anti pd l1  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Reduced YTHDF2 inhibits PD-L1 expression by stabilizing m 6 A-containing SPOP mRNA in colorectal cancer

doi: 10.1038/s41419-026-08615-2

Figure Lengend Snippet: A , B Experimental design for the humanized mouse model ( A ). NSG mice were injected subcutaneously with 1 × 10 6 sh-NC or sh-YTHDF2 RKO cells. 7 days after inoculation, mice received intravenous injection of 3 × 10 6 activated PBMCs. The tumor volumes (left) and mice weights (right) were monitored after cell implanting every 3 days ( n = 5) ( B ). When tumors reached the size limit (2 cm), mice were sacrificed. C Representative images of subcutaneous tumors formed by sh-NC or sh-YTHDF2 RKO cells (left). Tumor masses were weighed after harvesting (right). D GO analysis of significantly upregulated and down-regulated genes identified by RNA-seq data in sh-NC and sh-YTHDF2 RKO cells. The abscissa represented enrichment score, and the ordinate represented pathways. The size of the circles represented the number of genes enriched, and the color depth of the circles represented the significance levels. Red arrows indicated immune-related pathways. E GSEA showed the pathways of differentially expressed genes altered by YTHDF2. The abscissa represented genes arranged in order of fold change, and the ordinate represented enrichment score. F Flow cytometric analysis of the percentage of CD4 + (left) or CD8 + (right) tumor infiltrating-lymphocytes. G Western blots analysis of YTHDF2 and PD-L1 protein expression level in paired subcutaneous tumors (left). GAPDH was included as the loading control. The quantifications of YTHDF2 (middle) and PD-L1 (right) were shown. The hypothesis test for significance between two groups utilized the Student’s t -test. Results were analyzed with ANOVA for three or more groups. N.S., no statistical significance, * P < 0.050, ** P < 0.010, and *** P < 0.001. Values are mean ± SEM. i.v intravenous injection, NC negative control, NSG non-obese diabetic, severe combined immunodeficiency gamma, PBMCs peripheral blood mononuclear cells, s.c subcutaneous injection.

Article Snippet: Immunohistochemistry (IHC) was performed on samples using YTHDF2 antibody (ab246514, Abcam, Cambridge, MA, UK), PD-L1 antibody (13684, CST, BOS, USA), or CD8 (ab235951, Abcam, Cambridge, MA, UK).

Techniques: Injection, RNA Sequencing, Western Blot, Expressing, Control, Negative Control

Journal: Cell Death & Disease

Article Title: Reduced YTHDF2 inhibits PD-L1 expression by stabilizing m 6 A-containing SPOP mRNA in colorectal cancer

doi: 10.1038/s41419-026-08615-2

Figure Lengend Snippet: A qRT-PCR analysis of YTHDF2 and CD274 in siRNAs (NC or si-YTHDF2) transfected CRC cell lines (RKO, upper; LoVo, lower). HPRT1 served as the internal control. B Western blots (left) analysis of YTHDF2 and PD-L1 in siRNAs (NC or si-YTHDF2) transfected CRC cell lines (RKO, upper; LoVo, lower). GAPDH was included as the loading control. The quantifications of YTHDF2 (middle) and PD-L1 (right) were shown. C PD-L1 was visualized by flow cytometry (left) in RKO (upper) and LoVo (lower) cells treated with siRNAs (NC or si-YTHDF2). The quantification was shown (right). D Representative TEM images of purified exosomes from sh-NC and sh-YTHDF2 RKO cells. Scale bar, 100 nm. Western blots analysis of PD-L1 in the purified exosomes from sh-NC and sh-YTHDF2 RKO cells ( E ). The quantification was shown ( F ). CD63 was included as the loading control. G , H Tissue sections from 88 CRC patients were immunohistochemically stained for YTHDF2 and PD-L1. Representative IHC images were shown ( G ). Patients were divided into two groups based on median PD-L1 IHC score. Scale bars, 200 μm and 100 μm. The quantification was shown ( H ). I The correlation between YTHDF2 and CD274 in 620 CRC tissues from the TCGA database. The abscissa represented the expression distribution of CD274 , and the ordinate represented the expression distribution of YTHDF2 . The density curve on the right represented the trend in distribution of YTHDF2 , the upper density curve represented the trend in distribution of CD274 . The value on the bottom represented the correlation P value and correlation coefficient. J , K Immuno-stain of PD-1 (fused to Ig-Fc) on RKO cells treated by siRNAs (NC or si-YTHDF2) ( J ). The green fluorescence represented the binding between PD-1 and PD-L1. Scale bars, 20 μm (upper), 10 μm (magnified). The quantification was shown ( K ). L T cell killing assay of RKO (left) and LoVo (right) cells after YTHDF2 knockdown. The expression correlation of two genes was analyzed with Spearman. The hypothesis test for significance between two groups utilized the Student’s t -test. Results were analyzed with ANOVA for three or more groups. N.S., no statistical significance, * P < 0.050, ** P < 0.010, and *** P < 0.001. Values are mean ± SEM. IHC immunohistochemistry, MFI mean fluorescence intensity, NC negative control, RFU relative fluorescence unit.

Article Snippet: Immunohistochemistry (IHC) was performed on samples using YTHDF2 antibody (ab246514, Abcam, Cambridge, MA, UK), PD-L1 antibody (13684, CST, BOS, USA), or CD8 (ab235951, Abcam, Cambridge, MA, UK).

Techniques: Quantitative RT-PCR, Transfection, Control, Western Blot, Flow Cytometry, Purification, Staining, Expressing, Immunostaining, Fluorescence, Binding Assay, Knockdown, Immunohistochemistry, Negative Control

Journal: Cell Death & Disease

Article Title: Reduced YTHDF2 inhibits PD-L1 expression by stabilizing m 6 A-containing SPOP mRNA in colorectal cancer

doi: 10.1038/s41419-026-08615-2

Figure Lengend Snippet: A qRT-PCR analysis of SPOP expression level in siRNAs (NC, si-YTHDF2, or si-SPOP) transfected CRC cell lines (RKO, upper; LoVo, lower). B Western blots (left) analysis of SPOP and PD-L1 expression level in siRNAs (NC, si-YTHDF2, or si-SPOP) transfected CRC cell lines (RKO, upper; LoVo, lower). The quantifications of SPOP (middle) and PD-L1 (right) were shown. C RNA m 6 A dot blot assay of siRNAs (NC or si-YTHDF2) transfected CRC cell lines (RKO and LoVo). Methylene blue staining served as the loading control. D The decay rate of SPOP mRNA was detected after 2 μmol/L ActD treatment at the indicated times through qRT-PCR. E MeRIP-qPCR (left) and RIP-qPCR (right) analysis of m 6 A levels and YTHDF2-binding ability in sh-NC or sh-YTHDF2 RKO cells. F Schematic showed the generation strategy for the pmirGLO luciferase reporters containing WT (upper) and Mut (lower; A to T) SPOP 3’UTR. G Luciferase activities of the indicated pmirGLO vector were measured in CRC cell lines (RKO, left; LoVo, right) with or without YTHDF2 knockdown. H A schematic diagram depicting the function of the YTHDF2-SPOP-PD-L1 axis in CRC. The hypothesis test for significance between two groups utilized the Student’s t -test. Results were analyzed with ANOVA for three or more groups. GAPDH was included as the loading control in western blots. HPRT1 served as the internal control in qRT-PCR. N.S., no statistical significance, * P < 0.050, ** P < 0.010, and *** P < 0.001. Values are mean ± SEM. 3’UTR 3’ untranslated region, ActD actinomycin D, EV empty vector, F-Luc firefly/renilla luciferase, IgG immune globulin G, IP immunoprecipitation, m 6 A: N 6 -methyladenosine, MeRIP methylated RNA immunoprecipitation, Mut mutated-type; NC: negative control, RIP RNA binding protein immunoprecipitation, WT wild-type.

Article Snippet: Immunohistochemistry (IHC) was performed on samples using YTHDF2 antibody (ab246514, Abcam, Cambridge, MA, UK), PD-L1 antibody (13684, CST, BOS, USA), or CD8 (ab235951, Abcam, Cambridge, MA, UK).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Dot Blot, Staining, Control, Binding Assay, Luciferase, Plasmid Preparation, Knockdown, Immunoprecipitation, Methylation, RNA Immunoprecipitation, Negative Control, RNA Binding Assay