Journal: Cell Death & Disease

Article Title: KEAP1 promotes anti-tumor immunity by inhibiting PD-L1 expression in NSCLC

doi: 10.1038/s41419-024-06563-3

Figure Lengend Snippet: A HEK293T cells were transfected with SF-EV (empty vector with SF-tag) and SF-KEAP1 (KEAP1 with SF-tag), then cell lysates were pulled down with S-beads and analyzed by mass spectrometry. The protein names and corresponding peptide numbers were listed in the column. B and C Cell lysates of H1299 and H23 were immunoprecipitated with IgG, KEAP1 or PD-L1 antibodies, and endogenous interactions of KEAP1 and PD-L1 were tested by immunoblotting. D and E HEK293T cells were transfected with SF-KEAP1 and PD-L1-SF (PD-L1 tagged with SF), respectively. Cell lysates were pulled down with S-beads, and immunoblotting was performed using PD-L1 or KEAP1 antibodies to test the interaction of KEAP1 and PD-L1. F GST pull-down assay with purified GST-KEAP1 and PD-L1-Flag, and immunoblotting was performed to assess the interaction of KEAP1 and PD-L1. G Immunofluorescence (IF) staining for KEAP1 (green), PD-L1 (red), and nuclei (DAPI, blue) was performed in H1299 and H23 cells. Scale bars, 10 μm. H – J SF-tag full-length and truncated KEAP1 constructs were transfected into HEK293T cells, lysates were pulled down with S-beads to elucidate the interacting domain of KEAP1 to PD-L1.

Article Snippet: The compounds and their sources are as follows: MK-2206 (#S1078, Selleck), ML385 (#S8790, Selleck), DMSO (#PWL064, Meilun), Chloroquine (#S6999, Selleck), MG132(#S2619, Selleck), Cycloheximide (#S7418, Selleck), anti-PD-L1 (#A2004, Selleck), Human IgG1 kappa, Isotype Control (#HY-P99001, MCE), purified GST-KEAP1 protein (#HY-P75899, MCE), purified PD-L1-Flag protein (#HY-P70663, MCE), Coomassie Blue Staining Solution (#P0017F, Beyotime), Reactive Oxygen Species Assay Kit (#S0033S, Beyotime).

Techniques: Transfection, Plasmid Preparation, Mass Spectrometry, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, Immunofluorescence, Staining, Construct

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing