Fig 2: Construction and identification of NOX4GV 358Lv-RhoA shRNA stable cell lines. (A) The NOX4GV 358Lv cell line was transfected with a lentivirus containing RhoA shRNA. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. Original magnification, 200×; (B) RT-qPCR analysis of Nox4 mRNA in the NOX4 shRNA stable cell line. (C) WB analysis of the NOX4 protein levels in the NOX4GV 358Lv-RhoAi group. (D) Cell proliferation detected by the MTS assay. (E,F) Cell apoptosis levels detected by the TUNEL assay. Black arrows: typical apoptotic cells. Percentage of apoptotic cells = apoptotic cells/cell count per high-power field; 10 different fields of vision were randomly selected from each group. Original magnification, 200×; (G,H) Cell scratch assay. Original magnification, 100×. The data are presented as the means ± SEMs of three replicates. *p < 0.05, versus the NOX4GV 358Lv-con group; #p < 0.05, versus the NOX4GV 358Lv-RhoAi group.

Fig 3: UA intervention effects on the mRNA and protein expression of the NOX4/ROS and RhoA/ROCK1 signaling pathways. (A,B) RT-qPCR analysis of Nox4, Rhoa, Rock1, Alphasma, Col1a1, mmp1, and Timp1 mRNA expression levels in each group. (C–E) WB analysis of the protein expression levels in each group. (F) ROS contents in each group detected by the DCFH-DA assay. The data are presented as the means ± SEMs of three replicates. *p < 0.05, versus the CON group; #p < 0.05, versus the NOX4GV 358Lv group.

Fig 4: The regulatory effects of NOX4 and RhoA as well as the UA intervention effects on the migration and invasion of HSCs after TGF-ß1 treatment (10 ng/mL) for 48 h. (A) Cell proliferation detected by the MTS assay. (B,C) Cell apoptosis levels detected by the TUNEL assay. Black arrows: typical apoptotic cells. Original magnification, 200×. Percentage of apoptotic cells = apoptotic cells/cell count per high-power field; 10 different fields of vision were randomly selected in each group. (D,E) Cell scratch assay. Original magnification, 100×. (F,H) Cell invasion detected by the Transwell invasion system. Original magnification, 200×; (G) MTS indirect cell count curve. r = 0.9959. The data are presented as the means ± SEMs of three replicates. *p < 0.05, **p < 0.01 versus the CON group; #p < 0.05, ##p < 0.01 versus the NOX4GV 358Lv group. ?p < 0.05 versus the NOX4i group.

Fig 5: Construction and identification of stable cell lines. (A) The HSC-T6 cell line was transfected with a lentivirus containing NOX4 shRNA. Cells were observed under an optical microscope (left) and a fluorescence (GFP) microscope (right) in the same field of vision. (B) RT-qPCR analysis of Nox4 mRNA in the NOX4 shRNA stable cell line after TGF-ß1 treatment (10 ng/mL) for 48 h. (C) WB analysis of the NOX4 protein levels in the NOX4i group. (D) The HSC-T6 cell line was transfected with a lentivirus containing RhoA shRNA. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. (E) RT-qPCR analysis of Rhoa mRNA in the Rhoa shRNA stable cell line. (F) WB analysis of the RhoA protein levels in the RhoAi group. (G) The HSC-T6 cell line was transfected with a Nox4 overexpression lentivirus. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. (H) RT-qPCR analysis of Nox4 mRNA in the NOX4 overexpression stable cell line. (I) WB analysis of the NOX4 protein levels in the NOX4GV 358Lv group. Original magnification, 200×. The data are presented as the means ± SEMs of three replicates. *p < 0.05 versus the CON group (lentivirus containing a scramble sequence).