Fig 2: NUSAP1 orchestrates the Hippo pathway by stabilizing YAP1 protein. (A) Western blotting analysis to evaluate LATS1/2, YAP1, and YAP1 downstream target genes CTGF and CYR61 in lysates prepared from modified cell lines HGC-27 and BGC823. GAPDH was used as a loading control. (B, C) qRT-PCR analysis of YAP1 and its downstream target genes CYR61, FOXM1, EGFR, and AREG in modified HGC-27 and BGC823 cells. Relative expression is shown over GAPDH mRNA. (D) Western blotting analysis of YAP1 protein in modified BGC823 cells treated with CHX (25 µg/mL) for the indicated times. (E) The line graph shows YAP1 levels normalized to GAPDH at the indicated time points. (F) Immunofluorescence of YAP1 in modified BGC823 cells showing cellular localization. Student’s t test: *p<0.05, **p<0.01.

Fig 3: IRF2BP2 knockdown inhibits tumour growth in vivo. A, Xenograft tumours were harvested at the end of the experiment. After monitoring for 21 days, the growth curves of the xenograft tumours suggested that IRF2BP2 knockdown significantly inhibited tumour growth in vivo (mean ± SD, n = 6). B, The mRNA levels of CTGF were detected in six tumours by RT-qPCR (mean ± SEM, n = 6). C, The protein levels of IRF2BP2 and CTGF were detected in six tumours by Western blotting. *P < .05, **P < .01. CTGF, connective tissue growth factor; GC, gastric cancer; IRF2BP2, interferon regulatory factor 2 binding protein 2; RT-qPCR, real-time quantitative PCR

Fig 4: Inhibition of HDAC6 by TA prevents M2 macrophage polarization via suppressing TGF-ß1/Smad3 signaling in vivo. (A) Western blot analysis showed the levels of TGFßRI, p-Smad3, Smad3, p-TAK1, TAK1, CTGF, and GAPDH in peritoneum from different groups of mice. Expression levels of (B) TGFßRI, (C) p-Smad3, (D) Smad3, (E) p-TAK1, (F) TAK1, and (G) CTGF in different groups were quantified by densitometry and normalized with GAPDH, Smad3, TAK1 respectively. (H) Western blot analysis showed the levels of Twist, Snail, and GAPDH in peritoneum from different groups of mice. Expression levels of (I) Twist and (J) Snail in different groups were quantified by densitometry and normalized with GAPDH. (K) Representative micrographs of immunofluorescence staining of Twist on peritoneum from different groups of mice. (L) Quantization count of Twist-positive cells. Data were expressed as means ± SEM. *P<0.05, **P<0.01, ****P<0.0001. All scale bars = 20 µm. NS: P=0.05.

Fig 5: AuNPs decreased profibrotic cytokine levels in plasma and SW620 tumor.Notes: (A) Plasma level of TGF-ß1 was decreased after treatment with AuNPs (n=6, ***P<0.001). (B) Plasma level of CTGF was decreased (n=6, ***P<0.001). (C) The plasma level of VEGF was reduced after the administration of AuNPs (n=6, ***P<0.001). (D) Histologic images of the effect of AuNPs on TGF-ß1, CTGF, and VEGF expression in SW620 tumor (n=6). Scale bar, 100 µm. AuNPs decreased the expression of TGF-ß1, CTGF, and VEGF (***all P<0.001) in SW620 tumor. Student’s t-test was used for analysis, and error bars indicate SEM.Abbreviations: AuNPs, gold nanoparticles; CTGF, connective tissue growth factor; Ctrl, control; SEM, standard error of the mean; VEGF, vascular endothelial growth factor.