Fig. 3: HDAC2-AS2 released inside extracellular vesicles by HCC cells.

a qRT-PCR analysis of HDAC2-AS2 expression relative to the spike-in control λ polyA⁺ RNA in the TCS of MHCC97L and MHCC97H cells treated with 2 μg/mL RNase A alone or in combination with 0.1% Triton X-100 for 20 min. b Representative electron microscopy images of EVs. Scale bar: 100 nm. c NTA analysis of EVs. d Immunoblotting analysis of CD9, TSG101, GAPDH and VDAC1. Representative images are shown from three independent replicates. e EVs were identified using CD63 antibody, Iso isotype control. f qRT-PCR analysis of HDAC2-AS2 expression relative to spike-in control λ polyA⁺ RNA in the EVs from MHCC97L and MHCC97H cells treated with 2 μg/mL RNase A alone or in combination with 0.1% Triton X-100 for 20 min. UD undetectable. g and h qRT-PCR analysis of the HDAC2-AS2 levels relative to spike-in control λ polyA⁺ RNA in the EVs derived from MHCC97H Rab27A knockdown stable cell line (g), or from MHCC97H after treated with 5 μM GW4869 for 48 h (h). i qRT-PCR analysis of HDAC2-AS2 levels relative to spike-in control λ polyA⁺ RNA in the EVs of MHCC97L and MHCC97H cells that were treated with TGFβ (2.5 ng/mL) for 48 h. j MHCC97H cells were treated with 2.5 ng/mL TGFβ for 24 h. qRT-PCR analysis was performed to assess the expression of related genes relative to β-actin in ctrl and TGFβ-treated cells. k qRT-PCR analysis of HDAC2-AS2 expression relative to spike-in control λ polyA⁺ RNA in the EVs of HepG2 and HepG2.2.15 cells treated with RNase A (2 μg/mL) for 20 min. l Representative electron microscopy images of EVs isolated from plasma of healthy donors (HD, n = 9) and HCC patients (n = 14). Scale bar: 100 nm. m Immunoblotting analysis of CD9, VDAC1 expression in EVs isolated from plasma of HD and HCC patients. Representative images are shown from three independent replicates. n qRT-PCR analysis of HDAC2-AS2 expression relative to spike-in control λ polyA⁺ RNA in the EVs isolated from plasma of HD (n = 9) and HCC patients (n = 14). EVs were purified by ultracentrifugation (b–i, k–n). Error bars represent mean ± SEM for three independent replicates. Statistical analyses were determined by two-tailed Student’s t-test (a, f–k, n). Source data are provided as a Source Data file. EVs extracellular vesicles, HD healthy donors.