Fig. 6. LINC01569 functions in GMD through binding to YBX1.

(A) Stretched HSFBs were treated with TA and transfected with the control or LINC01569 siRNAs. Forty-eight hours after transfection, cells were UV cross-linked, and the cytosolic fraction was isolated. IP was performed using an anti-GR antibody or a rabbit IgG as negative control. The immunoprecipitated RNA was extracted and subjected to reverse transcription. The mRNA levels of EGR1, CITED2, and BMP7 were evaluated. (B and C) Cells as described in (A) were subjected to qRT-PCR (B) and Western blotting (C) analyses. qRT-PCR data were normalized to GAPDH mRNA expression. (D) Schematic diagram of predicted YBX1 binding sites within the LINC01569 RNA. (E) Cell lysate of HSFBs in the absence of TA were subjected to an RNA pulldown assay using biotin-labeled LINC01569, followed by Western blotting analysis for the indicated proteins. Proliferating cell nuclear antigen (PCNA) was used as the negative control. (F) Cells as described in (A) were lysed and subjected to RIP using an anti-YBX1 antibody or IgG control. The precipitated RNAs were determined using qRT-PCR primers specific for LINC01569, with hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) as the negative control. (G) HSFBs were transfected using empty vectors or those overexpressing LINC01569 and/or YBX1, followed by treatment with ACD (5 μg/ml). At the indicated time points, RNA was extracted, and mechanosensor expression was measured using qRT-PCR and normalized to the level of GAPDH. For (A), (B), and (G), data are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by Student’s t test for two groups or ANOVA for more than two groups. n.s., not significant.