p stat3 y705  (Cell Signaling Technology Inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor suppressor SLC9A2 inhibits colorectal cancer metastasis and reverses immunotherapy resistance by suppressing angiogenesis

doi: 10.1186/s13046-025-03422-7

Figure Lengend Snippet: SLC9A2 inhibits the migration and invasion of CRC cells by suppressing the STAT3 signaling pathway. ( A ) Pathway enrichment analysis was conducted to compare the SLC9A2-overexpressing CRC cells with the control group. ( B ) Western blot analysis was performed to measure the levels of SLC9A2, STAT3 and p-STAT3 Y705 in HCT116 cells transfected with either siRNA-NC or siRNA-SLC9A2. ( C ) Western blot analysis was performed to measure the levels of SLC9A2, STAT3 and p-STAT3 Y705 in HCT116-Hm cells overexpressing either Vector or SLC9A2. ( D ) HCT116 cells were transfected with either siRNA-NC or siRNA-SLC9A2, followed by treatment with or without 5 µM Stattic for 24 h. After 48 h, Western blot analysis was performed to evaluate the levels of STAT3 and p-STAT3 Y705 . ( E-F ) HCT116 cells were transfected with either siRNA-NC or siRNA-SLC9A2, and the siRNA-SLC9A2 group was subsequently treated with 5 µM Stattic for 24 h. Following a 48-hour incubation, nuclear-cytoplasmic separation was performed, and Western blot analysis was conducted to evaluate the levels of STAT3 and p-STAT3 Y705 ( E ). Immunofluorescence was used to assess the nuclear localization of p- STAT3 Y705 ( F ). ( G ) HCT116-Hm cells were transfected with either vector or SLC9A2. After 24 h, immunofluorescence was used to examine the nuclear localization of p-STAT3 Y705 . ( H ) Migration and invasion assays were conducted on HCT116 cells transfected with either siRNA-NC or siRNA-SLC9A2, and the siRNA-SLC9A2 group was subsequently treated with 5 µM Stattic for 24 h. ( I ) Wound healing assay was conducted on HCT116 cells transfected with either siRNA-NC or siRNA-SLC9A2, and the siRNA-SLC9A2 group was subsequently treated with 5 µM Stattic for 24 h. ( J ) Schematic diagram illustrating SLC9A2 knockdown and overexpression in PDOs from primary CRC and liver metastases to evaluate STAT3 activation. ( K ) Western blot analysis of p-STAT3 Y705 levels in PDOs with SLC9A2 knockdown from primary CRC (left) and overexpression from liver metastases (right). ( L ) Western blot analysis of p-STAT3 Y705 levels in mouse liver metastases as shown in Fig. C. Data in bar graphs indicate mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Multi-group analysis of variance ( B , H , I ), Student’s t test ( C , L )

Article Snippet: The p-STAT3 Y705 inhibitor Stattic (MCE, HY-13818) and the JAK1/2 inhibitor Ruxolitinib (MCE, HY-50856) were utilized in this study.

Techniques: Migration, Control, Western Blot, Transfection, Plasmid Preparation, Incubation, Immunofluorescence, Wound Healing Assay, Knockdown, Over Expression, Activation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor suppressor SLC9A2 inhibits colorectal cancer metastasis and reverses immunotherapy resistance by suppressing angiogenesis

doi: 10.1186/s13046-025-03422-7

Figure Lengend Snippet: Slc9a2 effectively reverses immune resistance in colorectal cancer by inhibiting angiogenesis. ( A ) Flowchart for the establishment of the MC38-R cell line demonstrating resistance to immunotherapy. MC38 cells that developed acquired resistance to immunotherapy were selected under low-dose PD-1 antibody pressure and designated as MC38-R. ( B ) Subcutaneous tumor models in C57BL/6 mice were constructed using both MC38-S (sensitive) and MC38-R cell lines ( n = 4/group). Subsequent intraperitoneal injection of PD-1 monoclonal antibody was conducted to evaluate the efficacy of immunotherapy based on tumor weight. ( C ) Three mice from each group depicted in Fig. 7B were selected for H&E staining, as well as IHC staining for CD8 and CD31, followed by statistical analysis. ( D ) MC38-R cells were employed to establish subcutaneous tumor models in C57BL/6 mice. Upon reaching a tumor volume of 100 mm³, an SLC9A2 overexpression plasmid was intratumorally injected, accompanied by intraperitoneal administration of anti-PD-1 and oral administration of Bevacizumab. ( E-G ) Following four weeks of MC38-R subcutaneous tumor implantation, the mice were sacrificed, and tumors were dissected for photography and weight measurement ( G ). The volume changes of tumor growth were analyzed for each group ( E ), alongside assessments of body weight variations in the mice ( F ). ( H ) VEGFA levels in tumor tissues from each group of mice were quantified using ELISA, with the control group mean normalized to 1 for statistical analysis and graphical representation. ( I-M ) Tumor tissues depicted in Fig. 7E were embedded in paraffin and subjected to H&E staining ( I ), as well as CD31 ( J ) and CD8 ( K ) immunohistochemistry. Additionally, TUNEL ( L ) and EdU ( M ) staining were performed to assess tumor proliferation and apoptosis. ( N ) Granzyme B (left) and IFNγ (right) levels in tumor tissues from each group of mice were quantified using ELISA, with the control group mean normalized to 1 for statistical analysis and graphical representation. ( O ) Map of the slc9a2 pcDNA plasmid and validation of the transfection efficiency in MC38. ( P ) Western blot analysis was conducted to determine the levels of VEGFA and p-STAT3 Y705 . Data in bar graphs indicate mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. Student’s t test ( B , C ), multi-group analysis of variance ( G , H , J , K , L , M , N )

Article Snippet: The p-STAT3 Y705 inhibitor Stattic (MCE, HY-13818) and the JAK1/2 inhibitor Ruxolitinib (MCE, HY-50856) were utilized in this study.

Techniques: Construct, Injection, Staining, Immunohistochemistry, Over Expression, Plasmid Preparation, Tumor Implantation, Enzyme-linked Immunosorbent Assay, Control, TUNEL Assay, Biomarker Discovery, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor suppressor SLC9A2 inhibits colorectal cancer metastasis and reverses immunotherapy resistance by suppressing angiogenesis

doi: 10.1186/s13046-025-03422-7

Figure Lengend Snippet: Ruxolitinib Augments the Effectiveness of Anti-PD-1 Immunotherapy against CRC. ( A ) MC38-R cells were used to establish subcutaneous tumor models in C57BL/6 mice. Starting on day 10, mice received intraperitoneal injections of anti-PD1, followed by oral administration of ruxolitinib starting on day 13. ( B-D ) Mice were sacrificed two weeks later, and tumors were harvested for photography and weight measurement ( B ). Tumor volume changes were analyzed for each group ( D ), along with assessments of body weight variations in the mice ( C ). ( E ) Western blot analysis was conducted to determine the levels of VEGFA and p-STAT3 Y705 in the tumor tissues. ( F-G ) Levels of VEGFA ( F ), IFNγ, and Granzyme B ( G ) in tumor tissues from each group of mice were quantified using ELISA. The mean of the control group was normalized to 1 for statistical analysis and graphical representation. ( H ) Tumor tissues were embedded in paraffin and subjected to H&E staining, as well as immunohistochemical analysis for CD8, CD31, Ki-67 and TUNEL staining. ( I ) Diagram illustrating the proposed mechanism by which SLC9A2 inhibits immune evasion in CRC. Data in bar graphs indicate mean ± SEM, and data were analyzed using multi-group analysis of variance. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: The p-STAT3 Y705 inhibitor Stattic (MCE, HY-13818) and the JAK1/2 inhibitor Ruxolitinib (MCE, HY-50856) were utilized in this study.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control, Staining, Immunohistochemical staining, TUNEL Assay