Journal: Autophagy

Article Title: PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells

doi: 10.1080/15548627.2023.2182594

Figure Lengend Snippet: PIP5K1C protein and phosphoinositide biosynthesis in WX8-sensitive and insensitive cells. (A) Immunoblots of the three PIP5K1 isozymes in WX8-sensitive and resistant human cell lines. Proteins were identified by a specific antibody and by their molecular weight relative to protein standards. PIP5K1B was not detected in any of the samples. (B) The indicated amounts of total cell protein from A375 and HFF1 cells were immunoblotted for PIP5K1C. Side by side comparison of 5 µg samples from A375 and HFF1 cells exposed for less time were quantified by densitometry. Ratios of PIP5K1C:TUBA are indicated. (C) PIP5K1C immunoblot of the same cell lines used to quantify PIKFYVE protein in . About 2X less PIP5K1C sample was applied to this gel as to gel in panel A. (D) The indicated cell lines were cultured for 24 h with either vehicle or 1 µM WX8. Total PtdIns(4,5)P 2 (pmoles/10 6 cells) was quantified by ELISA assays. (E) The percentage decrease of PtdIns(4,5)P 2 in different cells after WX8 treatment compared with cell viability as quantified by ATP loss. (F) The percentage change in the levels of PtdIns3P, PtdIns4P and PtdIns(4,5)P 2 in A375 cells cultured with 1 µM WX8 for 24 hours. (G) The percentage change in the levels of PtdIns3P, PtdIns4P and PtdIns(4,5)P 2 in A375 cells cultured for 72 h with no-target siRNA, or siRNA against PIP4K2C (si PIP4K2C ) either with or without 0.05 µM WX8. Phosphoinositides were quantified by ELISA assays.

Article Snippet: All lipid samples are sonicated at room temperature in a Diagenode’s Bioruptor 300 (high power setting, 30s ON, 30s OFF) for 10 min before being subjected to Mass ELISA assays for PtdIns3P (Echelon Biosciences, K-3300), PtdIns4P (K-4000E) and PtdIns(4,5)P 2 (K4500) according to the manufacturer’s instructions.

Techniques: Western Blot, Molecular Weight, Cell Culture, Enzyme-linked Immunosorbent Assay