PRMT1 - proficient ( - Dox ) as well as PRMT1 - depleted cells ( + Dox ) were further treated with the protein synthesis inhibitor cycloheximide ( CHX ) and harvested after 0 , 0 . 5 , 1 , 2 , 3 , 4 , and 6 hours . Cell lysates were subjected to immunoblotting using α - p14ARF and α - β - TUBULIN ( loading control ) antibodies and conventional ECL detection ( F ) . For precise quantification of the p14ARF protein stability in the - / + Dox conditions during the CHX time course , immunoblotting was performed with independent cell lysates and primary antibodies , as in ( F ) , which were then detected using fluorescence dye - coupled secondary antibodies and the LI - COR Odyssey system . The quantitative p14ARF signals were normalized to the corresponding quantitative β - TUBULIN signals and displayed in ( G ) . The CHX - untreated samples ( 0 hour ) were set to 100 % for each condition .

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

PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.


HeLa cells were transfected with the indicated siRNAs ( two control / non - targeting siRNAs and two PRMT1 - specific siRNAs ) and irradiated at 150 J / cm2 UVC or not irradiated . After 24 hours , the cellular distribution of endogenous p14ARF was determined by immunofluorescence ( IF ) staining using α - p14ARF antibody ( red , endogenous p14ARF ) and in the merge additionally DAPI ( blue , nuclei / DNA ) . Representative IF results are shown for two conditions ( siControl _ 4 and siPRMT1 _ 1 ) in ( A ) , with asterisks indicating exclusively nucleolar and arrowheads indicating not - exclusively nucleolar but additionally or predominantly nucleo - / cytoplasmic p14ARF localization . Scale bars : 15 μm . The subcellular distribution of p14ARF , as determined by IF , was quantified by cell counting ( percentage of cells ) for all conditions ( B ) .

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

HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, the cellular distribution of endogenous p14ARF was determined by immunofluorescence (IF) staining using α-p14ARF antibody (red, endogenous p14ARF) and in the merge additionally DAPI (blue, nuclei/DNA). Representative IF results are shown for two conditions (siControl_4 and siPRMT1_1) in (A), with asterisks indicating exclusively nucleolar and arrowheads indicating not-exclusively nucleolar but additionally or predominantly nucleo-/cytoplasmic p14ARF localization. Scale bars: 15 μm. The subcellular distribution of p14ARF, as determined by IF, was quantified by cell counting (percentage of cells) for all conditions (B).


H : CRISPR / Cas9 control ( CTR _ 1 and CTR _ 2 ) or PRMT1 - deleted ( KO _ 1 and KO _ 2 ) HeLa cell lines were irradiated at 150 J / cm2 UVC or not irradiated . After 4 hours , immunoprecipitations ( IP ) of endogenous p14ARF were performed from cell lysates using α - p14ARF antibody or IgG as negative control . IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies . Short and long exposure times are specified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC or not irradiated. After 4 hours, immunoprecipitations (IP) of endogenous p14ARF were performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are specified.


A : Kaplan - Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short - term ( < 500 days , left plot ) and long - term survival ( > 500 days , right plot ) .

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A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot).


B : Representative immunohistochemistry ( IHC ) stainings of PRMT1 ( brown ) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients . Based on the immunostaining intensity and the percentage of stained tumor cells , all specimens were divided into three PRMT1 expression scores : not elevated , moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas . Right images are magnifications of the left images , as indicated by the black rectangles . Scale bars in the left images : 100 μm . Scale bars in the right images : 50 μm .

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B: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm.


E : Immunofluorescence ( IF ) stainings of the 75 PDAC specimens were performed using α - p14ARF ( red ) , α - CK8 / 18 ( green , staining ductal cells in normal pancreas and neoplastic cells in PDAC ) antibodies and DAPI ( blue , nuclei / DNA ) . Representative IF images are shown for normal human pancreatic tissue , two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells . Left and corresponding right images show the same tissue section . Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC . The last image pair shows a magnification of the image pair above ( indicated by the rectangle ) . Scale bars : 35 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 O O O O O O O O O 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: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm.


B : PaTu8988t cells were treated with 0 , 1 , 3 , and 10 μM gemcitabine ( GEM ) . After 48 hours , cell lysates were analyzed by immunoblotting using the indicated antibodies . The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal , as specified by the numbers below the blots , with the untreated condition ( - GEM ) set to 1 . The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot . C : PaTu8988t cells were treated with 3 μM gemcitabine ( GEM ) or left untreated ( 0 hour ) . After 0 , 6 , 12 , 24 and 48 hours , methylation of endogenous p14ARF was analyzed by immunoblotting using α - me - p14ARF , α - p14ARF and α - GAPDH ( loading control ) antibodies . The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal , as specified by the numbers below the blots , with the untreated condition ( 0 hour ) set to 1 .

O 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 O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PaTu8988t cells were treated with 0, 1, 3, and 10 μM gemcitabine (GEM). After 48 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot. C: PaTu8988t cells were treated with 3 μM gemcitabine (GEM) or left untreated (0 hour). After 0, 6, 12, 24 and 48 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-GAPDH (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (0 hour) set to 1.


D , PaTu8988t and MiaPaCa - 2 cells were treated ( + ) with 20 μM MS023 or left untreated ( - ) for 3 days . For the last 2 days , the cells were additionally exposed to 0 , 1 , or 3 μM gemcitabine ( GEM ) . Subsequently , cell death was quantified by flow cytometry ( in ( D ) ) using FITC - labeled AnnexinV and propidium iodide ( PI ) for four independent experiments ( mean ± SD , * : p - value ≤ 0 . 05 using Welch ´ s t - test ) .

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

D, PaTu8988t and MiaPaCa-2 cells were treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 0, 1, or 3 μM gemcitabine (GEM). Subsequently, cell death was quantified by flow cytometry (in (D)) using FITC-labeled AnnexinV and propidium iodide (PI) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using Welch´s t-test).


G : Apoptosis was quantified by flow cytometry ( G ) using FITC - labeled AnnexinV and propidium iodide ( PI ) for six independent experiments ( mean ± SD , * * : p - value ≤ 0 . 005 , * : p - value ≤ 0 . 05 , ns : not significant using Welch ´ s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Apoptosis was quantified by flow cytometry (G) using FITC-labeled AnnexinV and propidium iodide (PI) for six independent experiments (mean ± SD, **: p-value ≤ 0.005, *: p-value ≤ 0.05, ns: not significant using Welch´s t-test).


( A ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at the indicated hours post - infection ( h . p . i . ) . Three independent experiments were measured for quantification of p62 levels , and the blot shown is representative .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at the indicated hours post-infection (h.p.i.). Three independent experiments were measured for quantification of p62 levels, and the blot shown is representative.


( B ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at 6 h . p . i . Cycloheximide ( Cy . ) was added to mock infected cells at a final concentration of 50 µg / mL , for 4 hours . Cells were then pre - treated with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) for 14 hours prior to infection and kept under treatment throughout infection . MG132 was used at a final concentration of 20 µM and added to the media at the time of infection . The blot shown is representative of three independent experiments .

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

(B) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.


( A ) Cells were transfected with scrambled ( SCR ) or anti - LC3 ( LC3 ) siRNAs , and 48 h later infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . , and immunoblots were performed on lysates for p62 , GAPDH , and LC3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were transfected with scrambled (SCR) or anti-LC3 (LC3) siRNAs, and 48 h later infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i., and immunoblots were performed on lysates for p62, GAPDH, and LC3.


( B ) Cells were treated with 10 mM 3 - MA for 12 h , then infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . and lysates were used for immunoblots .

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

(B) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.


Triplicate samples of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell ( A ) or MOI of 50 pfu / cell ( B ) . Cells were pre - treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection . Cell - associated virus was collected at the indicated times post - infection , and virus titers were determined by plaque assay . Parallel infections were collected for Western blots of LC3 ( A ) or p62 ( B ) In ( B ) cell - associated virus was collected at 6 h . p . i . for plaque assay .

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

Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (A) or MOI of 50 pfu/cell (B). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay.


( C ) Infections were performed as in ( A ) , then 10 µg / mL each of E64d and Pepstatin A ( Pep . A ) were added to the media at the time of infection . Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH . ( D ) Infection at an MOI of 50 pfu / cell , with E64 / Pep . A as in ( C ) .

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

(C) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (D) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).


( A ) Triplicate samples of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell , and cell - associated virus was collected at the indicated times post - infection . NH4Cl ( 20 mM ) was added to the media at the time of infection ( solid line ) . Virus titers were then determined by plaque assay . ( B ) Infection as in ( A ) , carried out to 16 h to represent a multiple - cycle infection .

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

(A) Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, and cell-associated virus was collected at the indicated times post-infection. NH4Cl (20 mM) was added to the media at the time of infection (solid line). Virus titers were then determined by plaque assay. (B) Infection as in (A), carried out to 16 h to represent a multiple-cycle infection.


( C ) Triplicate H1 - Hela infections with PV at an MOI of 50 pfu / cell , with cell - associated virus collected at 6 h . p . i . ( D ) Cells were infected as in ( A ) , then NH4Cl was added to half of the samples at 3 . 5 hours post - infection , and cell - associated virus was collected immediately or at 7 h . p . i .

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(C) Triplicate H1-Hela infections with PV at an MOI of 50 pfu/cell, with cell-associated virus collected at 6 h.p.i. (D) Cells were infected as in (A), then NH4Cl was added to half of the samples at 3.5 hours post-infection, and cell-associated virus was collected immediately or at 7 h.p.i.


( E ) Cells were pre - treated for 14 hours with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) and kept under treatment throughout infection ( MOI = 0 . 1 pfu / cell ) . Cell - associated virus was collected at 6 or 12 h . p . i . and virus titers were determined by plaque assay . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were pre-treated for 14 hours with 0.1 µM bafilomycin A1 (Baf.A1) and kept under treatment throughout infection (MOI = 0.1 pfu/cell). Cell-associated virus was collected at 6 or 12 h.p.i. and virus titers were determined by plaque assay. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - HeLa cells were infected with PV at an MOI of 50 pfu / cell . Cells were pulsed at the indicated h . p . i with 35S - labeled methionine for 1 h then lysed . Lysates were run on SDS - PAGE . Expected viral proteins are labeled according to recognized banding patterns .

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

(A) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with 35S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns.


( B ) Triplicate plates of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell , virus RNA and host GAPDH RNA were measured by qRT - PCR . Virus RNA levels were normalized to GAPDH levels using the delta - Ct method . NH4Cl treatment was as described in Figure 4 , and Guanidine HCl ( 2 mM ) was added to the media at the time of infection . The data shown are pooled from three replicate experiments , and the titer of cell - associated virus collected at 6h . p . i . from each replicate was determined by plaque assay .

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

(B) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH4Cl treatment was as described in Figure 4, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay.


( C ) Triplicate plates of 293T cells were treated with 20 mM 3 - MA for 2 hours prior to infection , and kept under treatment throughout infection . PV infections were done at an MOI of 0 . 1 pfu / cell . RNA levels and virus titers were analyzed as in ( B ) . * * p < 0 . 01 , * * * p < 0 . 0001 .

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

(C) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.


( A ) H1 - Hela cells were infected at an MOI of 50 pfu / cell , and half of the samples were treated with NH4Cl . Cells were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 or 6 h . p . i . , and lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected and the counts per minute ( CPM ) were analyzed for each fraction . Representative gradients from three independent experiments are shown .

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

(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown.


. ( B ) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis . Data shown are the averages from three independent experiments . (

O O O O O O O O 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) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. (


( C ) Three fractions representing the 150S and 75S peaks were pooled and run on SDS - PAGE , and the 35S - Methionine labeled bands were visualized . The bands are labeled according to expected relative migration pattern , and VP2 is identified by its absence in the 75S peak . The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - Hela cells were infected at an MOI of 50 pfu / cell , and half of the samples were treated with bafilomycin A1 as described in Figure 4 . Cells were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 h . p . i . , and lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected and the counts per minute ( CPM ) were analyzed for each fraction .

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(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in Figure 4. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction.


( B ) Three fractions representing the 150S and 75S peaks were pooled and run on SDS - PAGE as in Figure 6 . The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units . The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay . * p < 0 . 05 .

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(B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in Figure 6. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.


D . The model proteins consist of a fusion between Ura3p and GFP ( URA3sol , left ) and the amyloid - β - peptide of 42 residues ( Aβ ) ( URA3agg , right ) . After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci .

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D. The model proteins consist of a fusion between Ura3p and GFP (URA3sol, left) and the amyloid-β-peptide of 42 residues (Aβ) (URA3agg, right). After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci.


S values measured in different environments . In all cases the standard error is below 5 % . Color scale : purple in absence of uracil ( - Ura ) , white in the presence of uracil ( + Ura ) and orange in the presence of 5FOA ( + 5FOA ) . At 37 ° C , the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil , impeding the measurement of S . A bar of purple - white diagonal lines indicates a presumed value of S at 37 ° C without uracil .

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S values measured in different environments. In all cases the standard error is below 5%. Color scale: purple in absence of uracil (-Ura), white in the presence of uracil (+Ura) and orange in the presence of 5FOA (+5FOA). At 37°C, the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil, impeding the measurement of S. A bar of purple-white diagonal lines indicates a presumed value of S at 37°C without uracil.


B . Plots showing the distribution and relationship between the fractions of deposited or free Ura3p , its magnitude of effect on fitness ( α / β / γ ) and the selection coefficient ( S ) in different environments .

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B. Plots showing the distribution and relationship between the fractions of deposited or free Ura3p, its magnitude of effect on fitness (α/β/γ) and the selection coefficient (S) in different environments.


C . Average of the selection coefficient ( left ) and average of the cytosolic fluorescence ( equivalent to the fractional abundance of the free protein in URA3agg , right ) .

C. Average of the selection coefficient (left) and average of the cytosolic fluorescence (equivalent to the fractional abundance of the free protein in URA3agg, right).


Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance . A . Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30 ° C . B . Coefficient of variation and p - values ( Wilcoxon test ) of the total fluorescence measured from populations of URA3agg and URA3sol . C . Boxplot showing the distribution of foci fluorescence ( left ) or cytoplasmic fluorescence ( right ) , from the foci containing subset of a population of URA3agg grown at 30ºC . Coefficient of Variation and p - values of each boxplot are shown below . D . Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell . Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC .

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Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance. A. Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30°C. B. Coefficient of variation and p-values (Wilcoxon test) of the total fluorescence measured from populations of URA3agg and URA3sol. C. Boxplot showing the distribution of foci fluorescence (left) or cytoplasmic fluorescence (right), from the foci containing subset of a population of URA3agg grown at 30ºC. Coefficient of Variation and p-values of each boxplot are shown below. D. Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell. Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC.


E . Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30 ° C . F . Schematic showing that in our system , the phase separation - promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell . G . Schematic highlighting that higher / lower level of free protein inside the cell results in higher / lower Ura3p activity leading to the manifestation of a stronger / weaker phenotype . H . Boxplots of the cytoplasmic fluorescence normalized by the cell area ( FCYTOagg / cell area ) for individual cells of the URA3agg population in 5FOA . Light green box , cells with foci containing at least 70 % of the total fluorescence ( big foci ) . Dark green box , cells with foci containing less than 70 % of the total fluorescence ( small foci ) . I - J . Cell cycle arrest phenotype . Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content . The cells were incubated for 18 hours at 30 ° C without ( I ) and with 5FOA ( J ) .

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E. Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30°C. F. Schematic showing that in our system, the phase separation-promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell. G. Schematic highlighting that higher/lower level of free protein inside the cell results in higher/lower Ura3p activity leading to the manifestation of a stronger/weaker phenotype. H. Boxplots of the cytoplasmic fluorescence normalized by the cell area (FCYTOagg/cell area) for individual cells of the URA3agg population in 5FOA. Light green box, cells with foci containing at least 70% of the total fluorescence (big foci). Dark green box, cells with foci containing less than 70% of the total fluorescence (small foci). I-J. Cell cycle arrest phenotype. Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content. The cells were incubated for 18 hours at 30°C without (I) and with 5FOA (J).


( B ) Drug response curves upon treatment of organoid models with nutlin ‑ 3a , 5 ‑ FU , oxaliplatin , ibrutinib , panobinostat and entinostat ( biological replicates n = 3 , data are shown as mean ± SD ) .

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(B) Drug response curves upon treatment of organoid models with nutlin‑3a, 5‑FU, oxaliplatin, ibrutinib, panobinostat and entinostat (biological replicates n=3, data are shown as mean ±SD).


Drug response curves upon treatment with inhibitors for ( B ) EGFR ( afatinib , gefitinib ) , ( biological replicates n = 3 , data are shown as mean ± SD ) .

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Drug response curves upon treatment with inhibitors for (B) EGFR (afatinib, gefitinib), (biological replicates n=3, data are shown as mean ±SD).


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 ) .

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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 ) .

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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 images of human patient derived organoids ( PDOs ) from gastric cancer with and 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 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 ) .

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(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 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 .

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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 assay comparing the effects of varying amounts of K48 - and K63 - linked chains of different lengths on UBQLN2 LLPS at 30 μM UBQLN2 .

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 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 .

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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 ratio with Ub4 . The highlighted regions in the domain map are important for the homotypic interactions that drive UBQLN2 LLPS ( Dao et al , 2018 ) .

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 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 ) .

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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 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 .

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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 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 .

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 line ) are mapped onto the proximal Ub unit of Ub4 in panel A .

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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 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 .

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

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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 distance 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 all chain types , but clustered into two categories for clarity .

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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 and HOTag6 - Ub .

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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 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 .

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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 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 ) .

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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 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 .

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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 assay as a function of temperature at 30 μM UBQLN2 .

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


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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

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

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 cells exacerbates the decrease of colon length 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + 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 .

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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 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 ) .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 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 .

O O O O O O O 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

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

L 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 . Cell extracts from peritoneal macrophages of Elp3Control and Elp3 ∆ Mye mice treated or not with IL - 4 / IL - 13 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 .

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 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 .

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

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 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 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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

I-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 ) . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 . )

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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 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 .

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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 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 .

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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 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 .

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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 cell showing Rab7 ( red ) and 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 intensity in control GFP ( black ) , GFP - Rab34 ( green ) or GFP - Rab7 ( blue ) transfected cells . Note black line and 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 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 .

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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 fluorescence microscopy images of FLCN depleted or control cells , 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 ) 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 .

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(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 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 .

(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 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 .

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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 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 .

(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 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 .

(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 the Z - stack in figure 4B . Orange arrows show close LAMP1 / Rab34 association .

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


( 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 .

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(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 - 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 =

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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 - 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 =

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(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 and Rab34 in starved HeLa cells .

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


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 .

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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.


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 .

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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.


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 .

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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.


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 .

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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 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 .

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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 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 .

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

(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 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 .

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(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.