Journal: Oncogene

Article Title: EZH2 PROTACs outperform catalytic inhibitors in prostate cancer by targeting a methylation-independent function of PRC2

doi: 10.1038/s41388-025-03662-z

Figure Lengend Snippet: A A panel of PCa cell lines (LNCaP, C4-2B, LNCaP95, 22Rv1, and VCaP) and a normal prostate epithelial cell line (RWPE) were treated with PROTAC-6272 and EPZ-6438 with increasing doses from nM to µM for 48 h, and half inhibitory concentrations (IC 50 ) of proliferation rates were assayed by cell Titer Glo ( n = 6). B A panel of PCa cell lines was treated with DMSO, PROTAC-6272 (1 µM), and EPZ-6438 (1 µM) and incubated in an IncuCyte live imager chamber; images were taken every 2 h up to 9 days. Cell proliferation rates were analyzed by IncuCyte live imager software ( n = 6). C 22Rv1 cells were subjected to triplicate RNA-seq analysis upon 6 days of treatment with DMSO, PROTAC-6286, PROTAC-6272, and EPZ-6438 (1 µM). Heatmap shows combined differentially expressed genes identified ( | Log2FC | ≥0.585, adjusted p < 0.05) from treatment of PROTAC-6286, EPZ-6438, or PROTAC-6272 relative to DMSO. D VCaP cells were subjected to triplicate RNA-seq analysis upon 6 days of treatment with DMSO, PROTAC-6286, PROTAC-6272, and EPZ-6438. Heatmap shows combined differentially expressed genes identified ( | Log2FC | ≥0.585, adjusted p < 0.05) from treatment of PROTAC-6286, EPZ-6438, or PROTAC-6272 relative to DMSO.

Article Snippet: For the Incucyte assay, cells were seeded in 96-well plates and allowed to stabilize for 24 h. Cells were then treated with 1 μM of DMSO, PROTAC-6272, and EPZ-6438 and incubated in the Incucyte live imaging chamber (Sartorius).

Techniques: Incubation, Software, RNA Sequencing

Journal: Metabolism: clinical and experimental

Article Title: Mitochondrial phosphopantetheinylation is required for oxidative metabolism

doi: 10.1016/j.metabol.2025.156413

Figure Lengend Snippet: AASDHPPT is required for mitochondrial oxidative metabolism. (A) Whole cell lysates were separated by SDS-PAGE and immunoblotted for the denoted targets in clonal control, Aasdhppt -deficient, and siRNA-transfected cells. Data are representative of 3 biological replicates. (B–C) Cells were grown in either 25 mM glucose (B) or 10 mM galactose (C) in an IncuCyte ® system with images captured every 3 h for 4 days or 2 days respectively. Data represent the mean doubling time calculated from technical replicates of growth curves; error bars denote SEM **** = p < 0.0001. Data are representative of 1 experiment from n > 3 biological replicates. (D) Seahorse mitochondrial stress test for Oxygen Consumption Rate (OCR) of indicated cell lines expressing mtDSRed or PPT-GFP. Data are representative of 1 experiment from 3 biological replicates. Error bars represent SD. Significance determined at indicated time points using one-way ANOVA, **** = p < 0.0001 (E) Blue-native PAGE separation of isolated mitochondrial protein complexes from denoted cell lines expressing mtDSRed or PPT-GFP, followed by immunoblot with the indicated antibodies. Data are representative of n > 3 biological replicates. (F) Schematic of isotopomeric labeling patterns upon substitution of U 13 C-glutamine for unlabeled glutamine. Open circles indicate 12 C carbons, where circles with grey, red, or blue, indicate 13 C carbons. (G-H) Technical triplicate samples ( n = 3) of the indicated cell lines cultured in U 13 C-glutamine for the indicated time points, harvested, and analyzed via LC-MS. Steady state metabolite pool sizes and ratios are shown for the 4-h labeling time point. (I) Ratio of Citrate M + 5 to Citrate M + 4 at the four-hour time point. Error bars represent SEM (B, C), SD (D, G, H, I). ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 as determined by ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: Percent confluency was measured in an IncuCyte ® chamber (Sartorius) from sixteen images at 10× magnification every 3 h. Percent confluency curves were fit with a non-linear regression during the logarithmic growth phase using the exponential growth equation in GraphPad Prism to determine population doubling time.

Techniques: SDS Page, Control, Transfection, Expressing, Blue Native PAGE, Isolation, Western Blot, Labeling, Cell Culture, Liquid Chromatography with Mass Spectroscopy