Fig. 4

RPARP-AS1 elevated the lipid content and lipogenic enzyme expression in OS cells. A U2R and MG63 cells were exposed to various concentrations of glucose for 12 h after 50 min of starvation, and the results were determined by qRT-PCR and normalized to the 0 mM concentration. B, C Intracellular levels of FFAs (B, OD = 550 nm) and TGs (C, OD = 420 nm) were determined in OS cells in response to silencing (MG63) and overexpression (U2R) of RPARP-AS1. D, E Real-time PCR assay revealed the expression of FABP4, CD36, SCD1, ACSL1, ACLY, ACC1, FASN, and MAGL in MG63 or U2R cells stably transfected with si-NC, si-RPARP-AS1-1, si-RPARP-AS1-2 (D) or Vector, RPARP-AS1 (E). F Detection of the expression of lipogenic enzymes and the Akt/mTOR pathway-related proteins in MG63 or U2R cells after silencing or overexpression of RPARP-AS1. The levels of the indicated proteins were determined by Western blot. G Overexpression of SCD1 after silencing of RPARP-AS1. MG63 cells were transfected with pooled siRNAs containing two siRNAs for 24 h and then ectopically HA-tagged SCD1 for another 24 h. The mRNA and protein levels of RPARP-AS1 and SCD1 were detected by qRT-PCR and Western blot. H Ectopic expression of SCD1 counteracted the si-RPARP-AS1-mediated downregulation of FFAs and TGs. After silencing of RPARP-AS1 for 24 h, the MG63 cells were subsequently subjected to SCD1 overexpression. After 48 h, intracellular levels of FFAs and TGs were assayed using the respective assay kits. (I-K) Ectopic expression of SCD1 rescued the si-RPARP-AS1-mediated inhibition of MG63 cell proliferation, migration, and invasion. After silencing of RPARP-AS1 for 24 h, the MG63 cells underwent SCD1 overexpression. Then, cell proliferation, migration, and invasion were assayed by CCK8 assay after 72 h of SCD1 overexpression (I), or suspending cells into 6-well plates or 24-well plates for migration and invasion assay (J), colony formation assay (K) after 24 h of SCD1 overexpression. Scale bar, 100 μm. In A-E and G-K, a two-tailed unpaired Student’s t-test was used to determine statistical significance, with GAPDH or β-actin used as a loading control. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, n.s. = not significant, compared to NC or Vector