Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: Highly expressed ITGB4 in chemo-resistant BLCA is associated with immune resistance and poor prognosis. A , the intersection of gene sets associated with CAMs and gene sets related to the sensitivity of cisplatin treatment and the expression of PD-L1. B , the GSE268855 dataset analysis after overexpressing ITGB4 . C , TCGA-BLCA dataset correlation analysis, p values are shown in the figure. D and E , transfected HT-1376 and 5637 cells with shRNA for 72 h. The harvested cells were used for Western blot analysis ( D ) and RT-qPCR detection ( E ). F and G , HT-1376 and 5637 cells were transfected with overexpressed ITGB4 plasmid or empty vector for 72 h, and harvested cells were used for Western blot analysis ( F ) and RT-qPCR detection ( G ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. H and I , the IMvigor210 database analysis showed a negative correlation between the expression level of ITGB4 and CD8+ T cells in BLCA ( H ), and the GSE13507 database analysis showed a negative correlation between the expression level of CD8+ T cells and ITGB4 after treated PD-1 blockade therapy ( I ). p values are shown. J , MB49 cells were infected with lentivirus vectors expressing control or Itgb4 shRNAs. After 72 h, cells were harvested for Western blotting analysis. K and L , MB49 cells were infected with shControl or shItgb4 and subcutaneously injected into the left dorsal flank of C57BL/6 mice. Mice with subcutaneous MB49 tumors (n = 5/group) were treated with anti-PD-1 (10 mg/kg) or nonspecific IgG three times. The tumor growth curve was demonstrated in panel ( K ). The tumor volume on day 13 is shown in Figure ( L ). M and N , at the end of treatment, the tumors excised from the mice were dissociated and tumor tissues were harvested for immunofluorescence staining to detect the expression of CD8+ T cells. Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Expressing, Transfection, shRNA, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Infection, Control, Injection, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 activates MEK/ERK signaling in BLCA through tyrosine-1510 phosphorylation. A , KEGG enrichment analysis of ITGB4 RNA-seq in RT4 cells. The p values are shown in the figure. B , the HT-1376 and 5637 cells were transfected with indicated shRNAs for 72 h. Cells were harvested for Western blot analysis ( B , C ). D , HT-1376 and 5637 cells were transfected with wild-type (WT) -ITGB4 plasmid, ITGB4-Y1510A mutated plasmid or empty vector (EV) for 72 h, and the cells were harvested for Western blot analysis. ( D , E ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Phospho-proteomics, RNA Sequencing, Transfection, Western Blot, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 drives the upregulation of PD-L1 and mediates anti-PD-1 resistance via MEK/ERK signaling. A and B , transfected HT-1376 and 5637 cells with indicated shRNA for 72 h. The cells were then treated with either DMSO or LY3214996 (2 μM) for another 24 h. The cells were collected for Western blot analysis ( A ) and RT-qPCR detection ( B ). Data are presented as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C and D , HT-1376 and 5637 cells were transfected with indicated plasmids for 72 h. The cells were then treated with either DMSO or LY3214996 (2 μM) for another 24 h. Cells were collected for Western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. E – G , MB49 cells were transfected with indicated shRNAs for 72 h. The cells were then treated with or without LY3214996 (2 μM) and harvested for Western blotting analysis ( E ). MB49 cells were infected with shControl or shItgb4 and subcutaneously injected into the left dorsal flank of C57BL/6 mice. Mice with subcutaneous MB49 tumor (n = 5/group) were treated with either single-agent anti-PD-1 (10 mg/kg by intraperitoneal injection for three times) or dual-agent LY3214996 (100 mg/kg daily by oral gavage) and anti-PD-1 treatment (10 mg/kg by intraperitoneal injection for three times). The tumor growth curve is shown in Figure ( F ). The tumor volume on day 23 is shown in Figure ( G ). H and I , at the end of treatment, the tumors excised from the mice were dissociated and tumor tissues were harvested for immunofluorescence staining to detect the expression of CD8+ T cells. Data are presented as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Transfection, shRNA, Western Blot, Quantitative RT-PCR, Infection, Injection, Immunofluorescence, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 positively correlated with enfortumab vedotin target NECTIN4. A , the RNA-Seq analysis in the GSE268855 dataset after overexpressing ITGB4 ; B , the correlation analysis in the DepMap dataset. p values are shown in the figure. C , Pan-cancer data analysis in the DepMap database revealed the correlation between ITGB4 and NECTIN4 . p values were shown in the figure. D – G , HT-1376 and 5637 cells were transfected with shITGB4 or shControl for 72 h. The harvested cells were used for Western blot analysis ( D ) and RT-qPCR detection ( E – G ). H – K , HT-1376 and 5637 cells were transfected with overexpressed ITGB4 plasmid or empty vector for 72 h, and harvested cells were used for Western blot analysis ( H ) and RT-qPCR detection ( I–K ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. L , the tissue microarray of bladder cancer stained with ITGB4 and NECTIN4, respectively. The typical IHC images stained with ITGB4 and NECTIN4 are shown in the panel . M , the correlation of these two proteins is shown in the panel , and the p -value is indicated in the figure.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: RNA Sequencing, Transfection, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: BLCA with highly expressed ITGB4 demonstrates favorable therapeutic outcomes toward enfortumab vedotin. A , HT-1376 and 5637 cells were transfected with indicated shRNAs for 72 h. Cells were treated with continuous doses of enfortumab vedotin for 24 h and harvested for IC50 assays. B , HT-1376 and 5637 cells were transfected with the indicated plasmid for 72 h. Cells were treated with a continuous dose of enfortumab vedotin for 24 h and harvested for the IC50 assay. C and D , transfected HT-1376 and 5637 cells with indicated shRNAs for 72 h. The cells were treated with or without enfortumab vedotin (2 nM). Then, harvested cells for CCK-8 assay ( C ) and annexin V-PI assay ( D ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. E and F , HT-1376 and 5637 cells were transfected with indicated plasmid for 72 h, treated with or without enfortumab vedotin (2 nM), then CCK-8 assays ( E ) and annexin V-PI assays ( F ) were performed. Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. G – J , HT-1376 cells were transfected with indicated constructs for 72 h. After purinomycin selection, cells were collected and injected subcutaneously into nude mice. These mice were treated with or without enfortumab vedotin (3 mg/kg, single dose). Schematic for tumor implantation and treatment schedule for HT-1376 cell lines is shown in panel G . Tumor mass and tumor volume growth curves are shown in panel I and panel J . Data presents as mean ± SD with six replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Construct, Selection, Injection, Tumor Implantation

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 plays a vital role in BLCA sequential therapies. A mechanism diagram depicting that the highly expressed ITGB4 in cisplatin-resistant BLCA activated the MEK/ERK pathway through tyrosine-1510 phosphorylation, upregulated the expression level of PD-L1 in BLCA, and caused anti-PD-1 treatment resistance. Meanwhile, the overexpressed ITGB4 was positively correlated to NECTIN4, promoting the sensitivity of BLCA to enfortumab vedotin treatment.

Article Snippet: Immunohistochemistry was performed with primary antibodies against ITGB4 (#21738-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:1000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Phospho-proteomics, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: Highly expressed ITGB4 in chemo-resistant BLCA is associated with immune resistance and poor prognosis. A , the intersection of gene sets associated with CAMs and gene sets related to the sensitivity of cisplatin treatment and the expression of PD-L1. B , the GSE268855 dataset analysis after overexpressing ITGB4 . C , TCGA-BLCA dataset correlation analysis, p values are shown in the figure. D and E , transfected HT-1376 and 5637 cells with shRNA for 72 h. The harvested cells were used for Western blot analysis ( D ) and RT-qPCR detection ( E ). F and G , HT-1376 and 5637 cells were transfected with overexpressed ITGB4 plasmid or empty vector for 72 h, and harvested cells were used for Western blot analysis ( F ) and RT-qPCR detection ( G ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. H and I , the IMvigor210 database analysis showed a negative correlation between the expression level of ITGB4 and CD8+ T cells in BLCA ( H ), and the GSE13507 database analysis showed a negative correlation between the expression level of CD8+ T cells and ITGB4 after treated PD-1 blockade therapy ( I ). p values are shown. J , MB49 cells were infected with lentivirus vectors expressing control or Itgb4 shRNAs. After 72 h, cells were harvested for Western blotting analysis. K and L , MB49 cells were infected with shControl or shItgb4 and subcutaneously injected into the left dorsal flank of C57BL/6 mice. Mice with subcutaneous MB49 tumors (n = 5/group) were treated with anti-PD-1 (10 mg/kg) or nonspecific IgG three times. The tumor growth curve was demonstrated in panel ( K ). The tumor volume on day 13 is shown in Figure ( L ). M and N , at the end of treatment, the tumors excised from the mice were dissociated and tumor tissues were harvested for immunofluorescence staining to detect the expression of CD8+ T cells. Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Expressing, Transfection, shRNA, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Infection, Control, Injection, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 activates MEK/ERK signaling in BLCA through tyrosine-1510 phosphorylation. A , KEGG enrichment analysis of ITGB4 RNA-seq in RT4 cells. The p values are shown in the figure. B , the HT-1376 and 5637 cells were transfected with indicated shRNAs for 72 h. Cells were harvested for Western blot analysis ( B , C ). D , HT-1376 and 5637 cells were transfected with wild-type (WT) -ITGB4 plasmid, ITGB4-Y1510A mutated plasmid or empty vector (EV) for 72 h, and the cells were harvested for Western blot analysis. ( D , E ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Phospho-proteomics, RNA Sequencing, Transfection, Western Blot, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 drives the upregulation of PD-L1 and mediates anti-PD-1 resistance via MEK/ERK signaling. A and B , transfected HT-1376 and 5637 cells with indicated shRNA for 72 h. The cells were then treated with either DMSO or LY3214996 (2 μM) for another 24 h. The cells were collected for Western blot analysis ( A ) and RT-qPCR detection ( B ). Data are presented as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C and D , HT-1376 and 5637 cells were transfected with indicated plasmids for 72 h. The cells were then treated with either DMSO or LY3214996 (2 μM) for another 24 h. Cells were collected for Western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. E – G , MB49 cells were transfected with indicated shRNAs for 72 h. The cells were then treated with or without LY3214996 (2 μM) and harvested for Western blotting analysis ( E ). MB49 cells were infected with shControl or shItgb4 and subcutaneously injected into the left dorsal flank of C57BL/6 mice. Mice with subcutaneous MB49 tumor (n = 5/group) were treated with either single-agent anti-PD-1 (10 mg/kg by intraperitoneal injection for three times) or dual-agent LY3214996 (100 mg/kg daily by oral gavage) and anti-PD-1 treatment (10 mg/kg by intraperitoneal injection for three times). The tumor growth curve is shown in Figure ( F ). The tumor volume on day 23 is shown in Figure ( G ). H and I , at the end of treatment, the tumors excised from the mice were dissociated and tumor tissues were harvested for immunofluorescence staining to detect the expression of CD8+ T cells. Data are presented as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Transfection, shRNA, Western Blot, Quantitative RT-PCR, Infection, Injection, Immunofluorescence, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 positively correlated with enfortumab vedotin target NECTIN4. A , the RNA-Seq analysis in the GSE268855 dataset after overexpressing ITGB4 ; B , the correlation analysis in the DepMap dataset. p values are shown in the figure. C , Pan-cancer data analysis in the DepMap database revealed the correlation between ITGB4 and NECTIN4 . p values were shown in the figure. D – G , HT-1376 and 5637 cells were transfected with shITGB4 or shControl for 72 h. The harvested cells were used for Western blot analysis ( D ) and RT-qPCR detection ( E – G ). H – K , HT-1376 and 5637 cells were transfected with overexpressed ITGB4 plasmid or empty vector for 72 h, and harvested cells were used for Western blot analysis ( H ) and RT-qPCR detection ( I–K ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. L , the tissue microarray of bladder cancer stained with ITGB4 and NECTIN4, respectively. The typical IHC images stained with ITGB4 and NECTIN4 are shown in the panel . M , the correlation of these two proteins is shown in the panel , and the p -value is indicated in the figure.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: RNA Sequencing, Transfection, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: BLCA with highly expressed ITGB4 demonstrates favorable therapeutic outcomes toward enfortumab vedotin. A , HT-1376 and 5637 cells were transfected with indicated shRNAs for 72 h. Cells were treated with continuous doses of enfortumab vedotin for 24 h and harvested for IC50 assays. B , HT-1376 and 5637 cells were transfected with the indicated plasmid for 72 h. Cells were treated with a continuous dose of enfortumab vedotin for 24 h and harvested for the IC50 assay. C and D , transfected HT-1376 and 5637 cells with indicated shRNAs for 72 h. The cells were treated with or without enfortumab vedotin (2 nM). Then, harvested cells for CCK-8 assay ( C ) and annexin V-PI assay ( D ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. E and F , HT-1376 and 5637 cells were transfected with indicated plasmid for 72 h, treated with or without enfortumab vedotin (2 nM), then CCK-8 assays ( E ) and annexin V-PI assays ( F ) were performed. Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. G – J , HT-1376 cells were transfected with indicated constructs for 72 h. After purinomycin selection, cells were collected and injected subcutaneously into nude mice. These mice were treated with or without enfortumab vedotin (3 mg/kg, single dose). Schematic for tumor implantation and treatment schedule for HT-1376 cell lines is shown in panel G . Tumor mass and tumor volume growth curves are shown in panel I and panel J . Data presents as mean ± SD with six replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Construct, Selection, Injection, Tumor Implantation

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 plays a vital role in BLCA sequential therapies. A mechanism diagram depicting that the highly expressed ITGB4 in cisplatin-resistant BLCA activated the MEK/ERK pathway through tyrosine-1510 phosphorylation, upregulated the expression level of PD-L1 in BLCA, and caused anti-PD-1 treatment resistance. Meanwhile, the overexpressed ITGB4 was positively correlated to NECTIN4, promoting the sensitivity of BLCA to enfortumab vedotin treatment.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Phospho-proteomics, Expressing