Fig 2: miR-144 inhibits ESCC by targeting TIGAR. (A) Dual luciferase reporter gene assays revealed that miR-144 effectively weakened the luciferase activity of TIGAR wild-type by ~50%, while miR-144 did not affect the luciferase activity of TIGAR with mutant type 3′UTR. (B) Cell Counting Kit-8 analysis revealed that the proliferation of EC9706 cells was significantly inhibited when TIGAR was inhibited by si-TIGAR, when compared with the blank control. (C) Tumor colony formation assay revealed that the proliferation of EC9706 cells was significantly inhibited when TIGAR was inhibited by si-TIGAR, when compared with the blank control. (D) Flow cytometric analysis revealed that the apoptosis rate of EC9706 cells was significantly increased when TIGAR was inhibited by si-TIGAR, when compared with the blank control. (E) Quantification of the flow cytometric analysis assay. *P<0.05 vs. blank group. miR, microRNA; TIGAR, TP53-inducible glycolysis and apoptosis regulator; 3′UTR, 3′untranslated region; si, small interfering; NC, negative control.

Fig 3: Urolithin A leads to stabilization of p53 and expression of the p53 target genes p21 and TIGAR. WT and p53−/− HCT 116 cells were treated with 30 µM urolithin A for the indicated periods of time. Total cell lysates were then subjected to immunoblot analysis for p53, p21, TIGAR and tubulin as loading control. Representative pictures and compiled densitometric analyses from three independent experiments are depicted (mean ± SD, *P < 0.05, ANOVA, Tukey’s post-test). The bands of WT and p53 knockout lysates originate from one membrane; the line indicates that interjacent bands of no interest (i.e. standard, empty space) were cut out.

Fig 4: Knockdown of TIGAR by siRNA reduces the antiproliferative properties of urolithin A. (A) HCT116 WT cells were left untreated (ctrl) or transfected with siRNA (200 nM final concentration, scrambled (scr) or TIGAR-specific sequences #1–3, as indicated). After recovery and 24-h treatment with solvent (0.1% DMSO) or 30 µM urolithin A cells were lysed and subjected to western blot analysis for TIGAR, p21 and actin as loading control. Representative blots and densitometric analyses of three biological replicates are depicted. (B) HCT116 WT cells were transfected with the siRNAs (scr/mix#1–3) as indicated. Eight hours after transfection, cells were seeded into 96-well plates and treated with different concentrations of urolithin A. After 72-h incubation, a biomass staining via crystal violet was performed. Residual biomass was plotted against the concentration of urolithin A, and IC50 values were derived from the fitted curve (n = 3, mean ± SD, *P < 0.05; scr versus si). (C) HCT WT cells were transfected with scrambled siRNA or siRNAs (mix #1–3) targeting TIGAR, seeded in 96-well plates and treated with urolithin A and oxaliplatin at the indicated concentrations for 72 h. After biomass staining isobolograms for 50% reduction in biomass were drawn and combinatorial indices determined. Presented data are representative for three independent experiments with consistent results.

Fig 5: Schematic representation of glycolysis impaired by PRMT5 inhibition in STSs. The red and green arrows show the decrease and increase observed upon GSK595 treatment, respectively. Adapted from Grasmann et al. Biochim Biophys Acta Rev Cancer. 2019. G6PC, glucose-6-phosphatase; HK, hexokinase; PFK, phosphofructokinase; FBP1, fructose-1,6-bisphosphatase 1; TIGAR, TP53-inducible glycolysis and apoptosis regulator; PPP, pentose phosphate pathway; GK, glycerol kinase; PCK1, cytosolic isoform of phosphoenolpyruvate carboxykinase; PCK2, mitochondrial isoform of phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PYK, pyruvate kinase; GLUD, glutamate dehydrogenase; GA, glutaminase; LDHD, D-lactate dehydrogenase; and TCA cycle, tricarboxylic acid cycle.