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: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 5 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK1 is blocked. (a) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in Nec-1-treated HK2 cells. (b) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK1 was blocked, wogonin failed to further suppress the protein levels of KIM-1. (c) Real-time PCR showed that when RIPK1 was blocked, wogonin failed to further decrease IL-6 in mRNA level. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. *Po0.05, **Po0.01, ***Po0.001 compared with the control. ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01, $$

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 6 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK3 is disrupted. (a) Western blot analysis and quantitative data of RIPK3 in HK2 cells. Results showed that RIPK3 was downregulated by transfection of RIPK3 shRNA plasmid. (b) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in RIPK3 knockdown HK2 cells. (c) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK3 was knockdown, wogonin failed to further decrease the protein levels of KIM-1. (d) Real-time PCR identified that when RIPK3 was disrupted, wogonin failed to further decrease mRNA levels of TNF-α and IL-6. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. **Po0.01,***Po0.001 compared with the control. #Po0.05, ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01,$$$Po0.001 compared with RIPK3 EV group. Cis, cisplatin; Wog, wogonin; EV, empty vector; KD, knockdown.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 9 Wogonin prevents RIPK1-mediated necroptosis in cisplatin nephropathy. (a) Electron microscope. Results clearly demonstrated that wogonin largely reduced nuclear structure damage and abnormal cell organelle content in injured kidney. (b) Western blot analysis of RIPK1/RIPK3/MLKL signaling. Results showed that treatment of wogonin significantly suppressed the activation of RIPK1/RIPK3/MLKL axis in cisplatin-injured kidney. Data represent the mean ± s.e.m. for 6–8 mice. ***Po0.001 compared with control. ##Po0.01, ###Po0.001 compared with model. Cis, cisplatin; Wog, wogonin.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Microscopy, Western Blot, Activation Assay, Control