Fig 1: FSS activates p38α MAPK and NF-κB signaling.a,b, OVCAR3 were exposed to static or 11 dynes/cm2 FSS conditions for 24 hr. Protein expression (a) and quantification (b) of p38α MAPK-associated signaling molecules. Significance evaluated by Student’s t test. c,d, NF-κB p65 immunofluorescence of recovered OVCAR3 (c) and quantification of the nuclear-to-cytoplasmic (N:C) ratio of NF-κB signal (d). Significance evaluated by Student’s t-test. e,f, F-actin and pHSP27 immunofluorescence (e) and the associated quantification of F-actin intensity (f). Significance evaluated by Student’s t-test. g, 24 hr FSS stimulation results in a downregulation of MUC15, activation of p38α MAPK, HSP27, and NF-κB signaling, and increased cytoskeletal remodeling. h, Protein expression of the OVCAR4 parental, MUC15-overexpressing vector control (MUC15-OEScr), MUC15-overexpressing OVCAR4 (MUC15-OE), MUC15 shRNA control (shMUC15Scr), and MUC15 knockdown OVCAR4 (shMUC151/shMUC152 ) cell lines. i, Phopsho-kinase array and quantification of suspected FSS signal transducers in MUC15 knockdown and scrambled shRNA control OVCAR4. j,k, Boyden chamber migration assay (j) and quantification of the number of migrated cells per FOV (k) of MUC15-transduced OVCAR4. Significance evaluated by one-way ANOVA, multiple comparisons.
Fig 2: FSS induces a mesenchymal-like phenotype and downregulates MUC15 in ovarian cancer cells.a, Fold change of MUC15 mRNA expression in sheared OVCAR3 and OVSAHO cells compared to static controls (1 dynes/cm2, 5 dynes/cm2, 11 dynes/cm2; 24 hr; n = 3). b, MUC15 immunofluorescence of recovered OVCAR3 from static and sheared conditions (11 dynes/cm2, 24 hr). c, Kaplan-Meier curve showing overall survival in late-stage, high-grade papillary serous ovarian cancer patient cohort with either low or high MUC15 mRNA expression. Significance evaluated by Mantel-Cox test. d, MUC15 immunohistochemistry and quantification of MUC15 fold change between stage I, II, and III HGSC patients. Significance evaluated by one-way ANOVA, multiple comparisons. e,f, Protein expression and quantification of MUC15 and EMT markers in static and sheared OVCAR3 (e) and OVSAHO (f) cells (11 dynes/cm2, 24 hr). Significance evaluated by Student’s t-test. g, Phospho-kinase array and the associated quantification of suspected signal transducers in static and sheared OVCAR3 cells (11 dynes/cm2, 24 hr). h, Protein expression of suspected FSS-associated signaling molecules in static and sheared OVCAR3 (11 dynes/cm2, 24 hr).
Fig 3: GPCRs and MUC15 mediate FSS mechanotransduction in ovarian cancer cells.a, Pathway analysis based on RNA-seq of static and sheared OVCAR3 (11 dynes/cm2, 24 hr). b, Protein expression of OVCAR3 cells exposed to static or 11 dynes/cm2 FSS conditions for 30 min, 1 hr, or 24 hr. c, mRNA heat map showing the expression fold change of suspected mechanosensitive signaling molecules for pairs of static and sheared OVCAR3 (11 dynes/cm2, 1 hr). d,e, Protein expression of MUC15 and p38α MAPK-associated signaling molecules (d) and EMT markers (e) in static and sheared OVCAR3 (11 dynes/cm2, 24 hr) with or without YM-254890, a Gαq inhibitor. f,g, Boyden chamber migration assays (f) and quantification of the number of migrated cells per FOV (g) of static and sheared OVCAR3 (11 dynes/cm2, 24 hr) with or without YM-254890. Significance evaluated by one-way ANOVA, multiple comparisons. h,i F-actin immunofluorescence (h) and quantification F-actin intensity (i) of OVCAR3 (11 dynes/cm2, 24 hr) with or without YM-254890. Significance evaluated by one-way ANOVA, multiple comparisons. j, Protein expression of static or sheared MUC15-OEScr and MUC15-OE OVCAR3 cells (11 dynes/cm2, 24 hr). k, Schematic of the proposed ascitic FSS mechanotransduction pathway. I, MUC15 and PLCβ1 immunofluorescence in OVCAR3. m, MUC15 and PLCβ1 co-immunoprecipitation assay in OVCAR3. n, N-terminal GFP-MUC15 fusion protein promoter and gene sequence (top). Fluorescent intensity of recovered conditioned medium from static and sheared GFPCyto and GFPMUC15 (11 dynes/cm2, 24 hr; bottom). Significance evaluated by Welch’s t test.
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