B , Confocal of wild - type PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean distributed in Golgi normalized to cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated other U18 - treated sgRNA groups .

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B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.


H . Control or GFP - DHHC3 expressing tsA201 fixed stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) relative to cytoplasm .

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H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.


M . Confocal of control ( top row ) , NPC1I1061T ( middle row ) , NPC1I1061T treated with sgDHHC3 ( bottom row ) expressing P4M , LAMP1 CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


C . Confocal of control NPC1I1061T fibroblasts immunolabeled for ACBD3 . Black orange squares show expanded views of anti - ACBD3 in Golgi endomembrane compartments , respectively . D . Analyses of ACBD3 distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 ) , normalized to cytoplasm .

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C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.


I . Confocal of control sgACBD3 HEK293 - CAS9 expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in Golgi in endomembrane compartments . J . Quantitative analyses of P4M - YFP mean at Golgi ( left graph ) or endomembranes ( right graph ) normalized to cytoplasm . * represents a significant difference ( P < 0 . 05 ) between control other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated other U18 - treated sgRNA groups . For left graph ; Control : n = 19 ; U18 : n = 13 ; sgPI4K3B : n = 12 ; sgPI4K3B + U18 : n = 11 ; sgPI4K2A + sgPI4K3B : n = 12 ; sgPI4K2A + sgPI4K3B + U18 : n = 12 ; sgACBD3 : n = 15 ; sgACBD3 + U18 : n = 16 . For right graph ; control : n = 149 ; U18 : n = 251 ; sgACBD3 : n = 138 ; sgACBD3 + U18 : n = 190 . Please note that ' control ' ' U18 ' datasets in Fig 5J are identical to those in Fig . 4C . Data for CRISPR experiments were collected on same days .

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I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.


A . Confocal of control , NPC1I1061T ACBD3 - deleted NPC1I1061T fibroblasts expressing P4M - YFP . Black orange squares show expanded views of PtdIns4P in Golgi endo / lysosomal compartments respectively .

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A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.


C . Confocal of control , NPC1I1061T ACBD3 - deleted NPC1I1061T fibroblasts immunolabeled with mTORC1 antibody . Bottom show expanded views of mTORC1 immunolabeling in regions indicated by dashed squares in main . dashed lines represent outline of each . D . of mTORC1 mean integrated density normalized bycell in control , NPC1I1061T ACBD3 - deleted NPC1I1061T fibroblasts . Control : n = 15 ; NPC1I1061T : n = 14 ; NPC1I1061T + sgACBD3 : n = 11 .

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C. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell. D. Quantification of mTORC1 mean integrated density normalized bycell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.


B . Confocal of tsA201 expressing PI4KIIα - GFP . were treated with either vehicle ( control ) , U18 , or U18 PF for 24h prior imaging . Bottom panels show an enlarged view of enzyme in a perinuclear . C . of PI4KIIα - GFP mean in Golgi normalized to cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . D , E . Same as ( B , C ) but in tsA - 201 transfected with PI4KIIIβ - GFP . Control : n = 18 , U18 : n = 22 , U18 + PF : n = 23 .

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B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.


Confocal of tsA201 were treated with either vehicle ( control ) , U18 , or U18 PF for 24h prior imaging . Bottom panels show an enlarged view of enzyme in a perinuclear . of mean in Golgi normalized to cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . F , G . Same as ( B , C ) but in tsA - 201 transfected with P4M - YFP . Control : n = 39 , U18 : n = 42 , U18 + PF : n = 32 . H , I . Same as ( F , G ) but in wild - type SCAP - / - CHO . Control : n = 12 , U18 : n = 11 , SCAP - / - + U18 : n = 10 .

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Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.


A . Confocal of control NPC1I1061T fibroblasts immunolabeled for Giantin CI - M6PR . Insets show expanded views of each immunolabeling in Golgi region ( upper insets ) or centered on endomembranes ( bottom inset , merged signals ) . Dashed grey lines represent outline of each . B . of mean of M6P Giantin colocalizing ( top graph ; Control : n = 23 ; NPC1I1061T : n = 18 ) of their mean ( bottom graph ; Control : n = 18 ; NPC1I1061T : n = 18 ) in control NPC1I1061T fibroblasts .

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A. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell. B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts.


C . Confocal of control NPC1I1061T fibroblasts expressing ARF1 - GFP . Insets show enlarged views of ARF1 in Golgi . Tubular structures were observed in NPC1I1061T fibroblasts as indicated by black arrows . D . of ΑRF - 1 in Golgi normalized to cytoplasm in control NPC1I1061T fibroblasts . Control : n = 10 ; NPC1I1061T : n = 11 .

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C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows. D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.


B . Results of sequential digestion workflow applied to a synthetic 7 - mix , compared to using trypsin alone in four replicates parallel digestion with trypsin , AspN , chymotrypsin GluC . A trypsin four replicate experiment shows a large overlap of four datasets with little gain . Parallel digestions with trypsin , AspN , chymotrypsin GluC demonstrates high complementarity but moderate gains over trypsin . Sequential digestion shows low overlap between four datasets largest gain in unique residue pairs . C . Gains of repeated ( trypsin only ) , parallel digestion sequential digestion for same data as shown in panel B .

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B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs. C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B.


D . Crosslinked peptides obtained by sequential digestion of a synthetic 7 - mix are smaller than their corresponding tryptic peptides .

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D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.


B . in vitro helicase loading demonstrates that an Mcm2 C - terminal deletion mutant supports assembly ( lanes 6 7 ) blocks formation of final helicase loading product ( lanes 8 9 ) .

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B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).


C . Overexpression of Mcm2 - 7ΔC2 shows that this mutant causes dominant lethality , indicating that C - terminus of Mcm2 is essential in survival .

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C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.


Plot showing average Z - scores of 210 endolysosomal genes plotted against negative log10 of P values from one - sample t - of whether average Z - score ≠ 0 . Green circles represent significantly upregulated genes ( Z > 0 P < 0 . 05 ) . Magenta circles represent significantly downregulated genes ( Z < 0 P < 0 . 05 ) . Grey circles represent genes whose expression is not significantly changed ( P > 0 . 05 ) . comparisons were between cancers normal from same tissue . Dashed shows position on Y - axis that corresponds to P = 0 . 05 . Positions of MCOLN1 ( red circle ) , VAC14 ( blue circle ) , MTM1 ( yellow circle ) are indicated . Inset , schematic showing that Mtm1 Vac14 regulate of PI ( 3 , 5 ) P2 , thereby , influence TRPML1 activity .

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Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.


Unsupervised hierarchical clustering of Pearson ' s coefficients of pairwise correlation of gene expression reveal 4 indicated clusters . Violin plots of average Z - scores of genes that belong to four clusters derived from hierarchical clustering . thick horizontal dashed lines in each sample set represent median ; thin dashed lines represent quartiles ; polygons represent density estimates of data extend to extreme values . Statistical employed were 1 - sample t - or Wilcoxon signed rank to examine significance of differences from a hypothetical value of 0 .

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Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters. Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0.


GSEA conducted on endolysosomal genes ranked on basis of increasing Z - scores . Positions of CLEAR target genes are indicated .

GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated.


Bar graph showing relative expression of indicated genes in indicated types . Values were normalized to respective ' HN31 + HRAS shRNA ' average represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Western blots performed on extracts isolated from indicated probed with antibodies against Tfeb tubulin . Molecular weights are shown on left . Bar graph shows relative Tfeb in indicated samples . Values were normalized to ' HN31 + HRAS shRNA ' represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin. Molecular weights are shown on the left. Bar graph shows relative Tfeb levels in the indicated samples. Values were normalized to 'HN31+HRAS shRNA' and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Bar graph showing relative expression of MCOLN1 in indicated types . Values were normalized to HT1197 average represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graph showing the relative expression of MCOLN1 in the indicated cell types. Values were normalized to HT1197 average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Graphs showing changes in fura - 2 in of indicated genotypes in response to ML - SA1 GPN . Arrows serve as event markers that denote points of time at which drugs were added . Grey lines on top represent time at which bath Ca2 + was removed . values represent mean ± from 3 biological replicates .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 and GPN. Arrows serve as event markers that denote the points of time at which the drugs were added. Grey lines on top represent the time at which the bath Ca2+ was removed. All values represent mean±SEM from 3 biological replicates.


Graphs showing changes in fura - 2 in of indicated genotypes in response to ML - SA1 except that baseline ratios for both genotypes were adjusted to 0 at start of experiment . Lines on right indicate peak amplitudes of Ca2 + release in response to application of ML - SA1 . values represent mean ± from 3 biological replicates .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release in response to the application of ML-SA1. All values represent mean±SEM from 3 biological replicates.


Graphs showing changes in fura - 2 in of indicated genotypes in response to GPN . baseline ratios for both genotypes were adjusted to 0 at start of experiment . Lines on right indicate peak amplitudes of Ca2 + release Ca2 + release was evoked using GPN .

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Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to GPN. the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release Ca2+ release was evoked using GPN.


Peak amplitudes of Ca2 + release in of indicated genotypes in response to ML - SA1 ( E ) values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 (E) All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Peak amplitudes of Ca2 + release in of indicated genotypes in response to GPN ( F ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


of amplitudes of Ca2 + release in of indicated genotypes in response to ML - SA1 GPN . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Total Ca2 + release in of indicated genotypes in response to ML - SA1 GPN . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Bar graphs showing relative numbers in indicated types following indicated treatments . numbers were assessed after a 48 - hour period of growth in cultures seeded at equal densities . values represent mean ± . Data points represent values from biological replicates .

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Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments. Cell numbers were assessed after a 48-hour period of growth in cultures seeded at equal densities. All values represent mean±SEM. Data points represent values from biological replicates.


Representative showing Ki67 - labeling in xenografts generated from implantation of HN31 stably expressing control shRNA ( left ) or MCOLN1 shRNA ( right ) . Pseudocolored panels show representative distribution Ki67 positive or negative nuclei . Scale bars shown ( 0 . 5 mm ) apply to panels .

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Representative images showing Ki67-labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA (left) or MCOLN1 shRNA (right). Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei. Scale bars shown (0.5 mm) apply to all panels.


Bar graphs showing fraction ( left ) total number ( right ) of Ki67 labeled nuclei in indicated types . values represent mean ± . Values in bar graph on right were normalized to control mean . Data points represent values from independent eggs . Statistical employed was Student ' s t - .

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Bar graphs showing fraction (left) and total number (right) of Ki67 labeled nuclei in the indicated cell types. All values represent mean±SEM. Values in the bar graph on the right were normalized to control mean. Data points represent values from independent eggs. Statistical test employed was Student's t-test.


Bar graphs showing relative numbers in UMSCC - 22A - HRASWT following indicated perturbations . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graphs showing the relative cell numbers in UMSCC-22A-HRASWT cells following the indicated perturbations. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Kaplan - Meier curves showing survival of stratified on basis of MCOLN1 expression in BLCA HNSC whose tumors were wild - type for HRAS ( right ) or carried oncogenic HRAS mutations ( left ) . Statistical employed was Mantel - Cox log - rank .

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Kaplan-Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild-type for HRAS (right) or carried oncogenic HRAS mutations (left). Statistical test employed was the Mantel-Cox log-rank test.


Representative Western blots generated using extracts from of expressing oncogenic HRAS controls treated with ML - SI1 as indicated . antibodies used are indicated on right . Bar graph showing of Western blots shown in ( A ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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

Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from indicated types after indicated perturbations . antibodies used are indicated on right .

Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from indicated types after indicated perturbations . Bar graph showing of Western blots in ( C ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (C). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from indicated types after indicated perturbations . Bar graph showing of Western blots in ( D ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed were Student ' s t - ANOVA .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing relative of trpml mRNA in Drosophila larval brain extracts from control expressing dRasG12V in glia . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative of Drosophila 3rd instar larvae expressing indicated transgenes in glial . repo - GAL4 was used to drive expression of UAS - dRasG12V in glia . shown are volumetric reconstructions of confocal stacks . various anatomical structures of larval brain are annotated . VNC refers to ' ventral nerve chord ' , which is distinct from dorsal hemispheres of brain . GFP represents total glial membranes in larval brains . Scale bar , 100μm .

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Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells. repo-GAL4 was used to drive the expression of UAS-dRasG12V in glia. Images shown are volumetric reconstructions of confocal stacks. The various anatomical structures of the larval brain are annotated. VNC refers to the 'ventral nerve chord', which is distinct from dorsal hemispheres of the brain. GFP staining represents the total glial membranes in the larval brains. Scale bar, 100μm.


Bar graph showing relative glial volume in larvae of indicated genotypes . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graph showing relative glial cell volume in larvae of the indicated genotypes. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of indicated genotypes . He - GAL4 was used to drive expression of UAS - dRasG12V in macrophages . antibodies used are indicated on right . Bar graph showing of Western blots shown in ( D ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes. He-GAL4 was used to drive the expression of UAS-dRasG12V in macrophages. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using fat - extracts isolated from 3rd instar larvae of indicated genotypes . cg - GAL4 was used to drive expression of UAS - HRASG12V in larval fat - bodies . antibodies used are indicated on right . Bar graph showing of Western blots shown in ( F ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

Representative Western blots generated using fat-body extracts isolated from 3rd instar larvae of the indicated genotypes. cg-GAL4 was used to drive the expression of UAS-HRASG12V in larval fat-bodies. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Weighted mean univariate K - function curves of immunogold labeled GFP - HRASG12V under indicated conditions , n ≥ 15 for each condition . gold point patterns were analyzed with univariate K - functions expressed as L ( r ) - r . bar graph on right shows peak value Lmax of L ( r ) - r curve , which represents extent of GFP - HRASG12V nanoclustering . Values are normalized to DMSO average represent mean ± ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap .

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Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in inner leaflet of plasma is quantified as number of gold particles per 1μm2 region from electron . Values are normalized to DMSO average represent mean ± , n ≥ 15 for each condition . Statistical employed was Student ' s t - with Bonferroni post hoc correction to account for multiple pairwise comparisons .

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Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons.


extent of GFP - HRASG12V nanoclustering quantified by Lmax values in exposed to indicated perturbations . Values are normalized to DMSO average represent mean ± ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap .

The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in inner leaflet of plasma is quantified as number of gold particles per 1μm2 region from electron . but in exposed to indicated perturbations . Values shown are normalized to DMSO average .

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Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average.


Representative Western blots generated using extracts from HN31 stably expressing MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at bottom . antibodies used are indicated on right .

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Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from HN31 stably expressing MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at bottom . Bar graph showing of Western blots in ( F ) . values represent mean ± . Data points represent values from biological replicates . Statistical employed was ANOVA .

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Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA.


Bar graph showing relative of total in HN31 extracts exposed to indicated perturbagens Values represent mean ± . Data points represent values from biological replicates . Statistical employed were Student ' s t - ANOVA .

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Bar graph showing the relative levels of total in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing relative of free / total cholesterol in HN31 extracts exposed to indicated perturbagens Values represent mean ± . Data points represent values from biological replicates . Statistical employed were Student ' s t - ANOVA .

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Bar graph showing the relative levels of free/total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing relative of free / total cholesterol in larval brain extracts from of indicated genotypes Values represent mean ± . Data points represent values from biological replicates . Statistical employed were Student ' s t - ANOVA .

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Bar graph showing the relative levels of free/total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Confocal of MDCK expressing mCherry - D4H . Colocalization with cortical actin ( phalloidin ) indicates that mCherry - D4H is predominantly localized to plasma in DMSO treated . Within 24 hours of ML - SI1 treatment , mCherry - D4H redistributes to intracellular . Although minimal mCherry - D4H signal remained at plasma in ML - SI1 - treated , phalloidin signal ( cortical actin ) remained unchanged . Scale bar ( 10μm ) shown in panel on top right applies to panels . Bar graph showing of mCherry - D4H in DMSO or ML - SI1 - treated . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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

Confocal images of MDCK cells expressing mCherry-D4H. Colocalization with the cortical actin (phalloidin) indicates that mCherry-D4H is predominantly localized to the plasma membrane in DMSO treated cells. Within 24 hours of ML-SI1 treatment, the mCherry-D4H redistributes to intracellular punctae. Although minimal mCherry-D4H signal remained at the plasma membrane in ML-SI1-treated cells, the phalloidin signal (cortical actin) remained unchanged. Scale bar (10μm) shown in the panel on the top right applies to all panels. Bar graph showing quantification of mCherry-D4H punctae in DMSO or ML-SI1-treated cells. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Confocal of MDCK expressing mCherry - D4H LAMP1 - GFP treated with ML - SI1 . Scale bar ( 10μm ) shown in panel on top right applies to panels .

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Confocal images of MDCK cells expressing mCherry-D4H and LAMP1-GFP treated with ML-SI1. Scale bar (10μm) shown in the panel on the top right applies to all panels.


Confocal of control MCOLN1 shRNA - expressing HN31 treated with cholesterol sensor , filipin . Scale bar ( 10μm ) shown in panel on top applies to both panels . Bar graph showing of internalized filipin in HN31 after MCOLN1 knockdown . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Confocal images of control and MCOLN1 shRNA-expressing HN31 cells treated with the cholesterol sensor, filipin. Scale bar (10μm) shown in the panel on the top applies to both panels. Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Fraction of phase - separated GPMVs at indicated temperatures . Colors of lines indicate treatment conditions of MDCK prior to generation of GPMVs . Dashed vertical lines indicate miscibility of indicated populations of GPMVs . Values shown represent mean ± . Lines were generated by fitting data to sigmoidal functions .

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Fraction of phase-separated GPMVs at the indicated temperatures. Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs. Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs. Values shown represent mean±SEM. Lines were generated by fitting the data to sigmoidal functions.


Bar graph showing miscibility temperatures of GPMVs isolated from MDCK exposed to indicated conditions . values represent mean ± . Data points represent values from biological replicates . Statistical employed was Student ' s t - .

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Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


polarization ( FP ) competition derived affinity assessment of consecutive truncations of DAT C13 C - terminus as indicated by sequences . Bars indicate mean fold change relative to C13 ( Ki = 1 . 69 µM ) . Data is shown as mean with error - bars as with n = 6 . Representative binding curves are shown in EV1A .

Fluorescence polarization (FP) competition derived affinity assessment of consecutive truncations of the DAT C13 C-terminus as indicated by the sequences. Bars indicate the mean fold change relative to C13 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1A.


FP competition binding of C5 Tat - C5 to PICK1 with 5FAM - Tat - C5 ( 20 nM ) as tracer . Saturation binding curve is shown in EV1B .

FP competition binding of C5 and Tat-C5 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Saturation binding curve is shown in EV1B.


Backbone amide chemical shift changes ( ΔδHN ) of PICK1 - PDZ - C10 after addition of Tat - C5 addition of protease to cleave C10 from PDZ domain allow exchange . Numbers refer to residue number in PICK1 ( Uniprot : Q9EP80 ) . Red bars indicate a chemical shift larger than mean + SD .

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Backbone amide chemical shift changes (ΔδHN) of PICK1-PDZ-C10 after addition of Tat-C5 and addition of protease to cleave C10 from the PDZ domain and allow exchange. Numbers refer to residue number in PICK1 (Uniprot: Q9EP80). Red bars indicate a chemical shift larger than mean+SD.


1H - 15N - HSQC 2D spectra of PICK1 - PDZ - C10 ( orange ) following addition of Tat - C5 ( green ) subsequent addition of protease ( red ) to allow for exchange between C10 Tat - C5 .

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1H-15N-HSQC 2D spectra of PICK1-PDZ-C10 (orange) following addition of Tat-C5 (green) and subsequent addition of protease (red) to allow for exchange between C10 and Tat-C5.


Selectivity screen for Tat - C5 against a selection of 42 purified PDZ domains , performed with a fixed of Tat - C5 ( 10 µM ) in competition with PDZ domains their respective ligands . Data are normalized to binding in absence of peptide ( full ) . Dashed represents level of competition obtained for PICK1 . Screen for C5 can be seen in EV1C .

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Selectivity screen for Tat-C5 against a selection of 42 purified PDZ domains, performed with a fixed concentration of Tat-C5 (10 µM) in competition with PDZ domains and their respective fluorescent ligands. Data are normalized to binding in absence of peptide (full line). Dashed line represents the level of competition obtained for PICK1. Screen for C5 can be seen in EV1C.


Affinity gain of bivalent C5 peptides ( Pn - ( C5 ) 2 ) N - terminally conjugated with different PEG linkers as indicated . Bars indicate mean fold change relative to C5 ( Ki = 1 . 69 µM ) . Data is shown as mean with error - bars as with n = 6 . Representative binding curves are shown in EV1C .

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Affinity gain of bivalent C5 peptides (Pn-(C5)2) N-terminally conjugated with different length PEG linkers as indicated. Bars indicate the mean fold change relative to C5 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1C.


FP competition binding of C5 ( also used in C ) Tat - P4 - ( C5 ) 2 to PICK1 with 5FAM - Tat - C5 ( 20 nM ) as tracer .

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FP competition binding of C5 (also used in C) and Tat-P4-(C5)2 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer.


Representative confocal of SCMS expressing Tac - YFP - DATC24 , incubated with pre - complexed fluorescently labelled PICK1 unlabelled peptides as indicated ( 10 nM ) , scale bar 10 µm . Full curves shown in Fig S4 include representative shown here . SCMS derived competitive binding curves for peptides as indicated , red indicates 10 nM as shown in representative .

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Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with pre-complexed fluorescently labelled PICK1 and unlabelled peptides as indicated (10 nM), scale bar 10 µm. Full concentration curves shown in Fig S4 and include the representative images shown here.   SCMS derived competitive binding curves for peptides as indicated, red line indicates 10 nM as shown in representative images.


Representative confocal of SCMS expressing Tac - YFP - DATC24 , incubated with fluorescently labelled PICK1 subsequently unlabelled peptide as indicated ( 10µM ) . Scale bar 10 µm . Full curves shown in Fig EV2 include representative shown here . Efficacy of peptides as indicated to facilitate dissociation of PICK1 from SCMS , red indicates 10 µM as shown in representative .

Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with fluorescently labelled PICK1 and subsequently unlabelled peptide as indicated (10µM). Scale bar 10 µm. Full concentration curves shown in Fig EV2 and include the representative images shown here. Efficacy of the peptides as indicated to facilitate dissociation of PICK1 from SCMS, red line indicates 10 µM as shown in representative images.


exclusion chromatography ( SEC ) profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) presence of Tat - C5 ( purple , Absmax = 11 . 7 ml )

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Size exclusion chromatography (SEC) profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-C5 (purple, Absmax = 11.7 ml)


SEC profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) presence of Tat - P4 - ( C5 ) 2 ( blue , Absmax = 10 . 7 ml )

SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-P4-(C5)2 (blue, Absmax = 10.7 ml)


SEC profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) presence of TMR - Tat - P4 - ( C5 ) 2 ( blue ( abs280 nm ) , red ( abs544 nm ) , Absmax = 10 . 7 ml ) .

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SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of TMR-Tat-P4-(C5)2 (blue (abs280 nm), red (abs544 nm), Absmax=10.7 ml).


Representative SEC profiles of PICK1 ( 40 µM ) incubated with increasing of Tat - P4 - ( C5 ) 2 , colors indicate Gaussian fits of respective population , ( red ) / Void , ( blue ) dimeric PICK1 , ( green ) oligomeric PICK1 , ( orange ) unbound Tat - P4 - ( C5 ) 2 .

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Representative SEC profiles of PICK1 (40 µM) incubated with increasing concentration of Tat-P4-(C5)2, colors indicate Gaussian fits of respective population, (red) aggregates/Void, (blue) dimeric PICK1, (green) oligomeric PICK1, (orange) unbound Tat-P4-(C5)2.


of abs280 at Evol = 11 . 7 ml ( dimer - blue ) Evol = 10 . 7 ml ( tetramer - green ) as a function of ( 0 - 10 µM ) of Tat - P4 - ( C5 ) 2 . blue indicates tetramer fraction of total PICK1 .

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Quantification of abs280 at Evol = 11.7 ml (dimer - blue) and Evol = 10.7 ml (tetramer - green) as a function of concentration (0-10 µM) of Tat-P4-(C5)2. Light blue line indicates tetramer fraction of total PICK1.


SDS - PAGE sedimentation showing increased amounts of PICK1 in pellet ( P ) fraction with increasing concentrations of Tat - P4 - ( C5 ) 2 . PICK1 ~ 50kDa , Tat - P4 - ( C5 ) 2 ~ 10kDa . S denotes supernatant fraction .

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SDS-PAGE sedimentation assay showing increased amounts of PICK1 in the pellet (P) fraction with increasing concentrations of Tat-P4-(C5)2. PICK1~50kDa, Tat-P4-(C5)2~10kDa. S denotes supernatant fraction.


Small angle X - ray scattering ( SAXS ) derived scattering curves of a range of PICK1 on absolute scale show dependent aggregation . Pair distribution function ( pddf ) of PICK1 in absence of Tat - P4 - ( C5 ) 2 , indicating very large complexes present in sample .

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Small angle X-ray scattering (SAXS) derived scattering curves of a concentration range of PICK1 on absolute scale show concentration dependent aggregation. Pair distance distribution function (pddf) of PICK1 in absence of Tat-P4-(C5)2, indicating very large protein complexes present in sample.


SAXS derived scattering curves of a range of PICK1 in with Tat - P4 - ( C5 ) 2 on absolute scale show minor dependent aggregation . Pair distribution function ( pddf ) of a range of PICK1 in presence of Tat - P4 - ( C5 ) 2 , indicating a smaller than for PICK1 in absence of Tat - P4 - ( C5 ) 2 .

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SAXS derived scattering curves of a concentration range of PICK1 in complex with Tat-P4-(C5)2 on absolute scale show minor concentration dependent aggregation. Pair distance distribution function (pddf) of a concentration range of PICK1 in presence of Tat-P4-(C5)2, indicating a smaller complex than for PICK1 in absence of Tat-P4-(C5)2.


Representative confocal of hippocampal neurons showing penetration of TMR - Tat - P4 - ( C5 ) 2 TMR - Tat - C5 ( 5µM ) ( magenta ) , but not control TMR - C5 ( 20µM ) . was stained with DiO ( grey ) . Scale bars : 10µm 5µm on zooms / orthogonal views .

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Representative confocal images of hippocampal neurons showing membrane penetration of TMR-Tat-P4-(C5)2 and TMR-Tat-C5 (5µM) (magenta), but not the control TMR-C5 (all 20µM). The cell membrane was stained with DiO (grey). Scale bars: 10µm and 5µm on the zooms/orthogonal views.


Representative confocal of hippocampal neurons transduced with viral vector encoding GFP - PICK1 incubated with 5nM TMR - Tat - P4 - ( C5 ) 2 . Partial co - localization between TMR - Tat - P4 - ( C5 ) 2 ( red ) , GFP - PICK1 ( green ) GluA2 ( blue ) is seen in insert zooms . Experiment was done three times . Scale bars : 10µm 1µm on zooms .

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Representative confocal images of hippocampal neurons transduced with the viral vector encoding GFP-PICK1 and incubated with 5nM TMR-Tat-P4-(C5)2. Partial co-localization between TMR-Tat-P4-(C5)2 (red), GFP-PICK1 (green) and GluA2 (blue) is seen in insert zooms. Experiment was done three times. Scale bars: 10µm and 1µm on the zooms.


Representative immunoblots from hippocampal slices following treatment with Tat - P4 - ( C5 ) 2 or Tat - C5 ( bath application , 20µM ) .

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Representative immunoblots from hippocampal slices following treatment with Tat-P4-(C5)2 or Tat-C5 (bath application, 20µM).


Densitometric of immunoblots from C show reduced PICK1 : GluA2 Co - IP following incubation with Tat - P4 - ( C5 ) 2 but not Tat - C5 . Bar graphs show mean with error - bars as . ( One - way ANOVA followed by Dunnett ´ s multiple comparisons , * * * p ≤ 0 . 001 , n = 4 / group )

Densitometric analysis of immunoblots from C show reduced PICK1:GluA2 Co-IP following incubation with Tat-P4-(C5)2 but not Tat-C5. Bar graphs show mean with error-bars as SEM. (One-way ANOVA followed by Dunnett´s multiple comparisons test, *** p≤0.001, n = 4/group)


Representative immunoblots from hippocampal slices following Tat - P4 - ( C5 ) 2 bath application ( 20µM ) show reduction in phosphorylation level of S880 GluA2 both under basal condition after chemical LTD ( cLTD ) . Bar graphs show densitometric of immunoblots from ( E ) .

Representative immunoblots from hippocampal slices following Tat-P4-(C5)2 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD). Bar graphs show densitometric analysis of immunoblots from (E).


Representative immunoblots from hippocampal slices following Tat - C5 bath application ( 20µM ) show reduction in phosphorylation level of S880 GluA2 both under basal condition after chemical LTD ( cLTD ) . Bar graphs ( right panel ) show densitometric of immunoblots from ( G ) .

Representative immunoblots from hippocampal slices following Tat-C5 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD). Bar graphs (right panel) show densitometric analysis of immunoblots from (G).


Representative confocal of hippocampal neurons stained for surface GluA2 ( sGluA2 , blue ) internalized GluA2 ( iGluA2 , red ) . Treatment with Tat - P4 - ( C5 ) 2 ( 20 µM ) significantly increases constitutive , but not PMA induced internalization . Scale bars 10µm . of iGluA2 / sGluA2 within region of interest for neuron ( see I , top right corner ) . Tat - C5 did not increase GluA2 internalization ( see Appendix Fig S9D - F ) .

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Representative confocal images of hippocampal neurons stained for surface GluA2 (sGluA2, light blue) and internalized GluA2 (iGluA2, red). Treatment with Tat-P4-(C5)2 (20 µM) significantly increases the constitutive, but not PMA induced internalization. Scale bars 10µm. Quantification of iGluA2/sGluA2 within the region of interest for individual neuron (see I, top right corner). Tat-C5 did not increase GluA2 internalization (see Appendix Fig S9D-F).


Representative coronal slices from lumbar spinal cord show presence of both TMR - Tat - P4 - ( C5 ) 2 TMR - Tat - C5 peptides ( magenta ) 1 hour after i . t . administration in naïve mice . Scale bar 200µm . DH dorsal horn , VH ventral horn . Insert zooms of merged channels ( right panels ) , scale bar 50µm .

Representative coronal slices from lumbar spinal cord show the presence of both TMR-Tat-P4-(C5)2 and TMR-Tat-C5 peptides (magenta) 1 hour after i.t. administration in naïve mice. Scale bar 200µm. DH dorsal horn, VH ventral horn. Insert zooms of merged channels (right panels), scale bar 50µm.


Representative immunoblots of co - immunoprecipited PICK1 : GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i . t . injection of Tat - P4 - ( C5 ) 2 . Densitometric of immunoblots indicate a significant effect following Tat - P4 - ( C5 ) 2 treatment , graph show mean + / - points , unpaired t - , * * p ≤ 0 . 01 , n = 4 /

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Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i.t. injection of Tat-P4-(C5)2. Densitometric analysis of immunoblots indicate a significant effect following Tat-P4-(C5)2 treatment, graph show mean +/- SEM and individual points, unpaired t-test, **p≤0.01, n=4/group


Representative immunoblots of co - immunoprecipited PICK1 : GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i . t . injection Tat - C5 . Densitometric of immunoblots indicate no significant effect of Tat - C5 , graph show mean + / - points , unpaired t - , p = 0 . 10 , n = 4 /

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Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i.t. injection Tat-C5. Densitometric analysis of immunoblots indicate no significant effect of Tat-C5, graph show mean +/- SEM and individual points, unpaired t-test, p=0.10, n=4/group


Representative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880 - GluA2 total following 20 µM Tat - P4 - ( C5 ) 2 , Tat - C5 i . t . injections compared to saline injected . Densitometric of immunoblot indicates a significant effect only for Tat - P4 - ( C5 ) 2 ( graph show mean + / - points , One - way ANOVA followed by Dunnett ´ s multiple comparisons , * * p ≤ 0 . 01 , n = 5 - 6 / group as indicated by values )

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Representative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880-GluA2 total levels following 20 µM Tat-P4-(C5)2, and Tat-C5 i.t. injections compared to saline injected animals. Densitometric analysis of immunoblot indicates a significant effect only for Tat-P4-(C5)2 (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, ** p≤0.01, n=5-6/group as indicated by individual values)


Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 GluA2 surface level upon Tat - P4 - ( C5 ) 2 , Tat - C5 peptides incubation compared to untreated condition ( CTR ) . Densitometric of immunoblots show that reduction in mean surface for GluA2 in not significant ( graph show mean + / - points , One - way ANOVA followed by Dunnett ´ s multiple comparisons , n = 5 / group as indicated by values ) .

Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 and GluA2 surface level upon Tat-P4-(C5)2, Tat-C5 peptides incubation compared to the untreated condition (CTR). Densitometric analysis of immunoblots show that the reduction in mean surface for GluA2 in not significant (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, n=5/group as indicated by individual values).


Immunoblot of PICK1 following pull - down from lumbar spinal cord with Biotin - Tat - P4 - ( C5 ) 2 or Biotin following 20 µM i . t . administration demonstrating in vivo target engagement .

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Immunoblot of PICK1 following pull-down from lumbar spinal cord with Biotin-Tat-P4-(C5)2 or Biotin following 20 µM i.t. administration demonstrating in vivo target engagement.


i . t . administration ( 20µM ) of Tat - P4 - ( C5 ) 2 significantly reduced paw hypersensitivity of SNI mice in initiation stage ( 2 days after injury ) of spared nerve injury model at time points 1 3 hours ( n = 7 )

i.t. administration (20µM) of Tat-P4-(C5)2 significantly reduced ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model at time points 1 and 3 hours (n = 7)


i . t . administration ( 20µM ) of Tat - C5 did not affect paw hypersensitivity of SNI mice in initiation stage ( 2 days after injury ) of spared nerve injury model ( n = 8 )

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i.t. administration (20µM) of Tat-C5 did not affect ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model (n = 8)


Representative western blots ( top ) for GluA1 GluA2 following surface biotinylation of spinal cord slices . Densitometric ( bottom ) of immunoblots . We observed up - regulation of both GluA1 GluA2 surface level following SNI surgery compared to non - operated CTL ( unpaired t GluA1 : t ( 4 ) = 3 . 372 , GluA2 : t ( 4 ) = 3 . 991 , # p ≤ 0 . 05 , n = 3 / group ) . Tat - P4 - ( C5 ) 2 but not Tat - C5 significantly reduced SNI - induced GluA2 surface expression shows a strong tendency for GluA1 as well . Representative immunoblot shows unaltered PICK1 total level in total lysate in four conditions .

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Representative western blots (top) for GluA1 and GluA2 following surface biotinylation of spinal cord slices. Densitometric analysis (bottom) of immunoblots. We observed up-regulation of both GluA1 and GluA2 surface level following SNI surgery compared to non-operated animal CTL (unpaired t test GluA1: t(4)=3.372, GluA2: t(4)=3.991,#p≤0.05, n=3/group). Tat-P4-(C5)2 but not Tat-C5 significantly reduced the SNI-induced GluA2 surface expression and shows a strong tendency for GluA1 as well. Representative immunoblot shows unaltered PICK1 total level in the total lysate in the four conditions.


of lumbar spinal cord sagittal sections showing time course of TMR - Tat - P4 - ( C5 ) 2 ( magenta ) after i . t . administration ( 20 µM ) in SNI mice showing maximal accumulation in after 60 min . Scale bar 1000 µm 100 µm in Zooms .

Images of lumbar spinal cord sagittal sections showing the time course of TMR-Tat-P4-(C5)2 (magenta) after i.t. administration (20 µM) in SNI mice showing maximal accumulation in cells after 60 min. Scale bar 1000 µm and 100 µm in Zooms.


Overlay with immunohistochemical for neuronal marker NeuN ( yellow ) highlights strong neuronal tropism of TMR - Tat - P4 - ( C5 ) 2 ( magenta ) . Scale bar 100

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Overlay with immunohistochemical staining for the neuronal marker NeuN (yellow) highlights strong neuronal tropism of TMR-Tat-P4-(C5)2 (magenta). Scale bar 100 µm


Dose - dependent Von Frey in acute phase ( 2 days after injury ) of spared nerve injury model following i . t . administration of Tat - P4 - ( C5 ) 2 ( n = 8 / group ) did not affect paw hypersensitivity of SNI mice lasts up to 4h post injection .

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Dose-dependent Von Frey test in the acute phase (2 days after injury) of the spared nerve injury model following i.t. administration of Tat-P4-(C5)2 (n=8/group) did not affect ipsilateral paw hypersensitivity of SNI mice and lasts up to 4h post injection.


i . t . administration of Tat - P4 - ( C5 ) 2 ( 20 µM ) at day 14 28 after injury in maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in mice ( compared to time 0 pre - injection , D14 n = 8 / group / gender , D28 n = 6 , n = 7 ) .

i.t. administration of Tat-P4-(C5)2 (20 µM) at day 14 and 28 after injury in the maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in male and female mice (compared to time 0 pre-injection, D14 n=8/group/gender, D28 n=6 male, n=7 female).


Representative of kainate - induced cobalt uptake in spinal cord slices from p14 mouse pups treated with i ) saline , ii ) TNFα iii ) TNFα + Tat - P4 - ( C5 ) 2 . Scale Bar : 100 µm . of cobalt positive ( black soma ) . Each data point represents average 4 - 6 25 µm sections from a single 400 µm slice . Bars show means of 4 - 6 slices error bars represent . ( one - way ANOVA followed by Bonferroni ' s multiple comparisons , * P < 0 . 05 , * * P < 0 . 01 ) .

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Representative images of kainate-induced cobalt uptake in spinal cord slices from p14 mouse pups treated with i) saline, ii) TNFα and iii) TNFα + Tat-P4-(C5)2. Scale Bar: 100 µm. Quantification of cobalt positive (black soma) cells. Each data point represents the average 4-6 25 µm sections from a single 400 µm slice. Bars show means of 4-6 slices and error bars represent SEM. (one-way ANOVA followed by Bonferroni's multiple comparisons test, *P <0.05, **P<0.01).


Bar chart showing mean amplitude of field potential at lateral positions in lamina 1 recorded before approximately 30 minutes after peptide administration which was significantly reduced ( Wilcoxon matched pairs , * P = 0 . 0313 , W = - 26 , n = 7 mice , error bars : ) . chart to show data from ( E ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen that response was variable across mice with just over half showing a large reduction remaining mice only showing a relatively minor reduction ( with no reduction in one mouse ) . von Frey thresholds for mice before experiment are shown next to each . Bar chart showing mean amplitude of field potential at lateral positions in lamina 2 recorded before 30 minutes after peptide administration which was significantly reduced ( Wilcoxon matched pairs , * P = 0 . 0313 , W = - 26 , n = 7 mice , error bars : ) . chart to show data from ( G ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen there was reduction in but 1 mouse , although magnitude of reduction was again variable across mice . von Frey thresholds for mice before experiment are shown next to each .

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Bar chart showing the mean amplitude of the field potential at lateral positions in lamina 1 recorded before and approximately 30 minutes after peptide administration which was significantly reduced (Wilcoxon matched pairs, *P = 0.0313, W = -26, n = 7 mice, error bars: SEM). Line chart to show data from (E), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen that the response was variable across mice with just over half showing a large reduction and the remaining mice only showing a relatively minor reduction (with no reduction in one mouse). The von Frey thresholds for the individual mice before the experiment are shown next to each line. Bar chart showing the mean amplitude of the field potential at lateral positions in lamina 2 recorded before and 30 minutes after peptide administration which was significantly reduced (Wilcoxon matched pairs, *P = 0.0313, W=-26, n=7 mice, error bars: SEM). Line chart to show data from (G), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen there was reduction in all but 1 mouse, although the magnitude of the reduction was again variable across mice. The von Frey thresholds for the individual mice before the experiment are shown next to each line.


Bar chart showing that mean amplitude of incoming volley is reduced post administration ( Wilcoxon matched pairs , * P = 0 . 0156 , W = - 28 , n = 7 mice , error bar : ) . chart to show data from ( J ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen that response was variable across mice with half showing a large reduction remaining mice only a minor reduction .

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Bar chart showing that the mean amplitude of the incoming volley is reduced post administration (Wilcoxon matched pairs, *P = 0.0156, W = -28, n = 7 mice, error bar: SEM). Line chart to show data from (J), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen that the response was variable across mice with half showing a large reduction and the remaining mice only a minor reduction.


Amplitude of incoming volley expressed as a of baseline recording prior to peptide administration . In these 3 mice , a reduction in incoming volley at 30 minutes post - administration is seen . After this recording , remaining peptide was removed by washing spinal cord two further recordings made at 1 2 hours . Here it can be seen that incoming volley does not deteriorate further but stabilises or starts to return towards baseline confirming that initial reduction was not due to a deterioration in health of or damage to roots .

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Amplitude of the incoming volley expressed as a percentage of the baseline recording prior to peptide administration. In these 3 mice, a reduction in the incoming volley at 30 minutes post-administration is seen. After this recording, the remaining peptide was removed by washing the spinal cord and two further recordings made at 1 and 2 hours. Here it can be seen that the incoming volley does not deteriorate further but stabilises or starts to return towards baseline levels confirming that the initial reduction was not due to a deterioration in the health of the animal or damage to the roots.


Kaplan - Meier curves for overall survival ( OS ) in lung adenocarcinoma TCGA _ LUAD ( RASSF1 mRNA high / low cutoff FKPM 5 . 85 ) squamous carcinoma TCGA _ LUSC ( RASSF1 mRNA high / low cutoff FKPM 6 . 52 ) . Significance derived from log - rank .

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Kaplan-Meier curves for overall survival (OS) in lung adenocarcinoma TCGA_LUAD (RASSF1 mRNA high/low cutoff FKPM 5.85) and squamous cell carcinoma patients TCGA_LUSC (RASSF1 mRNA high/low cutoff FKPM 6.52). Significance derived from log-rank test.


Western blot with indicated antibodies of isogenic H1299 stably transfected with either empty vector pcDNA3 ( H1299control ) or RASSF1A ( H1299RASSF1A ) . Bottom : proliferation resazurin . ( n = 2 ) . Error bars represent mean ± S . E . M .

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Western blot with indicated antibodies of isogenic H1299 cells stably transfected with either empty vector pcDNA3 (H1299control) or RASSF1A (H1299RASSF1A). Bottom: cell proliferation resazurin assay. (n=2). Error bars represent mean ± S.E.M.


of lung tumours measured at day 30 after lung orthotopic injection either with H1299control ( n = 14 mice per group ; experimental groups of 6 8 different shading within groups represents two independent experiments ) or with H1299RASSF1A stably re - expressing RASSF1A , ( n = 12 mice per group ; experimental groups of 6 6 ) . of tumours was measured by MRI software ITK - SNAP . Statistical significance via 2 - Student ' s t - . Error bars represent mean ± S . E . M .

Size of lung primary tumours measured at day 30 after lung orthotopic injection either with H1299control (n=14 mice per group; experimental groups of 6 and 8 different shading within the groups represents two independent experiments) or with H1299RASSF1A cells stably re-expressing RASSF1A, (n=12 mice per group; experimental groups of 6 and 6). The size of primary tumours was measured by MRI software ITK-SNAP. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.


Visual assessment of metastatic events on ( left ) or ( right ) lungs , generated by H1299control or H1299RASSF1A at day 30 . Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A . Statistical significance via 2 - Student ' s t - . Error bars represent mean ± S . E . M .

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Visual assessment quantification of metastatic events on the ipsilateral (left) and or contralateral (right) lungs, generated by H1299control or H1299RASSF1A at day 30. Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.


Mass - spectrometry of purified from extracellular matrix isolated from H1299control H1299RASSF1A lung adenocarcinoma lines with summary of results . Only identified with two or more peptides were taken into consideration . resulting list of was restricted to ECM ( n = 3 ) .

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Mass-spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n=3).


Kaplan Meier plots depicting prognosis in TCGA _ LUAD ( lung adenocarcinoma ) with high low mRNA expression of ECM revealed by proteomics . Significance derived from log - rank . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of H & E for P4HA2 , ( bars ) of two independent regions of n = 5 H1299control n = 3 H1299RASSF1A lung tumours . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Hydroxyproline ( biolabs Inc . ) for total hydroxyproline activity of . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Western blot indicating of Collagen I in H1299control H1299RASSF1A .

Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells.


Western blot indicating of Collagen I in H1299control in presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination .

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Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.


of immunofluorescence of Collagen I in H1299control in presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination . H1299RASSF1A was used as negative control for comparison . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Immunofluorescence for Collagen I P4HA2 ( right graph ) in HOP92 after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Western blot analyses in HOP92 showing increased expression of collagen I after siRNA against RASSF1A .

Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.


Immunofluorescence for Collagen I P4HA2 ( right graph ) in HeLa after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Invasion of H1299control H1299RASSF1A through three - dimensional collagen matrix coated inserts , over 24 hours ( n = 3 ) . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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Invasion of H1299control and H1299RASSF1A cells through three-dimensional collagen matrix coated inserts, over 24 hours (n=3). Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of H1299 treated with siNT , siRNA against P4HA2 or in presence of prolylhydroxylase inhibitor DPCA ( 4μM ) , allowed to invade for 24 hours through a three - dimensional Matrigel matrix Boyden chamber . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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Representative images and quantification of H1299 cells treated with siNT, siRNA against P4HA2 or in the presence of the prolylhydroxylase inhibitor DPCA (4μM), and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of HOP92 stably transfected with shcontrol or shRASSF1A allowed to invade for 24 hours through a three - dimensional Matrigel matrix Boyden chamber . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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Representative images and quantification of HOP92 cells stably transfected with shcontrol or shRASSF1A and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


3D collagen contraction : 5x105 / ml were embedded into collagen rat tail I matrix ( 2mg / ml ) analysed after 4 days . circles indicates diameter of gel plugs at time 0 . Representative bright - field ( bottom ) with , showing effect of H1299RASSF1A or H1299control on collagen gel remodelling after pre - treatment with siRNA against P4HA2 , 4μM DPCA or combination as indicated . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

3D collagen contraction assay: 5x105/ml cells were embedded into collagen rat tail I matrix (2mg/ml) and analysed after 4 days. White circles indicates diameter of gel plugs at time 0. Representative bright-field images (bottom) with quantification, showing the effect of H1299RASSF1A or H1299control cells on collagen gel remodelling and after cells pre-treatment with siRNA against P4HA2, 4μM DPCA or combination as indicated. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Upper image : Cartoon of hanging drops method for spheroids formation embedding collagen rat tail I matrix ( 2mg / ml ) . Bottom : Representative immunofluorescence of YAP distribution its ( right bars ) by Pearson ' s coefficient for correlation of YAP DAPI co - in H1299control H1299RASSF1A 3D spheroids grown in 3D collagen matrix ( 2mg / ml ) . Scale bars 10µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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Upper image: Cartoon of hanging drops method for spheroids formation and embedding collagen rat tail I matrix (2mg/ml). Bottom: Representative immunofluorescence images of YAP distribution and its nuclear quantification (right bars) by Pearson's coefficient for correlation of YAP and DAPI co-staining in H1299control and H1299RASSF1A 3D spheroids grown in 3D collagen matrix (2mg/ml). Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Relative mRNA expression of P4HA2 YAP target genes CYR61 CTGF in H1299control in presence of siNT or siRNA targeting YAP1 . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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Relative mRNA expression levels of P4HA2 and YAP target genes CYR61 and CTGF in H1299control cells in presence of siNT or siRNA targeting YAP1. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative immunofluorescence of YAP distribution ( right ) its by Pearson ' s coefficient for correlation ( left ) of YAP DAPI co - in H1299control H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A with or without treatment with siP4HA2 or 4μM DPCA . ( Correlation , Pearson ' s coefficient ) . ( n = 3 , 300 per experiment ) Scale bars 10µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Representative immunofluorescence images of YAP distribution (right) and its nuclear quantification by Pearson's coefficient for correlation (left) of YAP and DAPI co-staining in H1299control and H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A cells with or without treatment with siP4HA2 or 4μM DPCA. (Correlation, Pearson's coefficient). (n=3, 300 cells per experiment) Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Bar graph representing of stiffness measured by atomic force microscopy ( Young modulus ) of ECM generated by H1299control or H1299RASSAF1A in vitro . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

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Bar graph representing quantification of stiffness measured by atomic force microscopy (Young modulus) of ECM area generated by H1299control or H1299RASSAF1A cells in vitro. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative atomic force microscopy ( AFM ) showing organization of extracellular matrix generated by H1299control H1299RASSF1A in 3D collagen matrices ( 2mg / ml ) .

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Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).


Bar graph representing of stiffness by atomic force microscopy ( Young modulus ) in of n = 5 H1299control n = 3 H1299RASSF1A lung tumours on day 30 . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

Bar graph representing quantification of stiffness by atomic force microscopy (Young modulus) in of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours on day 30. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative topographic of stiffness map of H1299control H1299RASSF1A lung tumours provided by AFM .

Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.


AFM of ECM fibre organization within lung tumours generated by H1299control H1299RASSF1A on day 30 .

AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.


Second harmonic generation ( SHG ) representative ( bars ) showing deposition organization of collagen fibres produced by H1299control H1299RASSF1A spheroids embedded in collagen I matrix ( 2mg / ml ) , treated with siNT , siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity . Red arrowheads show highly organized , long collagen fibres . Scale bars 20µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

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

Second harmonic generation (SHG) representative Images and Quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2mg/ml), treated with siNT, siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


SHG of organization ( bars ) of collagen fractions in lung tumours metastases at day 30 . Scale bars 10µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars 10µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative H & E ( bars ) of picrosirius red of two independent regions of n = 5 H1299control n = 3 H1299RASSF1A lung tumours . Scale bars 100µm , zoom 20µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

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Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Left : Representative of H1299 on 3D collagen wells with defined stiffness with NANOG ( green ) or DAPI ( blue ) . Scale bars 10µm . Right : of NANOG : DAPI co - localization ( upper graph ) total NANOG ( bottom graph ) . of co - localization ( n = 300cells / experiment ) is represented by Pearson ' s coefficient . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.