Phospho-CaMK1 alpha (Thr177) Antibody - #AF7381

Fig. 4. Molecular mechanism of cell cycle promotion in NPC. (A) Membrane depolarization in NPCs treated with Control, TBHP, TBHP + Gel, or TBHP + Gel + US (5 min/US cycle over 25 min) was quantified using membrane potential dye DiSC3(5). (B) KEGG enrichment analysis (TBHP + Gel + US versus TBHP) identified the top 10 up-regulated metabolic pathways, with dot-size reflecting metabolite counts, x axis as metabolite ratios, and color indicating enrichment significance (−log10P). (C) GSEA confirmed enrichment of cell cycle–related pathways in TBHP + Gel + US versus TBHP. (D) The mRNA sequencing heatmap showed the normalized expression of cell cycle–related genes in groups. (E) Flow cytometry graph of the cell cycle distribution (G0-G1, S, and G2-M phases) demonstrated a higher proportion of G2-M phase in TBHP + Gel + US versus TBHP + Gel (*P = 0.0321). (F) MTT assay showed enhanced cell viability in TBHP + Gel + US compared to TBHP or TBHP + Gel. (G) The intracellular Ca2+ concentrations (Fluo-4 fluorescence) under US exposure (10 min/ US cycle over 40 min) were determined. (H) Heatmap showed the normalized expression of CAMK related genes in Control-, TBHP-, TBHP + Gel–, and TBHP + Gel + US–treated NPCs, based on mRNA sequencing (n = 3). (I) WB images of CaMK1, P-CaMK1, CDK1/2, and P-CDK1/2 protein expression in NPCs from all groups. (J) Ben treatment reduced G2-M–phase cells (*P = 0.0377 versus TBHP + Gel + US) and suppressed (K) viability compared to TBHP + Gel + US. (L) WB showed TBHP + Gel + US–rescued COL2 expression and inhibited MMP13 up-regulation caused by TBHP, with these effects reversed by Ben. (M) WB confirmed that KN93 blocked CaMK1, P-CaMK1, CDK1/2, and CDK1/2 expression in TBHP + Gel + US. (N) KN93 reduced G2-M–phase cells (*P = 0.0273 versus TBHP + Gel + US), (O) diminished viability, (P) abolished COL2 restoration, and weakened MMP-13 suppression compared to TBHP + Gel + US group. (Q) Schematic: The regeneration is promoted by Ca2+-CaMK1-CDK1/2-cell cycle axis. Data represent means ± SD (three biological replicates). Statistical analysis: one-way ANOVA [(F), (K), and (O)], two-way ANOVA [(E), (J), and (N)].