Fig. 5
From: APC/CCDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression
TKTL1 reprograms R5P metabolism. a Progressive overexpression of TKTL1 in HEK293T cells and detection of TKT-TKT homodimers in cell lysates after removal of ectopically expressed TKTL1. The ratio of exogenous TKTL1 to endogenous TKTL1 is shown in the lower panel. b Affinity removal of TKTL1 from lysates of ccRCC tissues and their corresponding adjacent non-cancer tissues were achieved by TKTL1 antibodies; the remaining TKT levels in cleared lysates were determined. c Quantitation of relative TKTL1 levels at different cell cycle phases. The absolute levels of TKTL1 and TKT at G1/S and G2/M phases were calibrated to corresponding recombinant proteins. Data are presented by means ± SEM of three independent experiments. d TKTL1 was progressively overexpressed in HeLa cells (upper panel) and then relative (to endogenous) TKTL1 overexpression levels (middle panel) and corresponding cellular R5P levels were determined (lower panel). Data are shown by means ± SEM of three independent experiments. e Quantitation of relative TKTL1 levels in ccRCC tissues and their corresponding adjacent non-cancer tissues. The absolute levels of TKTL1 and TKT were calibrated to corresponding recombinant proteins. Data are presented by means ± SEM of three independent experiments. f Schematic diagram of the formation of the M1-form R5P from oxidative PPP (black) and M2-form R5P from non-oxidative PPP (green) when [1,2-13C] glucose was used as tracer. g Total R5P, M1 and M2 R5P, and S7P concentrations were determined in TKTL- or GFP-overexpressing HeLa cells and TKT-knockdown HeLa cells (n = 3 biologically independent experiments). The right panel shows the efficiency of TKTL1 overexpression and TKT knockdown. Mean values with SEM are reported, Student’s t test, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant vs the corresponding control group. h. Total R5P, M1 and M2 R5P, and S7P levels were compared between HeLa and TKTL1ΔD-box-knockin HeLa cells. Data are presented as mean ± SEM of three independent experiments, Student’s t test, *p < 0.05, ns not significant vs the corresponding control group. i, j The total (full-length bar), M1 (white-colored bar), and M2 (red colored bar) concentrations of R5P during cell cycle progression in (i) HeLa and (j) TKT-knockdown HeLa cells were determined. Five biological independent experiments are used to present the data. Student’s t test, ns not significant vs the corresponding control group. k Total, M1, and M2 R5P concentrations in HeLa and PFKFB3-knockout HeLa cells. Data are shown by means ± SEM of five independent experiments, Student’s t test, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant vs the corresponding control group. l M2 R5P concentrations in CDH1- or GFP-overexpressing HeLa cells and PFKFB3-knockout HeLa cells. Data are shown by means ± SEM of three independent experiments, Student’s t test, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant vs the corresponding control group. Full-length blots are presented in Supplementary Fig. 10
