Fig. 5: ATX-101 treatment reduces 6PGD activity and 6PGD and ENO1 protein levels and blocks ENO1 – PCNA interactions.
From: PCNA regulates primary metabolism by scaffolding metabolic enzymes
A–E 6PGD activity and protein levels measured in JJN3 cells and cell extracts exposed to no (black), ATX-101 (8 μM, orange), ATX-A (8 μM, brown) and Ebselen (20 μM, blue) treatment. A, B 6PGD activity (OD = 460 nm) are plotted as relative to untreated control, mean ± SEM. (A) 6PGD activity in cell extracts from untreated JJN3 cells after addition of ATX-101 and Ebselen immediately before measurements, n = 11. (B) 6PGD activity in cell extracts from JJN3 cells treated with ATX-101, ATX-A and Ebselen for 4 h, n = 5. (C, E) Lower panels: representative western blots (WB) showing β-actin and (C) 6PGD, (D) ENO1 and (E) PCNA levels in JJN3 cells 24 h after ATX-101 treatment. Upper panel: densitometric quantifications of (C) 6PGD, (D) ENO1 and (E) PCNA levels normalized to β-actin, presented as relative to untreated controls. The mean levels and the levels in each experiments, n = 4 (square, circle, triangle and diamond) are shown. *p < 0.05, ***p < 0.001, two tailed, paired t-test. Experimental details are listed in Supplementary Table S1. F Lower panels: WB showing 6PGD, GAPDH, ENO1, PCNA and H3 (Histone 3) levels in WT HAP1 cells treated with ATX-101 (20 μM) 1-4 times (t = 0, 4, 8, and 12 h) and harvested at t = 24 h. Upper panel: densitometric quantifications of ENO1, 6PGD, GAPDH and PCNA normalized to H3 levels, presented as relative to untreated control. G Proximity Ligation Assay (PLA) analysis of WT HAP1 cells untreated (left image) or treated with ATX-101 (20 μM) (four right images) 5 times. Cells were probed with α-ENO1 and α-PCNA (two left images), and in the PLA controls with only α-PCNA, only α-ENO1 or no primary antibodies (ab). Positive PLA signals (yellow spots), DAPI (blue). Scale bar: 10 µm.
