This section provides supplemental information and figures related to the study of interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Key points include: 1. **Supplemental Figures**: - **Figure 1**: nCounter mRNA expression levels of interferon receptors in three studied cell lines. - **Figure 2**: Functional validation of three stable reporter cell lines (DS244, DS263, DS381) using firefly and Renilla reporters to assess promoter induction and reporter expression. - **Figure 3**: Deletion of CRKL, PIK3CD, and p38 does not affect PD-L1 expression. - **Figure 4**: Validation of shRNA screen hits using qPCR. - **Figure 5**: ChIP analysis of IRF1 and STAT3 binding at the PD-L1 and PD-L2 promoters. - **Figure 6**: Transient luciferase reporter assay comparing PD-L2 promoter activity in M381 and M244 cells. - **Figure 7**: Surface flow cytometry analysis of PD-L1 and PD-L2 expression using a siRNA transduction approach. 2. **Supplemental Experimental Methods**: - **Construct Cloning**: Details on PCR amplification, cloning, and validation of constructs for transient and stable reporter cell lines. - **RNA Sequencing (RNA-seq)**: Methods for RNA-seq data generation, mapping, and differential gene expression analysis. - **Gene Set Enrichment and Transcription Factor Analysis**: Methods for gene set enrichment and transcription factor binding motif analysis. 3. **Supplemental Tables**: - **Table 1**: Primers used in cloning and ChIP methods. - **Table 3**: List of immune cell marker genes used for RNASeq analysis. - **Table 4**: List of immune cell marker genes for immunohistochemistry analysis. 4. **Supplemental References**: - Citations for key studies and references used in the research. This supplemental material supports the main findings and provides detailed methods and additional data to enhance the understanding of interferon receptor signaling pathways and their role in PD-L1 and PD-L2 expression.This section provides supplemental information and figures related to the study of interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Key points include: 1. **Supplemental Figures**: - **Figure 1**: nCounter mRNA expression levels of interferon receptors in three studied cell lines. - **Figure 2**: Functional validation of three stable reporter cell lines (DS244, DS263, DS381) using firefly and Renilla reporters to assess promoter induction and reporter expression. - **Figure 3**: Deletion of CRKL, PIK3CD, and p38 does not affect PD-L1 expression. - **Figure 4**: Validation of shRNA screen hits using qPCR. - **Figure 5**: ChIP analysis of IRF1 and STAT3 binding at the PD-L1 and PD-L2 promoters. - **Figure 6**: Transient luciferase reporter assay comparing PD-L2 promoter activity in M381 and M244 cells. - **Figure 7**: Surface flow cytometry analysis of PD-L1 and PD-L2 expression using a siRNA transduction approach. 2. **Supplemental Experimental Methods**: - **Construct Cloning**: Details on PCR amplification, cloning, and validation of constructs for transient and stable reporter cell lines. - **RNA Sequencing (RNA-seq)**: Methods for RNA-seq data generation, mapping, and differential gene expression analysis. - **Gene Set Enrichment and Transcription Factor Analysis**: Methods for gene set enrichment and transcription factor binding motif analysis. 3. **Supplemental Tables**: - **Table 1**: Primers used in cloning and ChIP methods. - **Table 3**: List of immune cell marker genes used for RNASeq analysis. - **Table 4**: List of immune cell marker genes for immunohistochemistry analysis. 4. **Supplemental References**: - Citations for key studies and references used in the research. This supplemental material supports the main findings and provides detailed methods and additional data to enhance the understanding of interferon receptor signaling pathways and their role in PD-L1 and PD-L2 expression.