Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: ERBB2 expression in different molecular types of MIBC. (A) Schematic diagram of consensus types of MIBC, including basal/squamous (Ba/Sq), luminal unstable (LumU), luminal nonspecified (LumNS), luminal papillary (LumP), neuroendocrine-like (NE-like), and stoma-rich. (B) ERBB2 mRNA expression in six MIBC consensus subtypes in the TCGA-MIBC dataset. (C) ERBB2 mRNA expression in luminal and basal cells in CCLE dataset. (D) The luminal and basal-type single-cell clusters were determined by their molecular markers (luminal: FOXA1 and GATA3; basal: CD44 and KRT5), and the distribution of ERBB2 mRNA expression in luminal and basal clusters is shown. (E) The luminal and basal bladder cancer tissues’ IHC were classified by their molecular marker (luminal: KRT20 and GATA3; basal: CD44 and KRT5/6), and ERBB2 protein expression was detected. (F) Quantification of ERBB2 immunohistochemistry protein levels in luminal and basal bladder cancer. (G) Western blot analysis showed the protein expression of ERBB2, KRT20, GATA3, CD44, and KRT5/6 in luminal (HT1376, RT4, and SW780) and basal (5637, J82, and T24) cells. (H) Quantitative chart of ERBB2, KRT20, GATA3, CD44, and KRT5/6 from the Western blot analysis. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.

Article Snippet: Human bladder cancer cells representing luminal type (SW780, HT1376, and RT4) and basal type (5637, T24, and J82) were purchased from Procell Life Science&Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Marker, Immunohistochemistry, Western Blot

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: The STAT3 pathway is compensatively activated in basal cells. (A) The diagram shows the GESA result based on the DEGs between basal/squamous and the other three luminal consensus subtypes. (B) The Venn diagram shows the common altered pathways from the GSEA results based on the DEGs determined between basal and luminal types of four molecular classification systems. (C, D) The heatmap shows the expression of genes positively correlated with STAT3 pathways in basal and luminal bladder cancer tissues and cell lines. (E) STAT3 mRNA expression levels in consensus types of TCGA-MIBC. (F) STAT3 mRNA expression levels in luminal and basal cells. (G) STAT3 protein levels in luminal and basal cells were detected by Western blot analysis. (H) The diagram shows the GSEA results based on the DEGs between RC48-exposed SW780 and control. The top 10 activated pathways are shown. (I) The diagram shows the correlation between ERBB2 and STAT3 in mRNA levels, with the PCC used for analysis. (J, K) In ERBB2-silenced luminal cells and ERBB2-overexpressing basal cells, Western blot analysis was performed to measure the expression changes of ERBB2, STAT3, and pSTAT3 (Tyr705). (L) After exposure to RC48 (HT1376 and SW780), the proteins ERBB2, STAT3, and pSTAT3 (Tyr705) were detected by Western blot analysis. (M) Control and RC48-exposed SW780 and HT1376 cells, or ERBB2-overexpressing T24 and 5637 cells, were fixed and incubated with antibodies against pSTAT3 (Tyr705) for immunofluorescence analysis. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.

Article Snippet: Human bladder cancer cells representing luminal type (SW780, HT1376, and RT4) and basal type (5637, T24, and J82) were purchased from Procell Life Science&Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Incubation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: STAT3 silencing and inhibition can increase the sensitivity of basal cells to RC48. (A) Western blot analysis validated the STAT3/pSTAT3 (Tyr705) protein expression in STAT3-silenced basal cells (T24 and 5637). (B) The percentage of surviving cells after exposure to RC48 (5 μg/ml) in STAT3-silenced basal cells and their corresponding control cells. (C) Colony formation assay revealed cell growth after exposure to RC48 (5 μg/ml) in STAT3-silenced cells and their corresponding control cells. (D) Dose–response assay of three STAT3 inhibitors in T24 and 5637 cells. (E) Western blot analysis revealed STAT3 and pSTAT3 (Tyr705) expression after exposure to different concentrations of ART. (F) The percentage of surviving cells after exposure to different concentrations of ART in basal (T24 and 5637) and luminal (SW780 and HT1376) bladder cancer cells. (G, H) Cell viability was assessed by combining different concentrations of RC48 and ART in T24 and 5637 cells. (I, J) The CCK-8 proliferation assay was performed on T24 and 5637 cells exposed to saline, a single drug (RC48 or ART), or a combination of both drugs. (K) The colony formation assay was conducted on T24 and 5637 cells exposed to saline, a single drug (RC48 or ART), or a combination of both drugs. **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001; ns, not significant.

Article Snippet: Human bladder cancer cells representing luminal type (SW780, HT1376, and RT4) and basal type (5637, T24, and J82) were purchased from Procell Life Science&Technology Co. Ltd. (Wuhan, China).

Techniques: Inhibition, Western Blot, Expressing, Control, Colony Assay, CCK-8 Assay, Proliferation Assay, Saline