Fig 1: KCNQ1OT1 enhanced OS growth by modulating ALDOA expression.a Overexpression efficacy of ALDOA in sh-KCNQ1OT1 OS cells (U-2OS and 143B) was detected by western blotting. b Overexpression of ALDOA partly reversed the suppressed effects of KCNQ1OT1-knockdown on the colony formation capability of U-2OS and 143B cells, values are means ± SD, **p < 0.01 (Student’s t-test). c and d Overexpression of ALDOA partly reversed the induced effect of KCNQ1OT1-knockdown on the apoptosis of OS cells (U-2OS and 143B). Values are means ± SD, *p < 0.05, **p < 0.01 (Student’s t-test). e Morphologic characteristics of the xenograft tumors from U-2OS/sh-Control group, U-2OS/sh-KCNQ1OT1 group and U-2OS/sh-KCNQ1OT1 + ALDOA group (n = 5). Scale bars = 1 cm. f Overexpression of ALDOA partly rescued the inhibitory effects of KCNQ1OT1-knockdown on the growth rate of U-2OS cells in vivo. The volumes of tumors were measured every 5 days; values are means ± SD, *p < 0.05 (Student’s t-test). g The ECAR in OS cells (MNNG-HOS and U-2OS) in different groups (sh-Control, sh-KCNQ1OT1, and sh-KCNQ1OT1 + ov-ALDOA) were determined. Values are means ± SD. h Quantification of the glycolytic capacity from Fig. 3g, **p < 0.01, ***p < 0.001 (Student’s t-test). i The OCR in OS cells (U-2OS and 143B) in different groups (sh-Control, sh-KCNQ1OT1, and sh-KCNQ1OT1 + ov-ALDOA) were determined. Values are means ± SD. j Quantification of maximal respiration from the Fig. 3i, **p < 0.01, ***p < 0.001 (Student’s t-test).
Fig 2: ALDOA was a direct target of miR-34c-5p.a Venn diagram showing the predicted target genes of ALDOA and KCNQ1OT1 from databases (miRDB, TargetScan, and StarBase). b and c The mRNA expression patterns of the ALDOA in predicted target miRNA mimics the treated or Control U-2OS and 143B cells, ***p < 0.001 (Student’s t-test). d Western blot showed the ALDOA expression in the U-2OS and 143B cells transfected with miR-34c-5p mimics or negative control. e The wild-type and the mutated sequences of the ALDOA mRNA 3’-UTR (mutation site: red). f and g The luciferase activity of the OS cells (U-2OS and 143B) in luciferase reporter plasmid containing wild-type ALDOA 3’-UTR (ALDOA-WT) and mutant ALDOA 3’-UTR (ALDOA-MUT) co-transfected with miR-34c-5p mimics or negative control was assessed, **p < 0.01, ***p < 0.001 (Student’s t-test). h and i RIP assays using antibodies against AGO2 or IgG were performed in cellular lysates from U-2OS and 143B cells. qRT-PCR showed the relative enrichment of ALDOA in cells transfected with miR-34c-5p or NC mimics, **p < 0.01, ***p < 0.001 (Student’s t-test).
Fig 3: G6PC3, ALDOA and CS exhibited inverse correlations with mir-122 in specimens from the indicated causes of death and Dual luciferase reporter analysis of CS in 293T cellsA. and B. Up-regulation of G6PC3, ALDOA and CS mRNA in human brain and heart specimens. C. Up-regulation of G6PC3, ALDOA and CS protein in rat brain and heart specimens. D. Dual luciferase reporter analysis of mir-122 and a reporter gene with predicted mir-122 target sequences (wildtype and mutant) in the CS 3'UTR in 293T cells.
Fig 4: MiR-34c-5p inhibition partly rescued the KCNQ1OT1 knockdown effect in OS cells.a Western blot showed the ALDOA expression in U-2OS and 143B cells transfected with miR-34c-5pinhibitor, sh-KCNQ1OT1, or negative control. b miR-34c-5-knockdown partly reversed the inhibitory effects of KCNQ1OT1-knockdown on the colony formation properties of U-2OS and 143B cells and silenced miR-34c-5 in wide type OS cells (U-2OS and 143B) also promoted their proliferation, values are means ± SD, *p < 0.05, **p < 0.01 (Student’s t-test). c and d miR-34c-5-knockdown partly reversed the induce effect of KCNQ1OT1-knockdown on the apoptosis of OS cells (U-2OS and 143B). Knockdown of miR-34c-5 inhibited apoptosis of wide OS cells. Values are means ± SD, *p < 0.05, **p < 0.01 (Student’s t-test). e Altered levels of ECAR in the OS cells (U-2OS or 143B) in different groups (sh-Control, sh-KCNQ1OT1, sh-KCNQ1OT1 + anti-miR-34c-5p and anti-miR-34c-5p). Values are means ± SD. f Altered levels of OCR in the OS cells (U-2OS or 143B) in different groups (sh-Control, sh-KCNQ1OT1, sh-KCNQ1OT1 + anti-miR-34c-5p and anti-miR-34c-5p). Values are means ± SD. g Morphologic characteristics of xenograft tumors from U-2OS/sh-Control group, U-2OS/sh-KCNQ1OT1 group and U-2OS/sh-KCNQ1OT1 + anti-miR-34c-5p group (n = 5). Scale bars = 1 cm. h Anti-miR-34c-5p partly rescued the inhibitory effects of KCNQ1OT1-knockdown on the growth rate of U-2OS cells in vivo. The volumes of tumors were measured every 5 days; values are means ± SD, *p < 0.05, **p < 0.01 (Student’s t-test).
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