Fig. 3

Lnc-LFAR1 regulates the expression of extracellular matrix genes in primary HSCs. a Microarray heat map demonstrates clustering of primary HSCs infected with shRNA-control (n = 2), lncRNA-shRNA1 (n = 2) and lncRNA-shRNA3 (n = 2). Hierarchical cluster analysis of significantly differentially expressed mRNAs: bright blue, under-expression; gray, no change; bright red, over-expression. b–e Microarray analyses, such as differential screening b, c, GO d, and KEGG pathway analysis e, were performed. f, g Primary HSCs at day 2 or day 12 were infected with two separated lentivirus-mediated shLFAR1 for 72 h and further treated with 10 ng ml⁻¹ TGFβ for additional 24 h. The expression of lnc-LFAR1, α-SMA, Col1α1, Col1α2, Col3α1, Col4α5, TGFβ, CTGF, TGFβR1, MMP2/9/10 and TIMP1 was detected by qRT-PCR f. The protein levels of α-SMA, Col1α1, TGFβ and MMP2 were detected by western blot. GAPDH was used as an internal control g. h Primary HSCs were transfected with siRNA for lnc-LFAR1 for 48 h and further treated with 10 ng ml⁻¹ TGFβ for additional 24 h. The expression of α-SMA and Col1α1 was determined by confocal microscopy. DAPI-stained nuclei blue; scale bar, 50 μm. i The RNA levels of lnc-LFAR1, α-SMA, Col1α1, Col1α2, Col3α1, Col4α5, TGFβ, CTGF, TGFβR1, MMP2/9/10 and TIMP1 were detected in primary HSCs infected with lenti-lnc-LFAR1 or lenti-control by qRT-PCR. j The protein levels of α-SMA, Col1α1, TGFβ and MMP2 were detected in lnc-LFAR1 over-expressed primary HSCs by western blot. GAPDH was used as an internal control. Uncropped blots of this figure accompanied by the location of molecular weight markers are shown in Supplementary Fig. 18. In f and i, the number of biological replicates for each experiment was n ⩾ 3. Data are presented as means ± s.e.m. P values were analyzed by one-way analysis of variance followed by post hoc comparison in f, and by Student’s t-test in i. */#P < 0.05, **/##P < 0.01. *P < 0.05 vs shRNA-control, #P < 0.05 vs shRNA-control + TGFβ in f; and *P < 0.05 vs LV-control in i