Fig 2: TK extract induced ERK1/2 phosphorylation in HaCaT cells. HaCaT cells were treated with the indicated concentration of TK extract, and the whole cell lysate proteins were used in western blot analysis. (a) GAPDH was used as a quantitative control. Phosphorylated forms of p38, ERK1/2, JNK, ERK5, and AKT were detected. (b) The p-ERK1/2 band was quantified with densitometric analysis and normalized to GAPDH. (*: p < 0.05, **: p < 0.01.)

Fig 3: Knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC-32 gene abolished GMC proliferation and ECM secretion in Thy-1N rats. LV-shFBXO28, LV-shTRAF6, LV-shERK5, LV-shMZF1 and LV-shRGC-32 were used to silence target genes, and then Thy-1N was induced 4 days later. A, The protein levels of FBXO28, TRAF6, p-ERK5, t-ERK5, MZF1, RGC-32, cyclin D2, PCNA, FN and collagen IV in the renal tissues of rats were detected using IB assay (at 5 hours for FBXO28, TRAF6, p-ERK5 and t-ERK5, at 10 hours for MZF1 and RGC-32, on 7 d for cyclin D2, PCNA, FN and collagen IV). B and C, The numbers of total glomerular cells in different groups of rats on 7 d after Thy-1N induction was observed using Haematoxylin and eosin under LM (Magnification, ×400). D, GMC proliferation and ECM accumulation in different groups of rats on 7 d after Thy-1N induction were evaluated by EM. E, The content of urinary protein (mg/24 h) of rats on day 7 after Thy-1N induction was detected. F, A schematic drawing for the pathway we revealed. **P < 0.01 vs Thy-1N group and LV-shCTR + Thy-1N group. Results from one representative experiment out of three are shown. Data are represented as means ± SD (n = 6 in each group)

Fig 4: The activation and roles of ERK5 in MZF1 and RGC-32 expression as well as GMC proliferation and ECM secretion in response to sublytic C5b-9. A and B, ERK5 phosphorylation in the renal tissues of Thy-1N rats (A) and in the GMCs exposed to sublytic C5b-9 (B) was examined using IB assay. *P < 0.05, **P < 0.01 vs 0 hour time-point. C, ERK5 phosphorylation in the renal tissues of Thy-1N and NRS rats at 5 hours was examined by IB analysis. **P < 0.01 vs NRS groups. D, Rat GMCs were treated in different groups for 5 hours and then ERK5 phosphorylation was detected using IB. **P < 0.01 vs other groups. E and F, Rat GMCs were transfected with pEGFP-N1/ERK5 or pEGFP-N1 for 48 hours and 72 hours and then the expression levels of ERK5, MZF1, RGC-32, cyclin D2, PCNA, FN and collagen IV were detected using IB (E, 48 hours). Meantime, cellular proliferation was also determined with CCK-8 assay (F, 72 hours). *P < 0.05, **P < 0.01 vs pEGFP-N1 group. G and H, Rat GMCs were transfected with shERK5 or shCTR for 48 hours followed by sublytic C5b-9 treatment for different time-points. The expression levels of p-ERK5, t-ERK5 at 5 hours, MZF-1 and RGC-32 at 10 hours, as well as cyclin D2, PCNA, FN and collagen IV at 18 hours were detected using IB (G). Additionally, GMC proliferation at 48 hours was also determined with CCK-8 assay (H). *P < 0.05, **P < 0.01 vs shCTR + sublytic C5b-9 group. Results from one representative experiment out of three are shown. Data are represented as means ± SD (n = 6 in vivo, n = 3 in each group for IB in vitro, n = 5 in each group for CCK-8)

Fig 5: MHJ-627 paradoxically upregulated the expression and phosphorylation of ERK5, possibly due to the stimulatory crosstalk of the ERK1/2 pathway. (a) Western blot image depicting the elevations in ERK5 protein expression and phosphorylation. Effect of MHJ-627 on the protein expression and phosphorylation of ERK5 was measured via Western blot analysis after HeLa cells were treated with compounds for 24 h; (b) quantitation of Western blot showing a paradoxical increase in ERK5 expression; (c) quantitation of Western blot showing a trend of increase in ERK5 phosphorylation; (d) the increase in ERK5 expression and phosphorylation was due to the compensatory action of ERK1/2. GAPDH was used as a loading control. Relative protein expression of the 0 µM control was set as 1. Western blot data were quantified using ImageJ software. Data are presented as mean ± SD. Each experiment was performed in duplicate and repeated at least three times. One-way ANOVA (* p < 0.05) was used for significance. All values were compared to the 0 µM control value to determine the significance.