Fig 1: (A) The results of RNA pulldown using SNHG18, its antisense RNA, and a negative control RNA, as analyzed using western blotting for α-enolase (ENO1). The input was the total protein used for RNA pulldown (mean ± SD, n = 3, *P < 0.05, Student's t-test). (B) The results of RIP assays assessed using real-time reverse transcription polymerase chain reaction (RT-PCR) for SNHG18. Quantitative RT-PCR (mean ± SD, n = 3, *P < 0.05, Student's t-test) (C) and western blotting (D) analysis of ENO1 expression in M059K cells silenced for SNHG18, in M059J cells overexpressing SNHG18, and in their control groups (mean ± SD, n = 3; *P < 0.05, Student's t-test). (E) M059K cells silenced for SNHG18 stained for nuclei [2-(4-amidinophenyl)-1H-indole-6-carboxamidine, blue fluorescence], and ENO1 (anti-ENO1 monoclonal antibody followed by fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, green fluorescence) (scale bar = 10 μm). (F) ENO1 expression in nuclear or cytoplasm extracts by western blotting.
Fig 2: SF2312 is a potent inhibitor of Enolase with mixed competitive and non-competitive kinetics(a) Chemical structure of PhAH, SF2312 (1), deoxy-SF2312 (2), with chiral centers indicated. (b) Enolase enzymatic activity was measured in lysates of the D423 cell line overexpressing human ENO1 and ENO2. Inhibitors were incubated with enzyme prior to addition of 5 mM substrate. Enzymatic activity was normalized to that in the absence of the inhibitor (y-axis, log2) and plotted as function of inhibitor concentration (nM, x-axis). Traces for ENO2 inhibited with SF2312, PhAH and deoxy-SF2312 are shown as red, blue and purple circles, respectively. Traces for ENO1 are shown crimson, light blue, and light purple diamonds. Each data point shows the mean ± S.D of N = 6 independent measurements. (c, d) Enolase activity (ENO2) was measured as a function of substrate (2-PGA) and inhibitor (c, SF2312; d, deoxy-SF2312) concentration. Data points show mean ± S.D of N = 5 independent measurements.
Fig 3: UPP1 knockdown increases the antitumor effect induced by 2-DG. (A) UPP1 knockdown enhances the inhibitory effects of 2-DG on cell proliferation. (B-C) UPP1 knockdown reinforces the degree of apoptosis induced by 2-DG. (D-E) UPP1 knockdown significantly decreases OCR and ECAR levels compared to the 2-DG group. (F-G) UPP1 knockdown significantly reduces lactic acid production and glucose uptake in H1975 cells. (H) Expression of ENO1 and LDHA in UPP1 knockdown cells with 2-DG, as determined by western blot. One-way ANOVA (Fig. 4A, B, F, G). The experiments were independently conducted three times. **P<0.01, ***P<0.001 vs. shNC+vehicle. ###P<0.001 vs. shNC+2-DG.
Fig 4: Non-selective toxicity of ENOBlock to ENO1-deleted glioma cells.A representative plate of cancer cells treated with ENOblock is shown in panel a, with quantification shown in panel b A plate treated with SF2312 is shown in panel c, with quantification shown in panel d. Cell were treated for 7 days. (b, d) D423 ENO1-deleted (red diamonds), D423 ENO1-rescued (blue squares) and LN319 ENO1 WT (grey circles) were treated with the indicated doses of ENOblock in panel b (N = 4 ± S.D) or SF2312 in panel d (N = 4 ± S.D). Cell density was quantified by crystal violet and expressed relative to vehicle control as a function of inhibitor concentration. At high concentrations, SF2312 selectively killed D423 ENO1-deleted cells as compared to D423 ENO1-rescued cells (p<0.05, Repeated Measures one-way ANOVA with Bonferroni correction). ENOblock failed to show such selectivity regardless of dose.
Fig 5: Silencing circ-ENO1 prohibited proliferation and facilitated apoptosis in LUAD cells.a Knockdown efficiency of circ-ENO1 by si-circ-ENO1#1, si-circ-ENO1#2, and circ-ENO1#3 in A549 and SPCA1 cells was determined by RT-qPCR analysis, with si-NC as negative control. b, c A549 and SPCA1 cells were transfected with si-NC, si-circ-ENO1#1, or si-circ-ENO1#2 for subsequent assays. CCK-8 and EdU assays were used to detect cell proliferation in each group. d TUNEL assay was used to detect cell apoptosis in each group. e Western blot assay was used to detect the expression levels of cleaved PARP, PARP, cleaved-caspase 3, caspase 3, cleaved-caspase 6, caspase 6, cleaved-caspase 9, and caspase 9, with GAPDH as normalized control. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-ENO1 antibody [EPR10863(B)]