Fig. 7

PFK158 treatment reduces cell viability in human patient-derived ascitic cells through autophagic degradation of PLA2G3. (A-B) Expression analysis of PLA2G3 levels in 9 patient-derived ascetic culture models. Densitometric analysis using Image J software was calculated, normalized to PCNA, and the fold change was provided beneath the panel. (C-D) Percent cell viability was assessed by MTT assay with an increasing concentration of PFK158 (0–20µM) in 5 ascitic cell models that express PLA2G3. IC50 value was calculated (A4832:IC50: 4µM, A7683:IC50: 9µM, JM076:IC50: 6.9µM, AM812:IC50: 4.1µM and KP263:IC50: 5.3µM). (E) Immunoblot analysis of A7683 cells upon treatment with 3 and 5µM PFK158 and (F) the mentioned other 3 ascites samples with 3µM PFK158 against PLA2G3, p62/SQSTM1and LC3BII. PCNA was probed for endogenous control. Image J software used for densitometric calculation, normalized to PCNA, and fold change was provided beneath the panel. (G) The percent ciliated cells in PFK158 treated A7683 and KP263 ascites cultures were quantified in 100 cells per field from the IF study and represented as mean ± SD (*p < 0.05, **p < 0.01 vs. control). (H-I) IF analysis of fluorescently tagged-acetylated α tubulin (red) to score the percent ciliated cells in A7683 and KP263 ascitic cells upon treatment with 3µM PFK158 for 24 h. DAPI was used to probe the nuclei. Images were captured using confocal microscopy. Scale Bar: 20 μm. (J-L) Percent cell viability was assessed by MTT assay with an increasing concentration of cisplatin (0–40µM) alone and combined with 1/2 IC50 concentration of PFK158 in the mentioned ascetic cell models and the shift in IC50 of cisplatin treatment was analyzed