A - E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs ( A - C ) and dermal HMVECs ( D - E ) were grown to 100 % confluence over 8 - μm - pore insert membranes . ECs were first treated with Vasculotide ( VT ) , PBS ( vehicle / negative control ) , Ang1 ( positive control ) , PEG - Cys ( polyethylene glycol backbone ) , T7c ( non - PEGylated CHHHRHSF peptides ) , or VT in the presence of 100 - fold molar excess of T7c . The concentrations used , 10 - 20 ng / mL VT and 200 - 400 ng / mL Ang1 , are estimated molar equivalents ( 0 . 71 - 1 . 43 nM ) . Thirty minutes later , ECs were stimulated with thrombin , 0 . 1 % BSA ( vehicle / negative control ) , or EDTA ( positive control ) . Another 30mins later , the amount of FITC - dextran diffusion into the lower chambers ( A , D ) provides a measure of endothelial permeability . CMTPX - labeled tumor cells ( TCs ) were then dispensed into inserts , and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h ( B - C ) or 28 h ( E ) reflects the efficiency of trans - endothelial TC migration . Representative fluorescent images ( 10 × ) of membrane undersides are shown in ( C ) , where DAPI - stained nuclei of ECs / TCs are shown in blue , ' extravasated ' CMTPX - labeled TCs are shown in red , and the # of CMTPX + pixels are shown numerically in red . Means ± SEM are shown ( A , B , D , E ) . Three experiments ( twice with HMVEC - LBl and once with HMVEC - DBl ) were run with n = 3 - 5 inserts ( independent biological replicates ) per group and analyzed by two - sampled unpaired t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A-C) and dermal HMVECs (D-E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10-20 ng/mL VT and 200-400 ng/mL Ang1, are estimated molar equivalents (0.71-1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B-C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, 'extravasated' CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3-5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.


A - H LM2 - 4luctumor cells were implanted intravenously into SCIDmice , generating predominantly lung - specific metastases in one experiment ( n = 5 - 6 ; A - D ) and predominantly lymphatic - specific metastases in another experiment ( n = 12 ; E - H ) . ( C and G ) Photographs of typical metastases in the lungs and caudal lymphatics , respectively . Kaplan - Meier survival analysis showed that Vasculotide ( VT ) significantly delayed mortality due to lungmetastases ( A ) but not lymphaticmetastases ( E ) . Quantitative analysis of in vivo bioluminescent images taken at 24DPI ( B ) showed that concurrent VT treatment effectively reversed the sunitinib ( SU ) - induced acceleration of lungmetastases ( D ) . With regard to lymphaticmetastases ( F , H ) , SU treatment did not accelerate their progression . Geometric means ± 95 % CI are depicted in ( D and H ) ; P values were derived by one - way ANOVA ( D ) and Kruskal - Wallis test ( H ) . The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5 .

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

A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.


A - D An experiment where HT29luc tumor cells were implanted intravenously into YFP - SCID mice . ( A ) Kaplan - Meier survival analysis . ( B ) Quantification of total metastatic burden in the liver and lymphatics , at 35 days post - implantation ( DPI ) , from whole - body bioluminescent imaging ( C ) . ( D ) Photograph of typical liver metastases .

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A-D An experiment where HT29luc tumor cells were implanted intravenously into YFP-SCID mice. (A) Kaplan-Meier survival analysis. (B) Quantification of total metastatic burden in the liver and lymphatics, at 35 days post-implantation (DPI), from whole-body bioluminescent imaging (C). (D) Photograph of typical liver metastases.


E - G An experiment where SN12luc tumor cells were implanted intravenously into YFP - SCID mice . ( E ) Kaplan - Meier survival analysis . ( F ) Quantification of total metastatic burden at 50DPI , mostly within the lungs , from whole - body bioluminescent imaging ( G ) .

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E-G An experiment where SN12luc tumor cells were implanted intravenously into YFP-SCID mice. (E) Kaplan-Meier survival analysis. (F) Quantification of total metastatic burden at 50DPI, mostly within the lungs, from whole-body bioluminescent imaging (G).


A , B In modified Boyden chamber experiments , confluent dermal HMVECs were stimulated with tumor cell - conditioned media ( TC - CM ) - that is , media conditioned by either LM2 - 4luc , SN12luc , or HT29luc tumor cells - and treated with 20 ng / mL Vasculotide vs . 400 ng / mL Ang1 ( positive control ) vs . PBS ( negative control ) . Meta - analyses of treatment effects on dextran permeability ( A ) and tumor cell migration ( B ) , where each datapoint represents the mean of an independent experiment , averaged over 2 - 3 biological replicates ( inserts ) per treatment group and normalized internally within that same experiment . Statistical significance of overall Ang1 or VT treatment effects was determined by paired t - tests , n = 11 ( A ) or 6 ( B ) experimental replicates . C Representative fluorescent images ( 10 × ) of ' extravasated ' CMTPX - labeled TCs ( red ) fixed on membrane undersides .

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

A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)-that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells-and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2-3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.C Representative fluorescent images (10×) of 'extravasated' CMTPX-labeled TCs (red) fixed on membrane undersides.


D LM2 - 4luc produced the highest levels of VEGF and IL - 8 , but lower levels of IL - 6 , compared to other TC types ; * * * P < 0 . 001 and * P < 0 . 05 ( unpaired t - tests ) .

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D LM2-4luc produced the highest levels of VEGF and IL-8, but lower levels of IL-6, compared to other TC types; ***P < 0.001 and *P < 0.05 (unpaired t-tests).


A - C Vasculotide ( VT ) did not alter the growth kinetics of orthotopic LM2 - 4luc tumors ; n = 8 mice per group . Sunitinib ( SU ) treatment significantly lowered primary tumor burden as measured by volume ( B : one - way ANOVA , P < 0 . 01 ) or bioluminescent activity ( C : one - way ANOVA , P < 0 . 001 ) .

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A-C Vasculotide (VT) did not alter the growth kinetics of orthotopic LM2-4luc tumors; n = 8 mice per group. Sunitinib (SU) treatment significantly lowered primary tumor burden as measured by volume (B: one-way ANOVA, P < 0.01) or bioluminescent activity (C: one-way ANOVA, P < 0.001).


D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors , as quantified by H & amp ; amp ; E histological staining .

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D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors, as quantified by H&amp;E histological staining.


E VT treatment did not alter the mean thickness of the CAIX + hypoxic edge between the viable periphery and necrotic core of a primary tumor ; IHC control and sample images taken at 5 × are shown .

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E VT treatment did not alter the mean thickness of the CAIX+ hypoxic edge between the viable periphery and necrotic core of a primary tumor; IHC control and sample images taken at 5× are shown.


F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs , without the corresponding effect on lymphatic metastases . Representative bioluminescent vs . photographic images of dissected lungs in a 24 - well plate are shown .

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F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs, without the corresponding effect on lymphatic metastases. Representative bioluminescent vs. photographic images of dissected lungs in a 24-well plate are shown.


G Human LM2 - 4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin , a cytoskeletal protein . Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules .

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G Human LM2-4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin, a cytoskeletal protein. Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules.


A Kaplan - Meier survival curves . Number of days gained in median survival due to treatments is listed in brackets . Yellow arrows mark the timepoints at which the bioluminescent images were taken .

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A Kaplan-Meier survival curves. Number of days gained in median survival due to treatments is listed in brackets. Yellow arrows mark the timepoints at which the bioluminescent images were taken.


B Bioluminescent images of tumor burden . C - D Total bioluminescent fluxes , at 35 days post - implantation ( while the ' SU _ 3 wk ' group was still receiving 60 mg / kg / day sunitinib treatment ) , were separately quantified from the thoracic ( C ) and abdominal ( D ) regions . Geometric means and P values from Mann - Whitney tests are depicted in ( C and D ) . Percentages listed below the x - axes in panels ( C and D ) refer to treatment - associated changes in the geometric mean bioluminescence , relative to the vehicle group .

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B Bioluminescent images of tumor burden.C-D Total bioluminescent fluxes, at 35 days post-implantation (while the 'SU_3 wk' group was still receiving 60 mg/kg/day sunitinib treatment), were separately quantified from the thoracic (C) and abdominal (D) regions. Geometric means and P values from Mann-Whitney tests are depicted in (C and D). Percentages listed below the x-axes in panels (C and D) refer to treatment-associated changes in the geometric mean bioluminescence, relative to the vehicle group.


A - F Primary human umbilical vein endothelial cells ( HUVECs ) or primary human dermal microvascular endothelial cells ( HMVEC - DBls ) were stimulated at 80 % confluency with Vasculotide ( treatment ) , Ang1 ( positive control ; ligand of Tie2 ) , PBS ( vehicle control ) , or VEGF ( negative control ; non - ligand of Tie2 ) . Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine . Vasculotide treatment did not lead to increases in the pTie2 / Tie2 ratio in HUVECs after 45 - min ( A - D ) or 15 - min ( E ) treatments , with ( E ) or without ( A - D ) serum / supplement starvation . Vasculotide also did not activate Tie2 receptors in HMVEC - DBls , whether in the presence or absence of serum , growth factor , and cytokine supplements ( F ) .

O O O O O O O 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 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 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

A-F Primary human umbilical vein endothelial cells (HUVECs) or primary human dermal microvascular endothelial cells (HMVEC-DBls) were stimulated at 80% confluency with Vasculotide (treatment), Ang1 (positive control; ligand of Tie2), PBS (vehicle control), or VEGF (negative control; non-ligand of Tie2). Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine. Vasculotide treatment did not lead to increases in the pTie2/Tie2 ratio in HUVECs after 45-min (A-D) or 15-min (E) treatments, with (E) or without (A-D) serum/supplement starvation. Vasculotide also did not activate Tie2 receptors in HMVEC-DBls, whether in the presence or absence of serum, growth factor, and cytokine supplements (F).


Healthy non - tumor - bearing mice ( n = 8 - 9 per group ) were treated for 1 week with vehicle ( Veh ) ; Vasculotide 250 ng / 2 days IP ( VT ) ; sunitinib 60 mg / kg / day PO ( SU ) ; or VT + SU . Tie2 immunoprecipitates from lung , kidney , or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti - Tie2 antibody ( ' Tie2 ' ) or an anti - phosphotyrosine antibody ( ' p - Tie2 ' ) . Densitometry analysis of immunoblots from ( A ) . pTie2 / Tie2 ratios were internally normalized to the vehicle group within each blot . Means ± SEM and P values ( unpaired t - tests ) are depicted .

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

Healthy non-tumor-bearing mice (n = 8-9 per group) were treated for 1 week with vehicle (Veh); Vasculotide 250 ng/2 days IP (VT); sunitinib 60 mg/kg/day PO (SU); or VT + SU. Tie2 immunoprecipitates from lung, kidney, or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti-Tie2 antibody ('Tie2') or an anti-phosphotyrosine antibody ('p-Tie2').Densitometry analysis of immunoblots from (A). pTie2/Tie2 ratios were internally normalized to the vehicle group within each blot. Means ± SEM and P values (unpaired t-tests) are depicted.


A - B Pull - down assay . Input ligands - rhAng1 ( A ) and VT ( B ) - were incubated with either protein A sepharose beads only , bead - conjugated mouse Tie2 - Fc ( mTie2 ) , or bead - conjugated human Tie2 - Fc ( hTie2 ) . The ' bound ' fraction shows the total amount pulled down by beads . The ' unbound ' fraction is 7 % of residual / free ligands remaining in the supernatant .

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A-B Pull-down assay. Input ligands-rhAng1 (A) and VT (B)-were incubated with either protein A sepharose beads only, bead-conjugated mouse Tie2-Fc (mTie2), or bead-conjugated human Tie2-Fc (hTie2). The 'bound' fraction shows the total amount pulled down by beads. The 'unbound' fraction is 7% of residual/free ligands remaining in the supernatant.


C Quantitative kinetics analysis by SPR . All analytes were run at increasing concentrations of 0 . 625 , 1 . 25 , 2 . 5 , 5 , and 10 nM , at 37 ° C and pH 7 . 4 . The positive controls - rhAng1 , BowAng1 , COMP - Ang1 - all bound strongly to immobilized hTie2 - Fc . There was no detectable binding of VT , or the negative control , rhVEGF , to immobilized hTie2 - Fc .

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C Quantitative kinetics analysis by SPR. All analytes were run at increasing concentrations of 0.625, 1.25, 2.5, 5, and 10 nM, at 37°C and pH 7.4. The positive controls-rhAng1, BowAng1, COMP-Ang1-all bound strongly to immobilized hTie2-Fc. There was no detectable binding of VT, or the negative control, rhVEGF, to immobilized hTie2-Fc.


( A ) Electrophoretic analysis ( under non - denaturing conditions ) of the association between the indicated IκBα species association and reconstituted nucleosome core particles ( NCP ) .

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(A) Electrophoretic analysis (under non-denaturing conditions) of the association between the indicated IκBα species association and reconstituted nucleosome core particles (NCP).


( B ) Quantification of the IκBα 60 kDa band ( upper panels ) and the 37 kDa band ( lower panels ) is shown as average and s . d . of three independent experiments .

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(B) Quantification of the IκBα 60 kDa band (upper panels) and the 37 kDa band (lower panels) is shown as average and s.d. of three independent experiments.


( D ) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2 - month - old WT mice ( each # represents a mouse ) .

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(D) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2-month-old WT mice (each # represents a mouse).


( G ) Pulldown assay using GST - SUMO IκBα protein and chromatin lysates from 2 - month - old WT mice villi .

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(G) Pulldown assay using GST-SUMO IκBα protein and chromatin lysates from 2-month-old WT mice villi.


( A ) Western Blot analysis of the crypt - derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times .

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(A) Western Blot analysis of the crypt-derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times.


( F ) Western Blot analysis of a cleavage experiment incubating modified H4 peptides ( P1 - P9 ) with soluble lysates from villi or crypts in the presence ( + ) or absence ( - ) of the commercial protease inhibitors cocktail . Lanes indicated as P correspond to the control peptide without lysate incubation . Notice the electrophoretic shift of peptides incubated with villus lysates ( compared with control peptides ) suggestive of post - translational modifications or binding to proteins absent from crypt lysates .

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(F) Western Blot analysis of a cleavage experiment incubating modified H4 peptides (P1-P9) with soluble lysates from villi or crypts in the presence (+) or absence (-) of the commercial protease inhibitors cocktail. Lanes indicated as P correspond to the control peptide without lysate incubation. Notice the electrophoretic shift of peptides incubated with villus lysates (compared with control peptides) suggestive of post-translational modifications or binding to proteins absent from crypt lysates.


WB analysis ( C ) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre - confluence or 7 days post - confluence . Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed .

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WB analysis (C) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre-confluence or 7 days post-confluence. Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed.


WB analysis ( E ) of MUC5AC in HT29 cells treated with the indicated protease inhibitors . Bars represent mean values ± standard deviation from 3 biological replicates

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WB analysis (E) of MUC5AC in HT29 cells treated with the indicated protease inhibitors. Bars represent mean values ± standard deviation from 3 biological replicates performed


WB analysis ( G ) of MUC5AC in HT29 cells transduced with sh - RNAs against trypsin ( T1 , T2 ) or chymotrypsin ( C1 , C2 ) . Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates

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WB analysis (G) of MUC5AC in HT29 cells transduced with sh-RNAs against trypsin (T1, T2) or chymotrypsin (C1, C2). Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed


( D , E ) Representative examples of dose - response curves and time courses . Lines through the dose - response curves are fits to the Michaelis - Menten equation ; time courses are fitted to linear equations . Shown are means and standard errors over at least three independent repeats .

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(D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats.


( F ) Fold increase ( positive values ) or decrease ( negative values ) in the initial uptake rates of the variants relative to WT GltPh . Circles are averages of technical triplicates . Bars are means over independent repeats ( as indicated ) . The dashed line marks a four - fold increase in the initial rate .

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(F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.


( A , B ) Schematic representations of the experimental conditions are shown on the far left . Data are shown for WT GltPh and select gain - of - function mutants , as indicated above the panels . Transition density plots ( top of each panel ) show the frequency of transitions between the EFRET values . The number of trajectories analyzed ( N ) and the population - wide mean frequency of transitions is shown on the panels . Representative 50 s sections of single - molecule EFRET trajectories ( middle ) with raw data in black and idealizations in brown . Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels . Dwell - time distributions for the OFS in blue and the IFS in red ( bottom ) . Mean dwell times are shown on the panels in corresponding colors . Black lines represent photobleaching survival plots normalized from 1 to 0 . Shown are means and standard errors over at least three independent repeats .

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(A, B) Schematic representations of the experimental conditions are shown on the far left. Data are shown for WT GltPh and select gain-of-function mutants, as indicated above the panels. Transition density plots (top of each panel) show the frequency of transitions between the EFRET values. The number of trajectories analyzed (N) and the population-wide mean frequency of transitions is shown on the panels. Representative 50 s sections of single-molecule EFRET trajectories (middle) with raw data in black and idealizations in brown. Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels. Dwell-time distributions for the OFS in blue and the IFS in red (bottom). Mean dwell times are shown on the panels in corresponding colors. Black lines represent photobleaching survival plots normalized from 1 to 0. Shown are means and standard errors over at least three independent repeats.


( A ) Changes in the initial rates of substrate uptake ( gray bars ) , transport domain dynamics ( black bars ) , and l - Asp dissociation constant ( white bars ) of the mutant GltPh variants relative to the WT transporter . The transition frequencies were measured under non - equilibrium transport conditions , and frequencies obtained in the presence of d , l - TBOA were subtracted from the data . Error bars represent standard errors for at least three independent measurements .

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(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.


( A ) Dwell - time distributions of the OFS ( blue ) and the IFS ( red ) for WT ( top ) and K290A ( bottom ) GltPh observed under equilibrium conditions in saturating Na + / l - Asp ( left ) . Lines are fits to three exponentials . Data are averages and standard errors of at least three independent measurements .

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(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements.


( B ) Representative EFRET trajectories of WT GltPh showing different transition frequencies . Raw data are in black , and idealizations are in brown . Scalebar is above the trajectories .

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(B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories.


( C ) Distribution of the WT ( top ) and the K290A GltPh ( bottom ) molecules with different mean transition frequencies ( white bars ) . N is the number of trajectories longer than 90 s used in the analysis . The stacked black bars are fractions of trajectories without transitions . Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0 . 04 s - 1 for the WT and 0 . 34 s - 1 for the K290A . Data are averages and standard errors of at least three independent measurements .

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(C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements.


( D ) 2D - histograms of the consecutive dwell lengths in the OFS ( top ) and the IFS ( bottom ) . From left to right : calculated distribution of dwell times randomly selected from the distributions in panel A ; measured distribution for WT GltPh and the K290A mutant .

(D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant.


( E ) Representative trajectories of the WT ( left ) and the K290A ( right ) GltPh molecules showing switching between dynamic modes . Raw data are in black and idealizations are in brown ( top ) . Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes . Survival plots for the OFS and the IFS ( middle ) . Solid lines are fits to a single ( IFS ) and double ( OFS ) exponentials . Dashed lines ( OFS only ) are rejected fits to single exponentials . Autocorrelation plots of the sequential dwell durations ( bottom ) .

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(E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom).


( A , B ) 2D - histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior . N is the number of traces used . Scale bar shows relative density normalized by the number of molecules . Above and to the right of each panel are stacked histograms of , respectively , the OFS and the IFS lifetimes of the analyzed molecules ( open bars ) and the photobleaching time of molecules showing no transitions ( black bars ) . Data from three independent measurements were combined for presentation . ( C ) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D - histograms ( yellow to red ) . The open blue bar represents the calculated mean OFS lifetime for WT GltPh Shown are means and standard errors of the mean of at least three independent measurements .

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(A, B) 2D-histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior. N is the number of traces used. Scale bar shows relative density normalized by the number of molecules. Above and to the right of each panel are stacked histograms of, respectively, the OFS and the IFS lifetimes of the analyzed molecules (open bars) and the photobleaching time of molecules showing no transitions (black bars). Data from three independent measurements were combined for presentation. (C) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D-histograms (yellow to red). The open blue bar represents the calculated mean OFS lifetime for WT GltPh Shown are means and standard errors of the mean of at least three independent measurements.


( B ) Representative live - cell fluorescent images of HeLa cells co - transfected with EGFP - TECPR1 and LAMP1 - mCherry and treated with 1 mM LLOMe for the indicated time . Scale bar = 10 µM .

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(B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and LAMP1-mCherry and treated with 1 mM LLOMe for the indicated time. Scale bar = 10 µM.


( D ) Representative live - cell fluorescent images of HeLa cells co - transfected with EGFP - TECPR1 and LAMP1 - mCherry and treated as indicated . Scale bars = 10 µM . ( E ) Quantification of the fold change in EGFP - TECPR1 lysosomal fluorescence from D . Grey points represent individual cells from three independent experiments . Red points represent the means of individual experiments ( n > 25 cells per experiment ) . Bars represent the mean ± SD from the three experiments . Significance was determined from biological replicates using a one - way ANOVA with Tukey ' s multiple comparisons tests . * * p = < 0 . 005 , * * * * p < 0 . 0001 , ns = not significant .

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(D) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and LAMP1-mCherry and treated as indicated. Scale bars = 10 µM. (E) Quantification of the fold change in EGFP-TECPR1 lysosomal fluorescence from D. Grey points represent individual cells from three independent experiments. Red points represent the means of individual experiments (n > 25 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey's multiple comparisons tests. ** p = <0.005, **** p < 0.0001, ns = not significant.


( B ) Representative live - cell fluorescent images of HeLa cells co - transfected with the indicated EGFP - TECPR1 deletion mutant and LAMP1 - mCherry before and after treatment with 1 mM LLOMe . Scale bars = 10 µM . To the right is the corresponding quantification of the fold change in EGFP - TECPR1 lysosomal fluorescence intensity after a 15 - minute treatment with vehicle or 1 mM LLOMe . Grey points represent individual cells from three independent experiments .

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(B) Representative live-cell fluorescent images of HeLa cells co-transfected with the indicated EGFP-TECPR1 deletion mutant and LAMP1-mCherry before and after treatment with 1 mM LLOMe. Scale bars = 10 µM. To the right is the corresponding quantification of the fold change in EGFP-TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments.


( A , B ) Representative live - cell fluorescent images of HeLa cells co - transfected with EGFP - TECPR1 and mCherry - Gal3 and treated with 1 mM LLOMe for the indicated time . Arrowheads in B indicate the appearance of TECPR1 ( green arrow ) and Gal3 ( magenta arrow ) . Scale bars = 10 µM for A and 1 µM for B . ( C ) Quantification of TECPR1 and Gal3 recruitment to damaged lysosomes . Data are presented as mean ± SD from 5 independent experiments ( each experiment represents a single cell with at least 3 individual lysosomes quantified ) .

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(A,B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and mCherry-Gal3 and treated with 1 mM LLOMe for the indicated time. Arrowheads in B indicate the appearance of TECPR1 (green arrow) and Gal3 (magenta arrow). Scale bars = 10 µM for A and 1 µM for B. (C) Quantification of TECPR1 and Gal3 recruitment to damaged lysosomes. Data are presented as mean ± SD from 5 independent experiments (each experiment represents a single cell with at least 3 individual lysosomes quantified).


( D ) Representative confocal image of a HeLa cell transfected with EGFP - TECPR1 , treated with LLOMe for 5 minutes , and immunostained for CHMP2A and LAMP1 . Scale bars = 10 µm for whole image and 1 µm for insets . Fluorescence intensity profiles of the indicated channels across the dotted lines are shown in the lower subpanel . ( E ) Representative confocal images of a HeLa cells transfected with EGFP - TECPR1 and TagBFP - TMEM192 ( a lysosomal / late endosomal protein ) , treated with 1 mM LLOMe for the indicated time , and immunostained for ALIX and Gal3 . Scale bars = 10 µm .

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(D) Representative confocal image of a HeLa cell transfected with EGFP-TECPR1, treated with LLOMe for 5 minutes, and immunostained for CHMP2A and LAMP1. Scale bars = 10 µm for whole image and 1 µm for insets. Fluorescence intensity profiles of the indicated channels across the dotted lines are shown in the lower subpanel. (E) Representative confocal images of a HeLa cells transfected with EGFP-TECPR1 and TagBFP-TMEM192 (a lysosomal/late endosomal protein), treated with 1 mM LLOMe for the indicated time, and immunostained for ALIX and Gal3. Scale bars = 10 µm.


( A ) Western blot analysis of LC3 lipidation status in wild type ( WT ) , FIP200 KO and ATG16L1 KO HeLa cells treated with the indicated concentrations of LLOMe for 30 minutes .

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(A) Western blot analysis of LC3 lipidation status in wild type (WT), FIP200 KO and ATG16L1 KO HeLa cells treated with the indicated concentrations of LLOMe for 30 minutes.


( B ) Quantification of LC3 - II levels in HEK , HeLa and MEF WT and ATG16L1 - deficient cell lines treated with and without 1 mM LLOMe for 30 minutes . Bars show mean ± SD from three biologically independent experiments represented as data points . Significance was determined from biological replicates using a Student ' s t tests . * p = 0 . 0177 , * * p = 0 . 0082 , * * * p = 0 . 0001 .

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(B) Quantification of LC3-II levels in HEK, HeLa and MEF WT and ATG16L1-deficient cell lines treated with and without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points. Significance was determined from biological replicates using a Student's t tests. *p = 0.0177, ** p = 0.0082, *** p = 0.0001.


( C ) Western blot analysis of LC3 lipidation status in WT , ATG7 KO and ATG16L1 KO HeLa cells ( top ) and WT , ATG5 - / - and ATG16L1Δ / Δ MEFs ( bottom ) treated with the indicated concentration of LLOMe for 30 minutes .

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(C) Western blot analysis of LC3 lipidation status in WT, ATG7 KO and ATG16L1 KO HeLa cells (top) and WT, ATG5-/- and ATG16L1Δ/Δ MEFs (bottom) treated with the indicated concentration of LLOMe for 30 minutes.


( F ) Western blot analysis of lyso - IP samples collected from HeLa ATG16KO cells treated with or without 1 mM LLOMe for 30 minutes . Bars show mean ± SD from three biologically independent experiments represented as data points .

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(F) Western blot analysis of lyso-IP samples collected from HeLa ATG16KO cells treated with or without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points.


( G ) Representative live - cell fluorescent images of HeLa cells co - transfected with TagBFP - TMEM192 , EGFP - ATG5 and mCherry - TECPR1 , before and after a 15 - minute treatment with 1 mM LLOMe . Scale bars = 10 µm for whole images and 2 µm for zoom . To the right is the corresponding quantification of the fold change in ATG5 / TECPR1 lysosomal fluorescence intensity after a 15 - minute treatment with vehicle or 1 mM LLOMe . Grey points represent individual cells from three independent experiments . Green / magenta points represent the means of individual experiments ( n > 25 cells per experiment ) .

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(G) Representative live-cell fluorescent images of HeLa cells co-transfected with TagBFP-TMEM192, EGFP-ATG5 and mCherry-TECPR1, before and after a 15-minute treatment with 1 mM LLOMe. Scale bars = 10 µm for whole images and 2 µm for zoom. To the right is the corresponding quantification of the fold change in ATG5/TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments. Green/magenta points represent the means of individual experiments (n > 25 cells per experiment).


( H ) Western blot analysis of LC3 lipidation status in HEK TECPR1 / ATG16L1 KO cells treated with 1 mMLLOMe for 30 minutes . To the right is the corresponding quantification of LC3 - II protein levels . Bars show mean ± SD from three or four biologically independent experiments which are represented as data points .

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(H) Western blot analysis of LC3 lipidation status in HEK TECPR1/ATG16L1 KO cells treated with 1 mMLLOMe for 30 minutes. To the right is the corresponding quantification of LC3-II protein levels. Bars show mean ± SD from three or four biologically independent experiments which are represented as data points.


( A ) Representative live - cell images of HEK cells treated as indicated and stained with LysoTracker Red . Nuclei were stained with Hoechst 33342 . Scale bars = 20 µm . ( B ) Quantification of LysoTracker Red puncta from A . Grey points represent individual cells from three independent experiments . Red points represent the means of individual experiments ( n > 100 cells per experiment ) .

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(A) Representative live-cell images of HEK cells treated as indicated and stained with LysoTracker Red. Nuclei were stained with Hoechst 33342. Scale bars = 20 µm. (B) Quantification of LysoTracker Red puncta from A. Grey points represent individual cells from three independent experiments. Red points represent the means of individual experiments (n > 100 cells per experiment).


C . Biotinylation of the centrosome and microtubules by V5 - BirA * - AURKA in different cell cycle stages . After 18 h biotin incubation , HEK293T : : V5 - BirA * - AURKA were fixed and stained for the fusion protein with V5 antibody , biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 5 μm .

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C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm.


D . Control and biotin - treated HEK293T cells stably expressing V5 - BirA * and V5 - BirA * - AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads . The initial sample ( initial ) and captured biotinylated proteins ( pulldown ) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin ( loading control ) .

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D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).


C . AURKA interacts with the centriolar satellite proteins PCM1 , CEP131 , CEP72 , CSPP1 , KIAA0586 / Talpid3 and CP110 . HEK293T cells were transiently transfected with FLAG - BirA * or FLAG - BirA * - AURKA . Following 18 h biotin incubation , cells were lysed , and biotinylated proteins were precipitated by streptavidin beads . The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG , CEP72 , CEP131 , PCM1 , CEP63 , CSPP1 , KIAA0586 / Talpid3 , CP110 , BBS4 and SSX2IP .

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C. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.


E . Endogenous interaction of PCM1 and AURKA . Lysates from synchronous and serum - starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control . The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA .

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E. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.


F . AURKA co - localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain . U2OS cells were co - transfected with GFP - PCM1 - FKBP and HA - Kif5b ( 1 - 269 a . a . ) - FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours . Cells that were not treated with rapamycin were processed in parallel as a control . Fixed cells were stained with antibodies against GFP , AURKA and gamma - tubulin . DNA was stained with DAPI . Cell edges are outlined . Scale bar , 10 μm . G . Quantification of pericentrosomal levels of AURKA and gamma tubulin for F . n > 25 cells per experiment . Data represent mean value from two experiments per condition ± SEM ( * * * * p < 0 . 0001 ; * * p < 0 . 01 ; Unpaired Student ' s t - test . ) .

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F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).


A , B . Representative images and quantification of basal body levels of ( A ) AURKA and ( B ) phospho - AURKA ( p - AURKA ) in control and PCM1 - depleted cells . RPE1 cells were transfected with control or PCM1 siRNA # 1 or siRNA # 2 for 48 h . Following 24 h serum starvation , cells were fixed and stained with the indicated antibodies . Centrosomal AURKA and p - AURKA fluorescence intensities were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . n > 500 cells per experiment . Data for ( A ) AURKA and ( B ) p - AURKA represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 , Unpaired Student ' s t - test . ) Scale bar , 10

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A, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μm


D . Cycloheximide chase experiment for quantification of AURKA half - life . Cells were transfected with control or PCM1 siRNA # 1 for 48 h , then treated with 200 nM cycloheximide along with serum starvation for indicated time points . AURKA intensities were quantified by immunoblotting for AURKA and vinculin ( loading control ) and AURKA levels were normalized to vinculin levels . Data represent mean value from three experiments per condition ± SEM ( * * p < 0 . 01 , Unpaired Student ' s t - test )

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)


A . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or MLN8237 . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO ( vehicle control ) or 0 . 5 μM MLN8237 in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm . B . Quantification of ciliogenesis efficiency for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * p < 0 . 01 ; ns , non - significant , Unpaired Student ' s t - test . ) . C . Quantification of cilium length for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 ; ns , non - significant , Unpaired Student ' s t - test . ) .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm. B. Quantification of ciliogenesis efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.).


D . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or tubacin . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm E . Quantification of ciliogenesis efficiency for D . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * p < 0 . 05 ; ns , non - significant , Unpaired Student ' s t - test ) .

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

D. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or tubacin. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test).


F . Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 . Cells were fixed and immunostained with antibodies against IFT88 , acetylated tubulin and gamma tubulin . DNA was stained with DAPI . Scale bar , 10 μm . G . Quantification of IFT88 centrosomal intensity and ciliary concentration for F . IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test)


H , I . Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 in serum stimulation medium for 2 h and 24 h . Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin , and percentage of ciliated cells were quantified . x - axis indicates the hours after serum stimulation . ( H ) n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM . ( I ) n > 95 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, I. Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237 in serum stimulation medium for 2 h and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( C ) Kaplan - Meier survival curves of mice treated with the indicated therapies ( n = 6 ) . Log rank ( Mantel - Cox ) statistical test was used for MAPKi vs MAPKi / BIBF1120 . * * * * P ≤ 0 . 0001 .

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

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001.


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( F ) Quantification of collagen fibers thickness ( n = 6 for control , BIBF and BRAFi / MEKi groups and n = 5 for BRAFi / MEKi + BIBF group ) . Two - way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Significance was calculated against the control group . Statistical significance of BIBF vs BIBF + BRAFi / MEKi was also calculated .

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

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated.


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( G ) Heatmap showing the differential expression of ECM and myofibroblast / CAF genes in mice treated with MAPK - targeted therapies with or without BIBF compared to control mice ( log2 ratio , n = 5 ) .

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

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5).


( H ) Heatmap and Western Blot showing the expression of ECM , myofibroblast / CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells . Heatmap represents the mean of expression of 3 independent experiments by RT - qPCR . ( I ) Heatmap showing the expression of ECM , myofibroblast / CAF and phenotype switch markers in human M238R cells treated with BIBF ( 2 µM , 72 h ) or vehicle alone by RT - qPCR ( n = 3 ) .

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

(H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3).


( B ) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 ( BIBF , 2 μM ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) ( 1µM ) , or with MAPKi ( 1 μM ) plus BIBF ( 2 µM ) by RT - qPCR and normalized to miR - 16 - 5p . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . One - way ANOVA was used for statistical analysis . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Significance was calculated against the control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated . ( C ) Expression of miR - 143 - 3p and miR - 145 - 5p in control mice and mice treated with the indicated therapies was quantified by RT - qPCR . Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice , with 2 sites of injections . One - way ANOVA was used for statistical analysis . * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 . Significance was calculated against the control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated .

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

(B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated.


( D ) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF - β ( 10 ng / mL ) or PDGF - BB ( 20 ng / mL ) by RT - qPCR and normalized to miR - 16 - 5p . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - test . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( E ) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib / BIBF1120 ( BIBF , 2 μM ) , the TGF - β receptor kinase inhibitor SB431542 ( SB , 10 µM ) , and the pan - AKT inhibitor GSK690693 ( GSK , 10 µM ) by RT - qPCR . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

(D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001.


( F , Phenotype switch / invasive / ECM markers were quantified in therapy - naïve ( N ) and resistant ( R ) PDX samples . The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, Phenotype switch/invasive/ECM markers were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.


( B ) M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 μM ) in the presence or the absence of LNA - based anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of the two . ECM markers RT - qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . One - way ANOVA was used for statistical analysis . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

(B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001.


( C ) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in ( A ) . ( D ) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors


( F ) Migration assay performed in Boyden chambers . Representative images showing migrating cells in the different conditions . The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

(F) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001.


( F ) Luciferase assay in HEK cells overexpressing miR - 143 or miR - 145 transfected with a plasmid harboring the WT or muted sequence of the miR - 143 and miR - 145 binding sites present in FSCN1 3 ' UTR . Each bar represents the mean ± SE of experiments performed at least in triplicate . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . P - values were calculated using Paired Student t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase assay in HEK cells overexpressing miR-143 or miR-145 transfected with a plasmid harboring the WT or muted sequence of the miR-143 and miR-145 binding sites present in FSCN1 3'UTR. Each bar represents the mean ± SE of experiments performed at least in triplicate. ***P≤0.001, ****P≤0.0001. P-values were calculated using Paired Student t-test.


( G ) RT - qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

(G) RT-qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ** P≤0.01, ***P≤0.001.


( B ) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi ( Vemurafenib , 3 μM ) in the presence or the absence of LNA - based anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of the two . Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments , Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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

(B) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( D ) Migration assay performed in Boyden chambers . Representative images showing migration of M238P cells treated with the indicated siRNAs . The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * * P ≤ 0 .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) Western Blot analysis of phenotype - switch markers on cell lysates from M238P cells transfected with the indicated siRNAs .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs.


BRAFi - resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( G ) Western Blot analysis of FSCN1 , phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells .

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BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells.


BRAFi - resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( H ) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * * * * P ≤ 0 . 0001 .

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

BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.


( A , M238P cells were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( B , M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) and MEKi ( Trametinib , 1 µM ) . ( A - B ) Images and quantification of cell area in cells stained for F - actin ( red ) and nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 cells per condition ) . Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


C ) M238P cells were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . D ) M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) and MEKi ( Trametinib , 1 µM ) . ( C - D ) Images and quantification of focal adhesions number in cells stained for pPaxillin ( green ) and nuclei ( blue ) . Focal adhesions ( FA ) number is represented as mean ± SD ( n ≥ 30 cells per condition ) . Each point represents the average number of focal adhesions per cell calculated for each field . Paired Student t - test has been used for statistical analysis . * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P . ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (C-D) Images and quantification of focal adhesions number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm.


( E - F ) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) Images and quantification of cell area in cells stained for F - actin ( red ) and nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 cells per condition ) . Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm . ( F ) Images and quantification of focal adhesions ( FA ) number in cells stained for pPaxillin ( green ) and nuclei ( blue ) . Focal adhesions number is represented as mean ± SD ( n ≥ 30 cells per condition ) . Each point represents the average number of focal adhesions per cell calculated for each field . Paired Student t - test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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(E-F) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (F) Images and quantification of focal adhesions (FA) number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


E ) M238P cells were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( E ) Effect of miR - 143 - 3p or miR - 145 - 5p overexpression on the expression of YAP / MRTF target genes assessed by RT - qPCR . Data are normalized to the expression in control cells . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 .

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E) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ****P≤0.0001.


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . Effect of FSCN1 downregulation on MRTFA ( F ) and YAP1 ( G ) nuclear translocation assessed by immunofluorescence in M238P . Data are represented as scatter plot with mean ± SD ( n ≥ 30 cells per condition ) . Each point represents the nuclear / cytoplasm ratio . Mann - Whitney U test was used for statistical analysis . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm.


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( H ) RT - qPCR analysis for the expression of MRTFA / YAP target genes in M238P cells transfected with the indicated siRNAs . Data are normalized to the expression in parental cells . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Scale bar 40 μm .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.


( A ) Rescue of growth phenotype . Wild - type ( WT ) or mdm10Δ cells were transformed with the empty plasmid pYX142 ( ) . In addition , mdm10Δ cells were transformed with pYX142 encoding MDM10 or MCP3 . Cells were grown to an OD600 of 1 . 0 and spotted on YPD or YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at the indicated temperatures .

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(A) Rescue of growth phenotype. Wild-type (WT) or mdm10Δ cells were transformed with the empty plasmid pYX142 (). In addition, mdm10Δ cells were transformed with pYX142 encoding MDM10 or MCP3. Cells were grown to an OD600 of 1.0 and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures.


( B ) Over - expression of Mcp3 partially rescues the mitochondrial morphology defect in mdm10Δ cells . Cells described in ( A ) expressing mitochondrially targeted GFP ( mtGFP ) were analysed by fluorescence microscopy . Typical images ( fluorescence and brightfield , BF ) of the four different strains are shown ( scale bar = 5 µm ) . The quantification shows the average percentage with standard deviation bars of three independent experiments ( n = 3 ; SD ; * , p < 0 . 05 and as indicated ) with at least 100 cells per experiment .

(B) Over-expression of Mcp3 partially rescues the mitochondrial morphology defect in mdm10Δ cells. Cells described in (A) expressing mitochondrially targeted GFP (mtGFP) were analysed by fluorescence microscopy. Typical images (fluorescence and brightfield, BF) of the four different strains are shown (scale bar = 5 µm). The quantification shows the average percentage with standard deviation bars of three independent experiments (n=3; SD; *, p < 0.05 and as indicated) with at least 100 cells per experiment.


( A ) Respiratory chain super - complex levels are slightly restored by over - expression of Mcp3 . Mitochondria isolated from the indicated strains were lysed in 1 % digitonin and subjected to a 4 - 8 % BN - PAGE . Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV .

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(A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV.


( B ) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over - expression . Mitochondria of the indicated cells were lysed in 1 % digitonin and subjected to a 6 - 13 % BN - PAGE and immunoblotting with antibodies against Tom40 ( left ) or Tob55 ( right ) . The assembled TOM complex and an unassembled Tom40 species at ca . 100 kDa ( arrowhead ) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex ( asterisk ) .

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(B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk).


( C ) Over - expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10 . Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis . The level of each phospholipid species in wild - type mitochondria was set to 100 % and the ERG / PL ratio to 1 . Relative changes in mitochondria from other cells are presented . The mean with standard deviations of three biological repeats with two technical repeats for each ( n = 6 ; SD ; * , p < 0 . 05 ) is given .

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

(C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given.