Journal: bioRxiv

Article Title: PDCD1 expression increases at elevated temperatures

doi: 10.1101/2025.05.22.652424

Figure Lengend Snippet: Up-regulation of PDCD1 transcription after heat shock in human cell lines and mouse tissues can be mediated by HSF1. ( A ) PDCD1 and HSPA1A (positive control for the heat shock response) transcript levels after heat shock (HS) were analyzed by RT-qPCR in human cell lines. ( B ) Pdcd1 and Hspa1 expression after heat shock (HS in vivo ) was analyzed by RT-qPCR in the mouse thymus, spleen, and heart. The readings were normalized against the mean of reference genes and presented versus Ctr in the spleen. Statistical significance versus corresponding Ctr is shown in the tables below. ( C ) HSF1 binding to the PDCD1 promoter was analyzed by ChIP-qPCR in the HL-60 cell line, untreated (Ctr) and after heat shock (HS at 43 °C). HSF1 binding to the negative locus and HSPA1A promoter were shown as negative and positive controls, respectively. ( D ) Western blot analysis of HSF1 levels in HL-60 cells: wild type (WT) and individual clones obtained after CRISPR/Cas9 editing (with HSF1 deficiency: #49, #215, #16, and unaltered: #18). ( E ) HSF1 binding to the PDCD1 promoter (analyzed by ChIP-qPCR) confirming the absence of heat-induced (HS at 43 °C) binding in the HSF1-deficient clone (#16). HSF1 binding to the negative locus and HSPA1A promoter were shown as negative and positive controls, respectively. ( F ) RT-qPCR analysis of PDCD1 and HSPA1A levels in WT and modified HL-60 cells. *** p < 0.0001, ** p < 0.001, * p < 0.05 (significance of differences).

Article Snippet: The cell pellet was resuspended in PBS, and CoraLite® Plus 488-conjugated antibodies were added: 0.4 μg anti-PDCD1 (Proteintech, Cat# CL488-66220; RRID:AB_2883287) or 0.4 μg mouse IgG1 isotype control (Proteintech, Cat# CL488-66360-1; RRID:AB_2934458).

Techniques: Positive Control, Quantitative RT-PCR, Expressing, In Vivo, Binding Assay, ChIP-qPCR, Western Blot, Clone Assay, CRISPR, Modification

Journal: bioRxiv

Article Title: PDCD1 expression increases at elevated temperatures

doi: 10.1101/2025.05.22.652424

Figure Lengend Snippet: Heat shock treatment can increase PDCD1 protein levels which may have functional consequences. ( A ) Western blot analyses in human cell lines and mouse lymph nodes heat-shocked (HS, 1 h at 43 °C; 42.5 °C in the case of NK-92 and 42 °C – mouse tissues) with indicated recovery time. HSPA1 and ACTB or GAPDH were used as positive controls for the HS response and loading controls, respectively. Graphs show the results of densitometric analyses (in the case of mouse tissues, the glycosylated form was not analyzed due to the overlap of the IgG signal detected by the secondary antibody). ( B ) Cell surface staining of PDCD1 was analyzed by flow cytometry in untreated (Ctr) and heat-shocked (HS, 1h at 43 °C and 12h recovery) cells. The counts (y-axis not to scale) from the blue squares areas are shown (as % of parent) in the table on the right. ( C ) Cytotoxicity of untreated and heat shocked NK-92 cells against T47D and CAL120 cells. The absorbance ratio: absorbance of the crystal violet stained target cells after 24 h co-culture with NK-92 was normalized versus absorbance of unattacked cells which is 1.0 (red dashed line). Boxplots represent the median, upper and lower quartiles, maximum and minimum. *** p < 0.0001, ** p < 0.001, *p<0.05 (significance of differences).

Article Snippet: The cell pellet was resuspended in PBS, and CoraLite® Plus 488-conjugated antibodies were added: 0.4 μg anti-PDCD1 (Proteintech, Cat# CL488-66220; RRID:AB_2883287) or 0.4 μg mouse IgG1 isotype control (Proteintech, Cat# CL488-66360-1; RRID:AB_2934458).

Techniques: Functional Assay, Western Blot, Staining, Flow Cytometry, Co-Culture Assay