A - B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle ( DMSO ) , NaB ( 100 mg kg − 1 / daily ) or DFZ ( 1 . 2 mg kg − 1 / daily ) for 3 weeks using the rotarod and weight test . Bar charts show the latency to fall or drop the weight of wt and dystrophic mice .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE B-ORGANISM O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O B-DISEASE B-ORGANISM O

A-B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle (DMSO), NaB (100 mg kg−1/daily) or DFZ (1.2 mg kg−1/daily) for 3 weeks using the rotarod and weight test. Bar charts show the latency to fall or drop the weight of wt and dystrophic mice.


A - I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ .

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A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.


J - K Representative blotting and bar chart with individual points showing the expression and / or phosphorylation of pAKT / AKT and COX2 in the gastrocnemius of the indicated six groups of mice .

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J-K Representative blotting and bar chart with individual points showing the expression and/or phosphorylation of pAKT/AKT and COX2 in the gastrocnemius of the indicated six groups of mice.


A - B Levels of AEA and 2 - AG in plasma samples of wt and mdx mice expressed as pmol mg − 1 of wet tissue weight .

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A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.


C - D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice .

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C-D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice.


E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice . F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH .

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E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice. F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH. D


A - C Bar charts with individual points showing the mRNA expression levels of Ulk , Pink and Becn1 measured in control and mdx mice treated with rimonabant ( 0 . 5 mg kg - 1 ) .

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A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).


D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice . E , F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH .

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D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice. E, F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH. D


A - B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).


A - F Bar chart with individual points showing the mRNA expression levels of Cb1 , Daglα , Daglβ , Magl , Napepld , and Faah in control ( vehicle , DMSO ) and / or GPR109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).


G - H Levels of AEA and 2 - AG were measured in C2C12 cells exposed to LPS ( 1 μg / ml ) or NaB ( 3 mM ) for 24 h . I Effect of ACEA ( 1 μM ) and rimonabant ( 1 μM ) on autophagosome formation measured in C2C12 cells .

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G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h. I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells. D


C - J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A - silenced C2C12 myoblasts exposed to LPS ( 1 μg / ml ) in the presence or absence of either NaB ( 3 mM ) , MK1903 ( 1 μM ) , rosiglitazone ( 1 μM ) . NaB was also tested in the presence or absence of either rosiglitazone ( 1 μM ) or T007 ( 1 μM ) .

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C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).


A - E Bar chart showing the mRNA expression levels of IL6 , COX2 , ULK 1 , ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor ( HD ) and five DMD donors ( D1 - D5 ) .

O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-DISEASE O O O O O O O

A-E Bar chart showing the mRNA expression levels of IL6, COX2, ULK 1, ATG13 and ATG4 mRNA in primary human myoblasts isolated from one healthy donor (HD) and five DMD donors (D1- D5).


( C ) Streptavidin Western blotting of total biotinylated proteins in APEX2 : RAB or APEX2 only Flp - In / T - REx HeLa cells .

(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.


( D ) APEX2 : RAB biotinylated proteins ( streptavidin ) are partially colocalized with EEA1 in HeLa cells , n = 2 independent experiments . Scale bars : 10µm or 5µm in enlarged views .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O

(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.


( C ) ProHits - viz generated dotblots of RAB effectors , GEFs , GAPs and sorting complexes . Green , blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins .

O O O O O O O B-EXP_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 B-EXP_ASSAY O O O O O O O

(C) ProHits-viz generated dotblots of RAB effectors, GEFs, GAPs and sorting complexes. Green, blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins.


Strumpellin and VPS35 are in close proximity to RAB5 and RAB21 . ( A ) Anti - biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35 . Lysates correspond to 1 % of input , n = 3 independent experiments . RAB21 can be seen interacting with various WASH and retromer complexes subunits .

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Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits.


GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous ( B ) VPS26 immunoblot Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous (B) VPS26 immunoblot Lysates correspond to 5% of input, n≥ 3 independent experiments.


GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and ( C ) FAM21 , Strumpellin and VPS35 immunoblots . Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and (C) FAM21, Strumpellin and VPS35 immunoblots. Lysates correspond to 5% of input, n≥ 3 independent experiments.


RAB21 actively pull - downs WASH and retromer complex subunits ( D ) Bacterially - purified and GTP - loaded GST : RAB21 pull - down of HeLa cell lysates followed by Strumpellin , FAM21 , CAPZα and VPS35 immunoblots . Ponceau staining reveals the purity of the GST and GST : RAB21 used for the pull - downs . n = 3 independent experiments .

B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments.


( E ) RAB21 colocalizes with the WASH and retromer complexes . Transiently expressing GFP : RAB21 HeLa cells were fixed and stained for endogenous EEA1 , FAM21 , WASH and VPS26 . Boxed region is magnified and single channels are depicted . Scale bars : 10µm or 5µm in enlarged view .

O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(E) RAB21 colocalizes with the WASH and retromer complexes. Transiently expressing GFP:RAB21 HeLa cells were fixed and stained for endogenous EEA1, FAM21, WASH and VPS26. Boxed region is magnified and single channels are depicted. Scale bars: 10µm or 5µm in enlarged view.


( F ) Per cell Pearson Correlation between RAB21 and the various markers . Error bars are SEM , n = 2 independent experiments .

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(F) Per cell Pearson Correlation between RAB21 and the various markers. Error bars are SEM, n=2 independent experiments.


( A ) Low RAB21 expression levels in two independent RAB21 KO cell populations . Immunoblot analysis of endogenous RAB21 , RAB5 , RAB7 and GAPDH .

O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH.


( B ) Endo - lysosomal compartments are unaffected by the loss of RAB21 . Immunofluorescences of APPL1 , TfR , RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations . Scale bars : 10µm , n = 3 independent experiments .

O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.


The stability of the retromer cargo sorting complex is affected in RAB21 KO cells . Immunoblot analysis of VPS35 , VPS26 , VPS29 , GAPDH and RAB21 in parental and the two RAB21 KO cell populations . Ratio of VPS35 , VPS26 and VPS29 integrated densities to GAPDH for four independent experiments ; SEM . * represents p < 0 . 05 Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The stability of the retromer cargo sorting complex is affected in RAB21 KO cells. Immunoblot analysis of VPS35, VPS26, VPS29, GAPDH and RAB21 in parental and the two RAB21 KO cell populations. Ratio of VPS35, VPS26 and VPS29 integrated densities to GAPDH for four independent experiments; SEM. * represents p <0.05 Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( B ) WASH complex proteins are not affected by RAB21 deletion . Immunoblot of Strumpellin , WASH , FAM21 , CAPZα , GAPDH and RAB21 . Ratio of WASH , FAM21 , Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments ; SEM . Data information Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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) WASH complex proteins are not affected by RAB21 deletion. Immunoblot of Strumpellin, WASH, FAM21, CAPZα, GAPDH and RAB21. Ratio of WASH, FAM21, Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments; SEM. Data information Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( C ) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( D ) Per cell Pearson Correlation between VPS35 and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : ( D ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (C) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (D) Per cell Pearson Correlation between VPS35 and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (D) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( E ) Average number of VPS35 puncta per cell ; SEM , n = 3 independent experiments . Data information Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (E) Average number of VPS35 puncta per cell; SEM, n=3 independent experiments. Data information Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( F - H ) RAB21 loss impairs endosomal recruitment of the WASH complex . ( F ) Immunofluorescence of endogenous WASH and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( G ) Per cell Pearson Correlation between VPS35 and EEA1 and ( H ) FAM21 and EEA1 ; SEM , n = 2 independent experiments . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-H) RAB21 loss impairs endosomal recruitment of the WASH complex. (F) Immunofluorescence of endogenous WASH and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (G) Per cell Pearson Correlation between VPS35 and EEA1 and (H) FAM21 and EEA1; SEM, n=2 independent experiments. Data information: scale bars represent 10µm or 2.5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( I - J ) RAB21 knockout reduces endosomal F - actin levels . ( I ) Immunofluorescence of endogenous F - actin using Alexa - 488 conjugated phalloidin and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( J ) Per cell Pearson Correlation between internal F - actin and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( A - C ) RAB21 is not required for CI - MPR or GLUT1 trafficking . ( A ) Immunofluorescence of endogenous CI - MPR and TGN46 or GLUT1 with LAMP1 in RAB21 - knockout cells . Boxed region is magnified . ( B ) Per cell Pearson Correlation between CI - MPR and TGN46 or ( C ) GLUT1 and LAMP1 ; SEM , n = 3 independent experiments . Data information : In ( A ) , scale bars represent 20µm or 5µm in enlarged views for CI - MPR , while they represent 10µm in enlarged views for GLUT1 . Statistical tests used : ( B ) - Unpaired t - tests ( C Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. Statistical tests used: (B) - Unpaired t-tests (C Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


MCT1 trafficking requires RAB21 . ( D ) Immunofluorescence of endogenous MCT1 in wild type or RAB21 - depleted cells . ( E ) Per cell integrated MCT1 intensity ( in Relative Fluorescence Unit ) ; SEM , n = 2 independent experiments . Data information : scale bars represent 20µm or 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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MCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. Data information: scale bars represent 20µm or 5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


( F ) RAB21 interacts with SLC3A2 . FLAG immunoprecipitation of FLAG : RAB21 and immunoblot of co - expressed SLC3A2 : HA . Lysates correspond to 5 % input , n = 3 independent experiments .

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(F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments.


( G - H ) RAB21 is required for appropriate SLC3A2 , Basigin and CD44 trafficking . ( G ) Antibody - uptake assays in parental or knockout RAB21 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Note that non - internalized antibodies were removed by an acid wash , which was not fully efficient for CD147 and CD44 , explaining plasma membrane labeling . ( H ) Percentage of cells with tubules in parental or RAB21 knockout cells ; SEM , n = 3 independent experiments . Data information scale bars represent 20µm or 5µm in enlarged views . Statistical tests used Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. Statistical tests used Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


( A ) Validation of WASH and retromer knockouts and SLC3A2 protein levels . Immunoblots of parental or FAM21 , VARP and VPS29 knockout cell populations .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations.


( B ) Antibody - uptake assays in parental or knockout FAM21 , VARP and VPS29 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Data information : ( B ) scale bars represent

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm


( C ) Percentage of cells with tubules in parental or in various knockout cells ; SEM , n = 3 independent experiments . Statistical tests used : ( C ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. Statistical tests used: (C) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( D ) Ratio of SLC3A2 integrated densities to GAPDH ; SEM , n = 3 independent experiments . Statistical tests used : ( D ) One sample T - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. Statistical tests used: (D) One sample T-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( E ) EEA1 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information scale bars represent 10µm or 2 . 5µm in enlarged views .

B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information scale bars represent 10µm or 2.5µm in enlarged views.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( F ) VPS26 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views .

B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information: scale bars represent 10µm or 2.5µm in enlarged views.


( G ) Per cell Pearson Correlation between GFP : RAB21 and EEA1 or VPS26 ; SEM , n = 3 independent experiments . Statistical tests used : ( G ) - Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments. Statistical tests used: (G) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM O O O O O O B-DISEASE I-DISEASE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O

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


a , Diagram of the cell cycle composed of G1 , S , G2 , and M phases ( not to scale ) . Phase durations were quantified by time - lapse fluorescence microscopy using a PCNA - mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min .

O O O O O B-EXP_ASSAY I-EXP_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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min.


b , Mean phase durations in RPE , U2OS , and H9 cell lines . Error bars represent standard deviations .

O O O O O O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O

b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.


c , Coefficient of variation ( CV ) of phase durations .

c, Coefficient of variation (CV) of phase durations.


d , Percentage of the total variation in cell cycle duration contributed by individual phases .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

d, Percentage of the total variation in cell cycle duration contributed by individual phases.


e , Correlations between individual cell cycle phase durations . Sample sizes were adequate to detect correlations n = 125 ( RPE ) , 130 ( U2OS ) , 113 ( H9 ) . R2 , square of Pearson correlation coefficient .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O

e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.


b , Shift in phase durations of RPE cells overexpressing Myc . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : Myc , n =

O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O

b, Shift in phase durations of RPE cells overexpressing Myc. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: Myc, n = 116


c , Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc . Number of cells : Myc , n =

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O O O

c, Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc. Number of cells: Myc, n = 116


e , Shift in phase durations of RPE cells treated with 50 ng / mL aphidicolin . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : aphidicolin , n =

O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O

e, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115


f , Pairwise correlation between cell cycle phase durations under aphidicolin treatment . Number of cells : aphidicolin , n =

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O

f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115


h , Shift in phase durations of RPE cells incubated at 34 ° C . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : 34 ° C , n =

O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Shift in phase durations of RPE cells incubated at 34°C. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: 34°C, n = 122


i , Pairwise correlation between phase durations for cells incubated at 34 ° C . Number of cells : 34 ° C , n =

O 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, Pairwise correlation between phase durations for cells incubated at 34°C. Number of cells: 34°C, n = 122


k , Shift in phase durations of RPE cells treated with NCS . , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : NCS , n = 119 .

O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O

k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.


l , Pairwise correlation between phase durations for cells treated with NCS . Number of cells : NCS , n = 119 .

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l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.


g , Shifts in phase durations for RPE cells treated with 2 μM CVT - 313 . A boxplot representing the distributions of durations in untreated cells is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov test . ( n = 117 cells ) .

O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).


h , Pairwise correlation between cell cycle phase durations upon treatment with CVT - 313 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.


j , Shifts in phase durations for RPE cells overexpressing cyclin D . A boxplot representing the distributions of durations in untreated cells is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov test . ( n = 113 cells ) .

O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells).


k , Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D . P indicates P - value .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O

k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.


b , Shift in phase durations of U2OS cells treated with NCS . , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : NCS , n =

O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O

b, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114


c , Pairwise correlation between phase durations for U2OS cells treated with NCS . Number of cells : NCS , n =

O O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O

c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114


e , Shift in phase durations of U2OS cells treated with 50 ng / mL aphidicolin . boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov test . Number of cells : aphidicolin , n = 153 .

O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O

e, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.


f , Pairwise correlation between cell cycle phase durations under aphidicolin treatment . Number of cells : aphidicolin , n = 153 .

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f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.


C . MA plot highlighting genes differentially expressed between state independent of strain ( glm state term , false discovery rate - adjusted P value ( FDR ) < 0 . 05 and log2FC > 1 ) . Core pluripotent TFs highlighted for each state ( maroon = naive ESCs , teal = formative EpiLCs ) .

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C. MA plot highlighting genes differentially expressed between state independent of strain (glm state term, false discovery rate-adjusted P value (FDR) < 0.05 and log2FC >1). Core pluripotent TFs highlighted for each state (maroon = naive ESCs, teal = formative EpiLCs).


H . Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G .

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H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.


A - D . Example expression patterns for select genes identified by applying a general linear model ( glm ) including state , genotype , and interaction terms . Dots represent individual biological replicates ( N = 3 ) ; lines highlight changes in mean values for replicates between each state ( log2FC > 1 and FDR < 0 . 05 ) . A . State dependent expression is exemplified by Nanog which showed no difference between strains . B . Expression of Zfp277 exemplifies strain - dependence being consistently higher in B6 . C & D . A significant genotype x state ( GXS ) interaction was identified for Nr5a2 ( C , a marker for pluripotency ) and Sox1 ( D , a marker for neuronal differentiation ) .

O O O O O B-EXP_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 O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O

A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).


D . Feature plot of expression gradients for indicated gene overlaid on UMAP from a . Three lineage markers that help to distinguish cellular identity are shown for five cell clusters .

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D. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.


E . Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains . Cells were labeled with anti - EpCAM ( top ) or biological replicates with anti - SOX1 antibody ( bottom ) . Gated population indicates percentages of EpCAM + ( Q3 ) or SOX1 + cells .

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E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.


B . Scatterplot of genomic locations of individual chromatin accessibility ( ca ) QTL ( x - axis , LOD > 5 ) versus location of ATAC peak being regulated ( y - axis ) for ESCs . Genetic effects that act locally ( n = 3 , 973 ) , i . e . in cis , fall on the diagonal line , while distal acting QTL ( n = 13 , 767 ) lie off the diagonal . Several prominent distal QTL hotspots appear as vertical lines . C . Similar to B , plotting expression ( e ) QTL position versus gene location ( LOD > 5 , n = 701 local - eQTL , n = 357 distal - eQTL ) .

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B. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). D


G . Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3 , ATAC , and RNA read depth from merged replicates . Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below .

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G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.


F . Left - Coverage profiles ( averaged biological replicates , N = 3 ) for ChIP factor occupancy at Cd59a on Chr 2 under trans - regulation by the Chr 13d QTL . Distal ca - QTL target region used for quantification is indicated by a black box with LOD score below . Right - Scatterplots for ChIP factors comparing quantitative level of modification / binding for independent replicates between B6 and D2 ESCs .

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F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.


G . The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 61 ) . H . TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal ( Pearson ' s r = - 0 . 64 ) .

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G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). I


I . Similar to F , coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932 , targeted by Chr 12 trans caQTL , showed greater occupancy of POU5F1 and increased open chromatin compared to D2 . Distal ca - QTL target region used for quantification is indicated by a black box with LOD score below .

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I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.


J . The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 66 ) . K . Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility ( Pearson ' s r = - 0 . 51 ) .

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J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D


A . Coverage profile for H3K4me3 , ATAC , P300 , and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility ( black boxes ) and gene expression ( Riok3 and Rmc1 ) . LOD scores are listed under each target locus .

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A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.


E . Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 ( left ) or D2 ( right ) . Dots represent mean values from biological duplicate experiments and lines connect means . Orange indicates loci for which H3K9me3 signal decreases in the KO ( p - value indicated on top , two - sided paired Wilcoxon test ) .

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E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).


H . MA - plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs ( n = 2 ) . Chr 4 QTL targets are indicated in magenta and increase expression in the KO .

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H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.


( B ) From top to bottom : experimental design of 4D induction , fluorescence pictures of the SVZ of a 4D + mouse and quantification of the proportion of RFP - ( black ) and RFP + ( red ) progenitors among B1 , C and A cells identified with markers as indicated .

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(B) From top to bottom: experimental design of 4D induction, fluorescence pictures of the SVZ of a 4D+ mouse and quantification of the proportion of RFP- (black) and RFP+ (red) progenitors among B1, C and A cells identified with markers as indicated.


( C ) From top to bottom : experimental design , fluorescence pictures of the SVZ of a 4D - ( left ) and 4D + ( right and insets magnified ) mice and quantification of the proportion of BrdU + among B1 , C and A cells ( identified as in B ) . Note that in 4D + mice quantification was restricted to the RFP + subpopulation ( red bars ) .

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(C) From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D- (left) and 4D+ (right and insets magnified) mice and quantification of the proportion of BrdU+ among B1, C and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars).


( D ) Quantification of the absolute number of B1 , C and A cells ( identified as in B ) in the SVZ of 4D - ( white ) and 4D + ( black ) mice regardless of RFP expression ( RFP + / - ) .

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(D) Quantification of the absolute number of B1, C and A cells (identified as in B) in the SVZ of 4D- (white) and 4D+ (black) mice regardless of RFP expression (RFP+/-).


From top to bottom : fluorescence pictures of the SVZ and absolute number of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated .

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From top to bottom: fluorescence pictures of the SVZ and absolute number of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated.


From top to bottom : fluorescence pictures of the SVZ and absolute number of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . Insets in ( C ) are magnified ( right ) with arrowheads pointing label - retaining NSC ( white ) or astrocytes ( empty ) .

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From top to bottom: fluorescence pictures of the SVZ and absolute number of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Insets in (C) are magnified (right) with arrowheads pointing label-retaining NSC (white) or astrocytes (empty).


From top to bottom : fluorescence pictures of the SVZ or proportions ( C - E ) of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . Arrowheads in ( D ) point cell doublets ( among RFP + protein - reteining cells in 4D + ) .

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From top to bottom: fluorescence pictures of the SVZ or proportions (C-E) of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Arrowheads in (D) point cell doublets (among RFP+ protein-reteining cells in 4D+).


From top to bottom : fluorescence pictures of OB ( E ) and absolute number or proportions of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . ( E ) GL = glomerular , EPL = external plexiform , MCL = mitral cell and GCL = granule cell layers .

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From top to bottom: fluorescence pictures of OB (E) and absolute number or proportions of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. (E) GL=glomerular, EPL=external plexiform, MCL=mitral cell and GCL=granule cell layers.


( B ) Fluorescence pictures ( left ) of immunolabeled GFP + RFP - ( 4D - ) and GFP + RFP + ( 4D + ) apical dendrites of superficial granule neurons and quantifications ( middle and right ) of spine density and total dendritic length ( box and whiskers ) and 3D - Sholl ( line graph profile ) of apical dendrites starting from the soma ( drawings ) as the mean number of intersections at 10 μm intervals .

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(B) Fluorescence pictures (left) of immunolabeled GFP+RFP- (4D-) and GFP+RFP+ (4D+) apical dendrites of superficial granule neurons and quantifications (middle and right) of spine density and total dendritic length (box and whiskers) and 3D-Sholl (line graph profile) of apical dendrites starting from the soma (drawings) as the mean number of intersections at 10 μm intervals.


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

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


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

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


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

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


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

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


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

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


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

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


A : Relative expression of ELDR in OSCC patient samples compared to adjacent non - tumor tissues ( N = 20 ) analyzed by qRT - PCR . Technical triplicates were used from each sample . 18S rRNA was used as an internal control . Data were analyzed by Student ' s t test . Small bars indicate standard error ( * , p < 0 . 05 , * * , p < 0 . 01 ; * * * p < 0 . 001 ) .

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A: Relative expression of ELDR in OSCC patient samples compared to adjacent non-tumor tissues (N=20) analyzed by qRT-PCR. Technical triplicates were used from each sample. 18S rRNA was used as an internal control. Data were analyzed by Student's t test. Small bars indicate standard error (*, p<0.05, **, p<0.01; *** p<0.001).


B : OSCC grade wise upregulation of ELDR expression was noted in the patient samples ( N = 20 ) . Correlation analysis showed signification positive correlation of ELDR expression with tumor grade ( R2 = 0 . 926 , significant P value = 0 . 01 ) . Pearson correlation coefficient and p value was calculated using socscistatistics . com . Well : well differentiated , Mod : moderately differentiated , Poor : poorly differentiated carcinoma . Data are represented as the mean ± SD ,

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B: OSCC grade wise upregulation of ELDR expression was noted in the patient samples (N=20). Correlation analysis showed signification positive correlation of ELDR expression with tumor grade (R2= 0.926, significant P value = 0.01). Pearson correlation coefficient and p value was calculated using socscistatistics.com. Well: well differentiated, Mod: moderately differentiated, Poor: poorly differentiated carcinoma. Data are represented as the mean ± SD,


C : Representative images of FISH analysis with an RNA probe of ELDR ( red ) and DAPI staining ( blue ) in formalin fixed paraffin embedded OSCC tissue . An unrelated probe was used as negative control . Magnification : 40X ; inset magnification 100X and scale bar 10 µm .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-DISEASE 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: Representative images of FISH analysis with an RNA probe of ELDR (red) and DAPI staining (blue) in formalin fixed paraffin embedded OSCC tissue. An unrelated probe was used as negative control. Magnification: 40X; inset magnification 100X and scale bar 10 µm.


D : Relative expression of ELDR in OSCC cell lines compared to normal oral keratinocytes ( NOK ) analyzed by qRT - PCR . 18S was used as internal control . Data were analyzed by Student ' s t test . Small bars indicate standard error ( * * , p < 0 . 01 ; * * * p < 0 . 001 ) . n = 4 biological replicates . Data are represented as the mean ± SD ,

O O O B-EXP_ASSAY O B-GENEPROD O B-DISEASE O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Relative expression of ELDR in OSCC cell lines compared to normal oral keratinocytes (NOK) analyzed by qRT-PCR. 18S was used as internal control. Data were analyzed by Student's t test. Small bars indicate standard error (**, p<0.01; *** p<0.001). n = 4 biological replicates. Data are represented as the mean ± SD,


A : Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points . Live cell numbers are presented . n = 4 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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: Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points. Live cell numbers are presented. n = 4 biological replicates. Data are represented as the mean ± SD,


B : Control or ELDR plasmid DNA overexpressed NOK ( 3 × 102 ) were seeded and allowed to form colonies . After two weeks , colonies were stained with crystal violet and counted . Representative images of colonies in control and ELDR overexpressed cells are presented . The right panel shows quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O 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: Control or ELDR plasmid DNA overexpressed NOK (3×102) were seeded and allowed to form colonies. After two weeks, colonies were stained with crystal violet and counted. Representative images of colonies in control and ELDR overexpressed cells are presented. The right panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 , JHU029 and JHU022 cells were transfected with control or ELDR siRNA ( 50nM ) , and cell proliferation was analyzed at the indicated time points . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

C: Cal27, JHU029 and JHU022 cells were transfected with control or ELDR siRNA (50nM), and cell proliferation was analyzed at the indicated time points. n = 3 biological replicates. Data are represented as the mean ± SD,


D : Cell lysates from control or ELDR overexpressing NOKs , control or Cal27 and JHU029 cells treated with two different ELDR siRNAs were subjected to Western blot analysis for PCNA expression using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control for normalization . The bottom panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

D: Cell lysates from control or ELDR overexpressing NOKs, control or Cal27 and JHU029 cells treated with two different ELDR siRNAs were subjected to Western blot analysis for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control for normalization. The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Cal27 and JHU029 cells were transfected with siELDR , ELDR plasmid DNA or both siELDR and ELDR plasmid DNA . Cell lysates were analyzed by Western blot for PCNA expression using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . The right panel shows quantitative representation of Western blot band intensities . n = 2 biological replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

E: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid DNA or both siELDR and ELDR plasmid DNA. Cell lysates were analyzed by Western blot for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,


A : Cal27 or JHU029 cells were transfected with control or ELDR siRNA , and EGFR mRNA expression was examined by qRT - PCR . 18S gene was used as internal control . n = 3 biological replicates and 3 technical repeates .

O O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O

A: Cal27 or JHU029 cells were transfected with control or ELDR siRNA, and EGFR mRNA expression was examined by qRT-PCR. 18S gene was used as internal control. n = 3 biological replicates and 3 technical repeates.


B : Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies . The membrane was reprobed with an antibody against Actin as an internal control . The same actin blot was used in Fig . 2D ( for PCNA ) . The bottom panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

B: Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA). The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 and JHU029 cells were transfected with siELDR , ELDR plasmid or both siELDR and ELDR plasmid . Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody . The membrane was reprobed with an antibody against Actin as internal control . The same actin blot was used in Fig . 2E ( for PCNA ) . n = 2 biological replicates .

O O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O

C: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid or both siELDR and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. The same actin blot was used in Fig. 2E (for PCNA). n = 2 biological replicates.


D : Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . The right panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

D: Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Cell lysates from control and ELDR - depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho - STAT3 ( pSTAT3 Y - 705 ) and total STAT3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Right panel shows a quantitative representation of Western blot band intensities . n = 2 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

E: Cell lysates from control and ELDR-depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho-STAT3 (pSTAT3 Y-705) and total STAT3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows a quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,


C : Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA , pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies . Bottom panel shows Western blot analysis of PUM - 1 ( an unrelated RNA binding protein ) .

O O O O O B-CELL_LINE O B-CELL_LINE O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

C: Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA, pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies. Bottom panel shows Western blot analysis of PUM-1 (an unrelated RNA binding protein).


D : Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates . Relative expression of ELDR ( left penal ) and PCAT1 ( right panel ) was analyzed by qRT - PCR . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

D: Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates. Relative expression of ELDR (left penal) and PCAT1 (right panel) was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


E : Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Right panel shows the quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

E: Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows the quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control , ELDR overexpressed and ELDR - depleted Cal27 cells were probed with an antibody against ILF3 ( red ) and DAPI ( blue ) . Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3 . Arrows indicate cytoplasmic expression . Magnifications 60X , scale bar 20 μ .

O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O

F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.


A : Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates . Relative mRNA expression of EGFR was analyzed by qRT - PCR . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

A: Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates. Relative mRNA expression of EGFR was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . n = 3 biological replicates .

O O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

B: Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. n = 3 biological replicates.


C : Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT - PCR . 18S was used as internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

C: Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT-PCR. 18S was used as internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


D : Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Bottom panels show quantitation . n = 4 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O 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: Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Bottom panels show quantitation. n = 4 biological replicates. Data are represented as the mean ± SD,


E : Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as internal control . Bottom panel shows quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD 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: Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. Bottom panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD B-CELL_LINE O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD 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: Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


G : Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho - RB and total RB using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ±

O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

G: Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho-RB and total RB using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD


H : Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested , fixed and stained with propidium iodide . DNA content was analyzed by flow cytometry . Results are represented cell population in G1 , S , and G2 / M phases of the cell cycle . Right panels show percentage of cell population in different phases of cell cycle . n = 4 biological replicates . Data are represented as the mean ± SD ,

O O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD,


A : Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

A: Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

B: Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 and JHU029 cells were transfected with control or mimic miR - 7 and relative expression of ELDR was analyzed by qRT - PCR . 18S rRNA was used as an internal control . n = 2 biological replicates ; n = 3 technical replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O 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: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD,


D : Cal27 and JHU029 cells were transfected with miR - 7 mimic , ELDR plasmid or both miR - 7 and ELDR plasmid . Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Representative bands were cropped from same gel . Right panels show quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O B-CELL_LINE O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Relative expression of miR - 7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O B-CELL_LINE O O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O 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: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


A : Body weight was measured in control and siELDR treated mice . n = 5 animal per

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O

A: Body weight was measured in control and siELDR treated mice. n=5 animal per group


B : Representative images of tumors in control and treatment groups .

B: Representative images of tumors in control and treatment groups.


C : Tumors were measured using a slide caliper and tumor volumes were calculated . Arrow indicates starting point siELDR treatment . n = 5 animal per group and tumor volume presented as the mean ±

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

C: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD