hours ( H ) resulting extracts were subjected to WB analyses .

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

Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 (H, , IL-13 (H) or by both cytokines (H). Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) (H, IL-13 (10 ng/ml) (H) or with both cytokines (H) for 8 hours (H) and the resulting cell extracts were subjected to WB analyses.


J Elp3 deficiency in myeloid impairs multiple signaling pathways triggered by IL - 4 I J ) Peritoneal macrophages isolated from indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) , I J ) , or for indicated periods of time ( I J ) resulting extracts were subjected to WB analyses .

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J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 I and J) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) , I and J), or for the indicated periods of time (I and J) and the resulting cell extracts were subjected to WB analyses.


L Elp3 promotes M2 macrophage polarization . Peritoneal macrophages isolated from indicated mouse genotypes were untreated or stimulated with IL - 4 / IL - 13 ( 10 ng / ml ) for 24 hours resulting extracts were subjected to WB analyses .

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L Elp3 promotes M2 macrophage polarization. Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting cell extracts were subjected to WB analyses.


B Defective induction of Pparγ by IL - 4 / IL - 13 upon Elp3 deficiency . extracts from peritoneal macrophages of Elp3Control Elp3 ∆ Mye mice treated or not with IL - 4 / IL - 13 were subjected to WB analyses .

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B Defective induction of Pparγ by IL-4/IL-13 upon Elp3 deficiency. Cell extracts from peritoneal macrophages of Elp3Control and Elp3∆Mye mice treated or not with IL-4/IL-13 were subjected to WB analyses.


A induction of Ric8b expression by IL - 4 relies on Elp3 . BMDMs from indicated genotypes were treated or not with IL - 4 ( 10 ng / ml ) up to 24 hours resulting extracts were subjected to WB analyses .

A The induction of Ric8b expression by IL-4 relies on Elp3. BMDMs from the indicated genotypes were treated or not with IL-4 (10 ng/ml) up to 24 hours and the resulting cell extracts were subjected to WB analyses.


B Elp3 promotes Ric8B mRNA translation . BMDMs from Elp3Control Elp3 ∆ Mye mice were treated by IL - 4 for 24 hours , followed by a treatment with 10 μg / ml puromycin for 5 minutes . To detect newly synthesized Ric8b , a Puro - PLA was conducted . Representative detecting Ric8b ( Red ) α - Tubulin + microtubules ( green ) in BMDMs are illustrated . On right , graph shows a of signals for Ric8b in α - Tubulin + areas . A random of six different areas were counted ( 6 technical replicates , mean + / - SD , Student ' s t - , * = p < 0 . 05 ) .

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B Elp3 promotes Ric8B mRNA translation. BMDMs from Elp3Control and Elp3∆Mye mice were treated by IL-4 for 24 hours, followed by a treatment with 10 μg/ml puromycin for 5 minutes. To detect newly synthesized Ric8b proteins, a Puro-PLA assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules (green) in BMDMs are illustrated. On the right, the graph shows a quantification of signals for Ric8b in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).


C Ric8b is a direct target of Elp3 . BMDMs from Elp3Control Elp3 ∆ Mye mice were infected with a lentiviral GFP construct ( negative control ) , Flag - Ric8b WT or with Flag - Ricb MUT , as indicated resulting extracts were subjected to WB analyses to assess Ric8b Elp3 . Gapdh was used as a loading control .

C Ric8b is a direct target of Elp3. BMDMs from Elp3Control and Elp3∆Mye mice were infected with a lentiviral GFP construct (negative control), Flag-Ric8b WT or with Flag-Ricb MUT, as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels. Gapdh was used as a loading control.


D E Ric8b promotes IL - 4 - dependent mTORC2 activation . Control or Ric8b - depleted BMDMs were treated or not with IL - 4 ( 10 ng / ml ) for indicated periods of time resulting extracts were subjected to WB analyses .

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D and E Ric8b promotes IL-4-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.


C Elp3 promotes mitochondrial translation . BMDMs from indicated genotypes were treated or not with IL - 4 / IL - 13 ( 10 ng / ml ) for indicated hours resulting extracts were subjected to WB analyses .

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C Elp3 promotes mitochondrial translation. BMDMs from the indicated genotypes were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated hours and the resulting cell extracts were subjected to WB analyses.


D Elp3 expression in myeloid maintains pool of - associated macrophages ( TAMs ) . number of TAMs , defined as CD301 + / F4 / 80 + among F4 / 80 + was quantified in intestinal crypts from both Apc + / Min Elp3Control Apc + / Min Elp3 ∆ Mye mice ( n = 5 mice per genotype ; mean + / - SD , Student T , * = p < 0 . 05 ) . Representative immunofluorescence analyses are illustrated .

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D Elp3 expression in myeloid cells maintains the pool of tumor-associated macrophages (TAMs). The number of TAMs, defined as CD301+/F4/80+ cells among F4/80+ cells was quantified in intestinal crypts from both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05). Representative immunofluorescence analyses are illustrated.


D . Northern blot of V . cholerae wild - type examined for expression of VcdRP monitored over growth in indicated media . solid triangle represents band corresponding to full vcdRP transcript , whereas open triangles correspond to different processed isoforms . Probing with 5S rRNA confirmed equal loading .

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D. Northern blot analysis of V. cholerae wild-type strain examined for the expression of VcdRP monitored over bacterial growth in the indicated media. The solid triangle represents the band corresponding to the full length primary vcdRP transcript, whereas the open triangles correspond to the different processed isoforms. Probing with 5S rRNA confirmed equal loading.


A . Northern blot of expression of VcdRP Spot42 sRNA monitored at low density ( OD600 of 0 . 1 ) high density ( OD600 of 2 . 0 ) , in absence ( - ) or presence ( + ) of cAMP ( 5mM f . c . ) supplemented externally in LB medium . solid triangle indicates band corresponding to full - transcript . Probing with 5S rRNA confirmed equal loading .

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A. Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density (OD600 of 0.1) and high cell density (OD600 of 2.0), in the absence (-) or presence (+) of cAMP (5mM f.c.) supplemented externally in LB medium. The solid triangle indicates the band corresponding to the full-length primary transcript. Probing with 5S rRNA confirmed equal loading.


B . Western blot ( top ) of CTX detected in secreted fractions of V . cholerae ΔhapR grown under AKI conditions carrying indicated plasmids . Coomassie - stained SDS gel ( bottom ) confirmed equal loading of fractions . normalized band intensities are indicated for each expression plasmid relative to pCtrl , which was set to 100 % .

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B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.


I - L . Relative intensities ( y - axis ) of E . coli strains harboring gene - specific translational reporters as in ( A - D ) or corresponding M2 * variant for each gene , combined with an empty control plasmid or vcdRP expression plasmids ( pVcdRP pVcdRP M2 ) measured using plate reader ( I - K ) or by Western blot ( L ) . of pCtrl ( for each reporter fusion ) was set to 1 .

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I-L. Relative fluorescence intensities (y-axis) of E. coli strains harboring the gene-specific translational reporters as in (A-D) or the corresponding M2* variant for each gene, combined with an empty control plasmid or vcdRP expression plasmids (pVcdRP and pVcdRP M2) measured using plate reader (I-K) or by Western blot analysis (L). The fluorescence of pCtrl (for each reporter fusion) was set to 1.


A . Immunoprecipitation of chromosomally tagged GltA : : HA combined with either an empty vector control ( pCtrl ) or SPA - tagged vcdP over - expression plasmid , grown in LB medium to exponential phase ( OD600 of 0 . 5 ) . SPA epitope contains 3 × FLAG calmodulin binding peptide sequences separated by a TEV protease cleavage site ( Zeghouf et al , 2004 ) . Therefore , samples corresponding to total input lysates before after subjecting to immunoprecipitation with anti - Flag antibody were loaded on a SDS - PAGE gel . Western blotting with anti - HA anti - Flag antibodies confirmed interaction of GltA with VcdP in vivo . RNAP served as loading control . solid triangles indicate corresponding sizes .

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A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.


B . Citrate synthase enzyme activity measurements performed on lysates . An empty vector control ( pCtrl ) or vcdP expression plasmids ( pVcdP pVcdP * ) were conjugated into V . cholerae wild - type , ΔvcdRP , ΔgltA or F383A GltA backgrounds . were grown in LB medium to stationary phase subsequently lysed . concentrations of these extracts were measured , served as input for . resulting colorimetric product at 412nm was proportional to enzymatic activity of citrate synthase present . enzyme kinetics were assayed at 15 second intervals for at least 30 minutes activity per milligram of lysate was determined from initial velocities .

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B. Citrate synthase enzyme activity measurements performed on cellular lysates. An empty vector control (pCtrl) or vcdP expression plasmids (pVcdP and pVcdP*) were conjugated into V. cholerae wild-type, ΔvcdRP, ΔgltA or F383A GltA backgrounds. Cells were grown in LB medium to stationary phase and subsequently lysed. The protein concentrations of these cellular extracts were measured, and served as input for the assay. The resulting colorimetric product at 412nm was proportional to the enzymatic activity of citrate synthase present. The enzyme kinetics were assayed at 15 second intervals for at least 30 minutes and the activity per milligram of lysate was determined from the initial velocities.


( a ) Widefield immunofluorescence of PFA fixed HeLa showing endogenous FLCN LAMP1 in ( top ) normal growth or ( bottom ) starvation conditions . Orange arrows highlight association of FLCN with LAMP1 positive membranes in peri - region . dotted shows periphery in starved condition . Scale bar = 10μm . ( b ) ( left ) of FLCN / LAMP1 association in growth starvation conditions . A is scored as positive if 5 or more discrete FLCN / LAMP1 were observed at 100x magnification using a widefield microscope . Data represents 60 from 3 independent experiments , error bars show S . E . M , * * * = p < 0 . 001 . ( right ) Western blot shows of phosphorylated of S6K 4EBP in whole extracts under same conditions .

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(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.


( c ) Western blot showing typical efficiency of FLCN knockdown in HeLa using a pool consisting of 4 oligonucleotides 2 independent oligonucleotides from pool . Graph shows relative FLCN expression ( n = 3 , error bars show S . E . M . )

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(c) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n=3, error bars show S.E.M.)


( d ) Representative confocal microscopy showing LAMP1 ( magenta ) distribution in HeLa under normal growth or starvation conditions in depleted of FLCN or control transfected with a non - targeting siRNA . location of Golgi ( green ) is highlighted by giantin . ( e ) Schematic illustrating application of cumulative distribution method for of lysosome distribution . ( f ) Graph showing cumulative distribution of LAMP1 in normal growth starvation conditions in HeLa . P - value is determined by extra sum of F - squares following non - linear regression curve fitting . Error bars show S . E . M . from 30 in 3 replicates . ( g ) Graph showing cumulative distribution of LAMP1 in starvation conditions for control transfected with non - targeting siRNA or depleted of FLCN . Error bars show S . E . M . from 30 in 3 replicates .

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(d) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non-targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining.(e) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.(f) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P-value is determined by the extra sum of F-squares test following non-linear regression and curve fitting. Error bars show S.E.M. from 30 cells in 3 replicates.(g) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non-targeting siRNA or cells depleted of FLCN. Error bars show S.E.M. from 30 cells in 3 replicates.


( a ) Representative spinning disk confocal image showing FLCN - GFP lysosomes ( Lysotracker - Red ) in a live FLCN - GFP / HA - FNIP2 transfected HeLa ( from Movie EV1 ) . Top panels show first frame of a 180 frame ( 1fps ) image series . Bottom panels show a maximum projection of image series to highlight mobile immobile components . Boxed magnified regions show typical peri - ( i ) peripheral ( ii ) regions in as well as a FLCN - GFP tubule ( iii ) Scale bar = 5μm .

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(a) Representative spinning disk confocal image showing FLCN-GFP and lysosomes (Lysotracker-Red) in a live FLCN-GFP/HA-FNIP2 transfected HeLa cell (from Movie EV1). Top panels show the first frame of a 180 frame (1fps) image series. Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components. Boxed and magnified regions show typical peri-nuclear (i) and peripheral (ii) regions in the cell as well as a FLCN-GFP tubule (iii) Scale bar = 5μm.


( b ) Spinning disk confocal image series of a region live FLCN - GFP / HA - FNIP2 transfected HeLa highlighting dynamic FLCN - GFP tubules ( Movie EV2 ) . First merge panel shows FLCN - GFP lysotracker - Red at T = 0 Subsequent panels show only FLCN - GFP at 30 second intervals .

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(b) Spinning disk confocal image series of a region live FLCN-GFP/HA-FNIP2 transfected HeLa cell highlighting dynamic FLCN-GFP tubules (Movie EV2). First merge panel shows FLCN-GFP and lysotracker-Red at T=0 Subsequent panels show only FLCN-GFP at 30 second intervals.


c ) Confocal image of a region of a methanol fixed FLCN - GFP / HA - FNIP2 transfected HeLa showing Rab7 ( red ) LAMP1 ( blue ) association with FLCN - GFP tubules . Scale bar = 1μm .

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c) Confocal image of a region of a methanol fixed FLCN-GFP/HA-FNIP2 transfected HeLa cell showing Rab7 (red) and LAMP1 (blue) association with FLCN-GFP tubules. Scale bar = 1μm.


( a ) Graphs showing cumulative distribution of LAMP1 in control GFP ( black ) , GFP - Rab34 ( green ) or GFP - Rab7 ( blue ) transfected . Note black symbols partially obscure blue .

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(a) Graphs showing cumulative distribution of LAMP1 intensity in control GFP (black), GFP-Rab34 (green) or GFP-Rab7 (blue) transfected cells. Note black line and symbols partially obscure blue.


( b ) GST - RILP pull - down from HeLa in normal starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions . is mean of 3 independent experiments . Error is S . E . M .

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(b) GST-RILP pull-down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is S.E.M.


( c ) Representative confocal microscopy of FLCN depleted or control , transfected with GFP - Rab34 . Scale bar = 10μm .

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(c) Representative confocal fluorescence microscopy images of FLCN depleted or control cells, transfected with GFP-Rab34. Scale bar = 10μm.


( d ) ( left ) Graphs showing mean GFP - Rab34 expression ( grey values ) cumulative distribution of LAMP1 ( right ) in FLCN depleted or control siRNA transfected HeLa expressing GFP - Rab34 . Error bars show S . E . M . . from 30 in 3 replicates . Scale bar = 10μm .

(d) (left) Graphs showing mean GFP-Rab34 expression (grey values) and cumulative distribution of LAMP1 intensity (right) in FLCN depleted or control siRNA cells transfected HeLa cells expressing GFP-Rab34. Error bars show S.E.M.. from 30 cells in 3 replicates. Scale bar = 10μm.


( e ) Western blot graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa . Error bars show S . E . M from 3 experiments , * * * = p < 0 . 001 .

(e) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells. Error bars show S.E.M from 3 experiments, *** = p<0.001.


( f ) Representative confocal microscopy showing LAMP1 ( magenta ) distribution in HeLa under normal growth or starvation conditions in depleted of Rab34 or control transfected with a non - targeting siRNA . Giantin is shown in green . Scale bar = 10μm . ( g ) Graphs showing cumulative distribution of LAMP1 in normal growth or starvation conditions for control or depleted of Rab34 using siRNA . ( h ) Graph showing of LAMP1 in cis - medial Golgi - region ( giantin overlap ) in indicated conditions . Error bars show S . E . M from 30 in 3 replicates , * * * = p < 0 . 001 .

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(f) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non-targeting siRNA. Giantin is shown in green. Scale bar = 10μm.(g) Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA.(h) Graph showing percentage of LAMP1 in cis-medial Golgi-region (giantin overlap) in the indicated conditions. Error bars show S.E.M from 30 cells in 3 replicates, *** = p<0.001.


( a ) ( left ) Representative confocal microscopy image showing methanol fixed HeLa transfected with GFP - Rab34 stained for LAMP1 . Linescan shows relative of GFP LAMP1 signals . Scale bar = 10μm .

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(a) (left) Representative confocal fluorescence microscopy image showing methanol fixed HeLa cell transfected with GFP-Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar = 10μm.


( b ) Maximum projection from a confocal Z - stack showing LAMP1 endogenous Rab34 in P - M fixed HeLa from normal starved conditions . Whole fields from which these panels are derived are shown in Figure EV4A . Boxes highlight regions shown in Figure 4C . Scale bar = 2μm .

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(b) Maximum intensity projection images from a confocal Z-stack showing LAMP1 and endogenous Rab34 in P-M fixed HeLa cells from normal and starved conditions. Whole cells and fields from which these panels are derived are shown in Figure EV4A. Boxes highlight regions shown in Figure 4C. Scale bar = 2μm.


( c ) Single confocal sections from Z - stack in figure 4B . Orange arrows show close LAMP1 / Rab34 association .

(c) Single confocal sections from the Z-stack in figure 4B. Orange arrows show close LAMP1/Rab34 association.


( d ) of Lysotracker - Red dynamics using PCC decay in HeLa transfected with either GFP or GFP - Rab34 . Data is from 10 per condition . Error bars show S . E . M .

(d) Analysis of Lysotracker-Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP-Rab34. Data is from 10 cells per condition. Error bars show S.E.M.


( e ) Live - spinning disk confocal microscopy image series at 10 sec intervals ( from Movie EV3 ) showing GFP - Rab34 positive compartment making dynamic contacts with lysosomes . Scale bar =

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(e) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV3) showing GFP-Rab34 positive compartment making dynamic contacts with lysosomes. Scale bar = 1μm


( f ) Live - spinning disk confocal microscopy image series at 10 sec intervals ( from Movie EV4 ) of FLCN - GFP mCherry - Rab34 in peri - region of a FLCN - GFP / HA - FNIP2 / mCherry - Rab34 transfected HeLa . Blue arrow highlights FLCN - Rab34 association moving on a linear trajectory . Yellow arrow highlights shorter saltatory movements . arrow highlights dynamic associations between distinct FLCN - GFP Rab34 positive structures . Scale bar =

(f) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV4) of FLCN-GFP and mCherry-Rab34 in the peri-nuclear region of a FLCN-GFP/HA-FNIP2/mCherry-Rab34 transfected HeLa cell. Blue arrow highlights FLCN-Rab34 association moving on a linear trajectory. Yellow arrow highlights shorter saltatory movements. White arrow highlights dynamic associations between distinct FLCN-GFP and Rab34 positive structures. Scale bar = 1μm


( g ) Confocal image showing localisation of endogenous FLCN Rab34 in starved HeLa .

(g) Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells.


from fixed GFP - Rab34 transfected showing contacts between GFP - Rab34 positive peri - membranes lysosomes . ( a ) Spinning disk confocal microscopy acquired at 160x magnification . Left panels show a maximum projection imaged of a Z - stack , sampled according to Nyquist criterion deconvolved . Right panels shows a 3D volume rendering of same data .

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Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(a) Spinning disk confocal microscopy images acquired at 160x magnification. Left panels show a maximum intensity projection imaged of a Z-stack, sampled according to the Nyquist criterion and deconvolved. Right panels shows a 3D volume rendering of the same data.


from fixed GFP - Rab34 transfected showing contacts between GFP - Rab34 positive peri - membranes lysosomes . ( b ) A projection of 4 planes from a structured illumination microscopy super - resolution Z - stack of same .

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Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(b) A projection of 4 planes from a structured illumination microscopy super-resolution Z-stack of the same cells.


from fixed GFP - Rab34 transfected showing contacts between GFP - Rab34 positive peri - membranes lysosomes . ( c ) A thin section electron microscopy image of peri - region of a GFP - Rab34 transfected HeLa . * indicates lysosomes .

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Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(c) A thin section electron microscopy image of the peri-nuclear region of a GFP-Rab34 transfected HeLa cell. * indicates lysosomes.


from fixed GFP - Rab34 transfected showing contacts between GFP - Rab34 positive peri - membranes lysosomes . ( d ) A cryo - immuno - EM image of a GFP - Rab34 transfected HeLa . GFP - Rab34 ( black dots ) is identified by immunostaining for GFP detection with gold conjugated A . lysosome is intimately associated with Rab34 positive membranes . Sites of contact are highlighted by arrows .

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Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.


( a ) Confocal immunofluorescence microscopy of HeLa transfected with Rab34 - dsRED - Mito , FLCN - GFP HA - FNIP2 . arrows highlight FLCN - GFP / HA - FNIP2 co - localisation distinct from Rab34 - mitochondria / FLCN - GFP localisation . Scale bar = 10μm .

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(a) Confocal immunofluorescence microscopy images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar = 10μm.


( b ) Confocal immunofluorescence microscopy of Rab34 - dsRED - Mito ( WT ) FLCN - GFP DENN only or ΔDENN transfected HeLa showing DENN domain dependent recruitment to mitochondria . Scale bar = 10μm .

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(b) Confocal immunofluorescence microscopy images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria. Scale bar = 10μm.


( c ) Western blot of GST - RILP pull down showing association of endogenous FLCN , Rab34 Rab7 with RILP . Input lane shows 1 % input extract .

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(c) Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.


( d ) Western blot of GST - RILP pull down from UOK257 showing DENN domain dependent association of FLCN with RILP N - terminal , Rab binding domain ( RBD ) C - terminal fragments .

(d) Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.


( e ) Western blot showing binding of His - tagged FLCN - DENN domain in E . coli extract to GST - RILP .

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(e) Western blot showing binding of His-tagged FLCN-DENN domain in E.coli extract to GST-RILP.


( f ) Western blot of GST - RILP pull down showing direct interaction between RILP GTP hydrolysis deficient ( Q111L ) Rab34 ( at 200nM , in presence of 1mM GTPγS ) FLCN - DENN domain ( 1μM ) , but not low nucleotide affinity ( T66N ) Rab34 . FLCN - DENN enhances association of RILP with Q111L Rab34 . Graph shows of relative RILP / Rab34 Q111L binding . Error bars show . * * = p < 0 . 01 .

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

(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.


( g ) Western blot of GST - RILP / Rab34 Q111L pull down ( Rab34 at 200nM ) showing that enhanced association of with RILP is achieved with low concentrations of FLCN .

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(g) Western blot analysis of GST-RILP / Rab34 Q111L pull down (Rab34 at 200nM) showing that enhanced association of with RILP is achieved with low concentrations of FLCN.


( h ) Western blot of GST - RILPpull down experiments with GTPγS loaded ( in presence of 1mM GTPγS ) GDP loaded ( in presence of 1mM GDP ) wildtype Rab34 , showing FLCN - DENN ( 500nM ) induced interaction with RILP .

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(h) Western blot analysis of GST-RILPpull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.


( i ) Western blot of a GFP - Trap immunoprecipitation experiment from HeLa showing that expression of HA - RILP promotes association of GFP - Rab34 HA - Rab7 .

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(i) Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.


( a ) Western blot showing endogenous Rab7 Rab34 expression in UOK257 UOK257 - 2 extracts relative of active forms as measured by GST - RILP pull down . HSC70 is used as a loading control . Graphs show of bound Rab7 / Rab34 from 3 independent experiments , error bars show S . E . M . , * = p < 0 . 05 .

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(a) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257-2 cell extracts and relative levels of the active forms as measured by GST-RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show S.E.M., * = p<0.05.


( b ) ( top ) Representative confocal immunofluorescence microscopy showing intracellular distribution of LAMP1 in UOK257 UOK257 - 2 . Dashed highlights periphery in UOK257 - 2 . Scale bar = 10μm . ( bottom left ) Graph showing cumulative distribution of LAMP1 in UOK257 vs UOK257 - 2 . Error bars show S . E . M . from 30 in 3 replicates . ( bottom right ) Graph showing Lysotracker - Red PCC decay with time from confocal movies of UOK257 UOK257 - 2 . Mean values from 5 . Error bars show S . E . M .

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(b) (top) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257-2 cells. Dashed line highlights cell periphery in UOK257-2 cells. Scale bar = 10μm. (bottom left) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257-2 cells. Error bars show S.E.M. from 30 cells in 3 replicates. (bottom right) Graph showing Lysotracker-Red PCC decay with time from confocal movies of UOK257 and UOK257-2 cells. Mean values from 5 cells. Error bars show S.E.M.


( c ) Widefield immunofluorescence showing early endosomes ( EEA1 ) Golgi ( golgin - 97 ) in UOK257 UOK257 - 2 . Scale bar = 10μm .

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(c) Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin-97) in UOK257 and UOK257-2 cells. Scale bar = 10μm.


( d ) Western blot showing FLCN FLCNΔDENN expression in stable UOK257 derived lines .

(d) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.


( e ) Representative widefield microscopy showing intracellular distribution of LAMP1 in UOK257 , UOK257 - FLCN UOK257 - FLCNΔDENN . Scale bar = 10μm . Graphs showing cumulative distribution of LAMP1 in UOK257 , UOK257 - FLCN UOK257 - FLCNΔDENN . Error bars show S . E . M from 30 in 3 replicates .

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(e) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Scale bar = 10μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Error bars show S.E.M from 30 cells in 3 replicates.


( f ) ( left ) Western blot showing endogenous Rab7 Rab34 expression in UOK257 , UOK257 - FLCN UOK257 - FLCNΔDENN relative of activity as measured by GST - RILP pull down . HSC70 is used as a loading control . ( right ) Graphs show of bound Rab7 / Rab34 from 3 independent experiments ( p < 0 . 05 , * * * = p < 0 . 001 ) .

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

(f) (left) Western blot showing endogenous Rab7 and Rab34 expression in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells and relative levels of activity as measured by GST-RILP pull down. HSC70 is used as a loading control. (right) Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments (p<0.05, *** = p<0.001).


( g ) Western blot showing endogenous RILP expression in indicated lines . Graph shows of RILP expression from 3 independent experiments ( * = p < 0 . 05 , * * * = p < 0 . 001 ) .

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

(g) Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (* = p<0.05, *** = p<0.001).


( A ) Immunoblotting for Dnmt1 , Dmnt3a Dmnt3b in spinal cords of AR - 97Q , AR - 24Q , wild - type mice . ( B ) Quantitative densitometry indicated that Dnmt1 spinal cord level was up - regulated in AR - 97Q mice ( n = 3 ) . Dnmt3a Dnmt3b spinal cord level was not significantly different among wild - type , AR - 24Q , AR - 97Q mice ( n = 3 ) .

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(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).


( C ) of Dnmts mRNA of spinal cord in wild - type , AR - 24Q , AR - 97Q mice using RT - qPCR ( n = 3 ) .

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(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).


( D E ) Representative immunohistochemical depicting expression of Dnmt1 , Dmnt3a Dmnt3b in spinal cords of AR - 97Q , AR - 24Q , wild - type mice of immunoreactivity for Dnmts ( n = 25 neurons from three mice of each group ) .

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(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).


( F ) Double - immunofluorescence for Dnmt1 1C2 in spinal motor neurons of an AR - 97Q mouse .

(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.


( G ) Immunoblotting for Dnmt1 of skeletal muscle of wild - type AR - 97Q mice . ( H ) of signal of Dnmt1 bands ( n = 3 ) .

(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).


( A ) Immunoblotting of NSC34 expressing AR - 24Q or AR - 97Q ( NSC24Q NSC97Q , respectively ) that were treated with DHT for Dnmt1 , Dnmt3a Dnmt3b , of signal intensities of bands corresponding to Dnmt1 , Dnmt3a Dnmt3b ( n = 3 ) .

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(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).


( B ) Relative mRNA of Dnmt1 in NSC24Q NSC97Q measured with RT - qPCR ( n = 3 ) .

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(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).


( C ) of Dnmt1 , Dnmt3a Dnmt3b in DHT - treated - untreated NSC97Q by Western blotting .

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(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.


( D ) RT - qPCR of Dnmt1 in DHT - treated - untreated NSC97Q ( n = 3 ) .

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(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).


( E , F ) Quantitative assessment of viability changes due to siRNA - mediated knock down of Dnmt1 , Dnmt3a or Dnmt3b in NSC97Q with DHT ( E ) or without DHT ( F ) using WST - 8 ( n = 3 ) .

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(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).


( G ) Quantitative assessment of viability of NSC97Q treated with DHT RG108 using WST - 8 ( n = 3 per group ) .

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(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).


( H ) Immunoblotting of NSC97Q treated with DHT RG108 for Dnmt1 , Dnmt3a Dnmt3b .

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(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.


( I , J ) viability with siRNA - mediated knockdown of Dnmts in DHT - untreated NSC97Q ( I ) DHT - treated NSC24Q ( J ) .

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(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).


( A - D ) Grip power ( A ) , ( B ) , rotarod performance ( C ) ( Two - way ANOVA with Tukey ' s ) survival rate ( D ) ( Kaplan - Meier log - rank ) of AR - 97Q mice treated with or without RG108 ( DMSO , n = 20 ; 0 . 5 mg / dL , n = 20 ; 2 . 0 mg / dL , n = 20 ) . parameters were significantly improved in AR - 97Q mice treated with 2 . 0 mg / dL RG108 compared with those treated with DMSO : * P < 0 . 05 ( grip ) ; * P < 0 . 05 ( ) ; * P < 0 . 05 ( rotarod ) ; * P < 0 . 05 ( survival ) .

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(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).


( E ) Representative photograph of a 13 - week - old AR - 97Q mouse treated with DMSO ( left ) an - matched AR - 97Q mouse treated with 2 . 0 mg / dL RG108 ( right ) . AR - 97Q mouse medicated with RG108 maintained muscle volume compared with DMSO - treated AR - 97Q mouse of same . compact masses ( arrow ) of mice head are cement covering surgical site .

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(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.


( F , G ) Immunoblots ( F ) relative intensities ( G ) of Dnmt1 - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).


( H , I ) Dnmt1 immunohistochemistry in spinal cord sections from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).


( A ) Immunohistochemistry of spinal cords of AR - 97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody . ( B ) of 1C2 - positive motor neurons in spinal cords ( n = 5 per group ) .

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(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).


( C ) Immunoblotting of spinal cords of AR - 97Q mice treated with or without RG108 for AR . ( D ) Densitometric analyses to quantify AR accumulation in spinal motor neurons of AR - 97Q mice treated with DMSO or RG108 ( n = 3 ) .

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(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).


( E ) Relative mRNA expression of human AR in AR - 97Q mice spinal cord with or without RG108 treatment ( n = 3 ) .

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(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).


Immunohistochemistry for ChAT in spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


quantitative of neuron sizes in spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

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quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


( H , I ) Western blot ( H ) relative signal intensities ( I ) of ChAT - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).


( A ) Immunoblotting of SH - SY5Y stably expressing AR - 24Q ( SH24Q ) or AR - 97Q ( SH97Q ) for Dnmt1 , Dnmt3a Dnmt3b . ( B ) Densitometry showing that Dnmt1 level was up - regulated in SH97Q compared with SH24Q ( n = 3 ) .

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(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b. (B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).


( C , D ) Immunoblots for Dnmts of SH97Q with or without DHT treatment ( C ) data ( D ) ( n = 3 ) .

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(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).


( E ) Seven genes with hyper - methylation in their CpG islands were identified by methylation array .

(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.


( F ) relative mRNA of CDC25B , GFRA3 , NPY , HES5 , SCTR , LEF1 - AS1 CABS1 , normalized to beta - 2 microglobulin , in DHT - treated SH24Q SH97Q were measured using RT - qPCR ( n = 3 ) .

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(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).


( G ) RT - qPCR indicated decrease of Hes5 mRNA level in DHT - treated SH97Q compared with DHT - untreated SH97Q ( n = 3 ) .

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(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).


( H ) Methylation - specific PCR of SH24Q SH97Q that were treated with DHT .

(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.


( I ) Hes5 immunoreactivity of spinal motor neurons in wild - type , AR - 24Q , AR - 97Q mice ( n = 3 ) .

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(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).


( J ) Hes5 mRNA in spinal cords of wild - type , AR - 24Q , AR - 97Q mice ( n = 3 ) .

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(J) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).


methylation of HES5 promoter region of SH97Q treated with DHT RG108 .

DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.


HES5 mRNA of SH97Q treated with DHT RG108 .

HES5 mRNA levels of SH97Q cells treated with DHT and RG108.


methylation of Hes5 promoter region in DHT - treated NSC97Q treated with or without RG108 .

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DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.


mRNA of Hes5 ( n = 3 ) in DHT - treated NSC97Q treated with or without RG108 .

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mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.


( E ) Hes5 mRNA in spinal cords of AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).


( F , G ) Hes5 immunoreactivity of spinal motor neurons in AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).


( A ) Viability of DHT - treated NSC97Q treated with Hes5 control siRNA measured by WST - 8 ( n = 3 ) .

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(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).


( B ) Immunoblots of DHT - administrated NSC97Q treated with Hes5 siRNA ( n = 3 ) .

(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).


( C ) Quantitative viability revealed that siRNA - mediated Hes5 knock down negated therapeutic effect of RG108 in NSC97Q that were treated with DHT ( n = 3 ) , as determined by WST - 8 .

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(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.


viability measured with WST - 8 of DHT - treated NSC97Q transfected with a mock plasmid or Hes5 vector ( n = 3 ) .

Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


viability measured with immunoblotting of DHT - treated NSC97Q transfected with a mock plasmid or Hes5 vector ( n = 3 ) .

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Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


( F ) of LDH release from DHT - treated NSC97Q transfected with a mock plasmid or Hes5 vector ( n = 4 ) .

(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).


( G ) Immunoblots of DHT - administrated NSC97Q transfected with a mock plasmid or Hes5 vector for AR . ( H ) AR aggregation as quantified by densitometry ( n = 3 ) .

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(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).


( A ) Immunoblots of NSC34 treated with Hes5 siRNA . ( B ) of of pSmad2 , Smad2 , phospho - IkBα heat shock factor - 1 ( Hsf1 ) ( n = 3 ) .

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(A) Immunoblots of NSC34 cells treated with Hes5 siRNA. (B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).


( C ) siRNA - mediated knockdown of Hes5 down - regulated Hes5 level ( n = 3 ) .

(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).


( D ) Western blots of Smad2 pSmad2 in NSC97Q with or without DHT treatment ( n = 3 ) .

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(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).


( E ) of Smad2 , pSmad2 , Hes5 in NSC97Q transfected with Hes5 vector ( n = 3 ) .

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(E) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).


( F ) Immunoblots of Smad2 , pSmad2 , Hes5 in cortical neurons treated with lentiviral vector containing AR24Q AR97Q ( n = 3 ) .

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(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).


( G ) Relative mRNA of Hes5 in cortical neurons expressing AR24Q AR97Q ( n = 3 ) .

(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).


( H ) Relative mRNA of Hes5 in motor neurons expressing AR24Q AR97Q ( n = 6 ) .

(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).


( I ) Western blots of Smad2 , pSmad2 , Hes5 in cortical neurons expressing AR97Q treated with mock plasmid or Hes5 - containing lentiviral vector .

(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Spinning disc confocal live imaging of Alexa 555 - EGF ( 555 - EGF ) shown as vesicle tracking with time represented as a colour spectrum . Tracking started 5 min after addition of 20 ng / mL 555 - EGF for indicated durations . Scale bar : 10 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . B : Immunofluorescence of EGFR following 5 or 15 min stimulation with 20 ng / mL EGF . Scale bar : 20 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : of EGFR vesicles in a perinuclear region ( within 30 μm diameter of centre of nucleus ) Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . D : were stimulated with 20 ng / mL 555 - EGF for 15 or 30 min before immunofluorescence against EGFR . Scale bar : 10 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : of of total EGFR vesicles that colocalise with 555 - EGF Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . F : stably expressing mCherry - Rab5 were stimulated with 20 ng / mL EGF for indicated times before immunofluorescence against EGFR . Scale bar : 10 μm .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . G : Pearson ' s colocalisation coefficient between mCherry - Rab5 EGFR Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Pearson's colocalisation coefficient between mCherry-Rab5 and EGFR Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . A : Control , sgAtg7 , or sgAtg16l1 were treated with 2 ng / mL EGF for 15 min . were then processed for using anti - EEA1 antibodies PI ( 3 ) P probe ( Alexa 488 - labelled 2XFYVE domains ) . To ensure specificity of probe , control were pre - treated with 5 mM 3 ' MA for 30 min . Scale bar : 10 μm . B : of PI ( 3 ) P + label per ( in A ) . C : Pearson ' s colocalisation coefficient between PI ( 3 ) P EEA1 ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3)P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . D : Control sgAtg7 were stimulated 20 ng / mL EGF for 5 or 15 min before immunofluorescence against EEA1 EGFR . Scale bar : 10 μm . E : Pearson ' s colocalisation coefficient between EEA1 EGFR ( in D ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . F : Endogenous VPS34 was immunoprecipitated from control sgAtg7 that were treated with 2 ng / mL EGF for 15 min then lysed in CHAPS - containing detergent buffer binding partners detected by western blotting . G : Densitometry analyses of coimmunoprecipitated with endogenous VPS34 ( in F ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting. G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : transiently expressing YFP - Galectins ( YFP - Gal3 , - Gal8 , or - Gal9 ) were stimulated with 2 ng / mL EGF for 15 min before fixation immunofluorescence against EEA1 . Scale bar : 10 μm . B : of of total EEA1 vesicles that colocalise with YFP - Galectins ( in A ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells transiently expressing YFP-Galectins (YFP-Gal3, -Gal8, or -Gal9) were stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A).Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : transiently expressing YFP - Gal8 were stimulated with 20 ng / mL Alexa 555 - EGF ( 555 - EGF ) for 15 min . Scale bar : 10 μm . D : of of YFP - Gal8 - labelled vesicles that colocalise with 555 - EGF in control or ATG7 - deficient ( in C ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.