Journal: Scientific Reports

Article Title: Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

doi: 10.1038/s41598-020-60409-4

Figure Lengend Snippet: DNMT1 protein levels are decreased by combination treatment of DNMTi and HDAC6i. ( A ) Ovarian cancer cell lines were treated as in Fig. and protein was extracted at Day 7 after treatment with IFN-gamma (IFN-γ+) (to assess MHC I and PD-L1 expression, in later figures) or control (IFN-γ -). Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( B ) The TykNu cell line was treated as in ( A ) and the protein synthesis cycloheximide added to cells on Day 7 for 0, 4, and 8 hours at 10 μM as indicated on the blot. Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( C ) Stable knockdowns of the HDAC6 protein were generated in the ID8 Trp53+/+ and Trp53−/− cell lines . Protein was extracted and immunoblots were run for the DNMT1 protein with B-actin as a loading control. Immunoblot membranes were probed for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( D ) Immunoblot showing knockdown of HDAC6 protein with a-Tubulin as a loading control. Protein was extracted and immunoblots were run for the HDAC6 protein with B-actin as a loading control. Immunoblots were probed for HDAC6 (131 kDa) and tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( E ) Ovarian cancer cell lines were treated as in Fig. and RNA was extracted at Day 7. qRT-PCR was run for DNMT1, DNMT3a, and DNMT3b and TBP was used as a reference gene. *p < 0.05 compared to Mock.

Article Snippet: The antibodies used for immunoblotting included: DNMT1 (Sigma, D4692), PD-L1 (ProSci, 4059), HDAC1 (Cell Signaling, 2062), HDAC2 (Cell Signaling, 2540), HDAC6 (Assay Biotech, C0026), acetyl-alpha Tubulin (Cell Signaling, 3971), alpha-Tubulin (Cell Signaling, 3873).

Techniques: Expressing, Control, Isolation, Western Blot, Generated, Knockdown, Quantitative RT-PCR

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: Summary of the tumor immunohistochemical findings in the patients with TCL ( n = 16).

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Immunohistochemical staining, Silver Staining

Journal: Frontiers in Immunology

Article Title: Comprehensive immune profiling reveals that Orbivirus infection activates immune checkpoints during acute T cell immunosuppression

doi: 10.3389/fimmu.2023.1255803

Figure Lengend Snippet: PD-L1 expression increases on CD4 + , CD8 + , and CD14 + cells at day 7 post-BTV infection. Sheep PBMC obtained prior to infection (D0) and at day 3, 7 and 15 post-BTV infection (DPI) were stained with anti-CD4, -CD8, -CD14 and –PD-L1 antibodies and percentage of PD-L1 + cells within each PBMC subpopulation determined by flow cytometry. (A-C) Representative dot-plots at day 0, 3, 7, and 15pi for PD-L1 expression in (A) CD4 + , (B) CD8 + , and (C) CD14 + cells. Fluorescence minus one (FMO) and isotype controls were used to set PD-L1 gates. ( Supplementary Figure 2 ). Percentage of PD-L1 + cells within (A) CD4 + , (B) CD8 + , and (C) CD14 + cells at day 0, 3, 7, and 15pi are plotted. *p<0.05; ** p<0.01; ***p<0.001. One-way ANOVA with Fisher’s LSD post-test.

Article Snippet: For PD-L1 staining, rabbit anti-bovine PD-L1 polyclonal antibody (reference KP1917B-100 from Kingfisher Biotech) was used.

Techniques: Expressing, Infection, Staining, Flow Cytometry, Fluorescence