Extended Data Fig. 5: SNHG6 promotes cholesterol biosynthesis and proliferation via mTORC1 signaling in liver cancer cells.
(A) IF staining of mTOR-LAMP2 in Huh7 cells. shScr/shSNHG6 Huh7 cells were subjected to cholesterol starvation and cholesterol replenishment (50 μM, 2 h). Scale bar, 10 μm. (B) Quantification of the colocalization of mTOR-LAMP2 in (A). Data are mean values ± S.D. n = 21 cells per condition. Two-way ANOVA. (C) IB detection of indicated proteins. Vector/ SNHG6 were transfected into HepG2 cells for 48 h, and lysosomes were purified from HepG2 cells, followed by IB detection using indicated antibodies. (D) Co-IP detection of RagA-Raptor interaction. SFB-Raptor, His-RagA plasmid, siCtrl and siRNA-SNHG6 were transfected into HepG2 cells as described conditions for 48 h. (E) Co-IP detection Raptor-RagA interaction in Huh7 cells. shScr/shSNHG6 Huh7 cells were transfected with SFB-Raptor plasmids for 48 h, followed by and cholesterol stimulation (50 μM, 2 h). (F) IB detection of indicated proteins in shScr/shSNHG6 Huh7 cells that were subjected to cholesterol stimulation (50 μM, 2 h). (G,H) IB detection of indicated proteins. HepG2 cells (G)/ Huh7 cells (H) overexpressing vector or SNHG6 were treated with cholesterol stimulation (50 μM, 2 h), followed IB detection for indicated proteins. (I-N) HepG2 cells overexpressing vector or SNHG6 were subjected to rapamycin treatment (100 nM) for 24 h. (I) IB detection of indicated proteins. (J-L) The mRNA levels of SREBF2, SQLE, HMGCS1 were detected by qRT-PCR. (M) Relative cholesterol content in HepG2 cells. (N) MTT assay of HepG2 cells with or without rapamycin. Data are mean values ± S.D. Two-way ANOVA. (O-Q) Vector/SNHG6 was transfected to PMH for 48 h. (O) IB detection of indicated protein expression. (P) qPCR analysis of Srebf2, Hmgcs1, Sqle and SNHG6 expression. (Q) Relative cholesterol concentration in PMH. Data are presented as mean values ± S.D. Two-sided Student’s t-test.
