a311  (Vazyme Biotech Co)

Journal: Genes & Diseases

Article Title: Nuclear and cytoplasmic USP30-AS1 coordinately regulate breast cancer progression through HnRNPF/p21 and EZH2/c-Myc/p21 axes

doi: 10.1016/j.gendis.2025.101684

Figure Lengend Snippet: USP30-AS1 knockdown inhibits breast cancer cell proliferation both in vitro and in vivo . (A) The knockdown efficacy of USP30-AS1 siRNA and shRNA was validated by qRT-PCR analysis. (B) The effect of USP30-AS1 knockdown on MDA-MB-231 cell proliferation was evaluated by CCK-8 assays. (C) The impact of USP30-AS1 knockdown on the proliferative capacity of MDA-MB-231 cells was assessed by colony formation assays. (D) Flow cytometry analysis was performed to determine the effect of USP30-AS1 knockdown on the cell cycle progression of MDA-MB-231 cells. (E) EdU assays were conducted in MDA-MB-231 cells with USP30-AS1 knockdown. (F) A xenograft tumor experiment was conducted using three MDA-MB-231 cell lines: shNC, shUSP30-AS1#1, and shUSP30-AS1#2. (G) Tumor growth and tumor weight were assessed every two days. (H) The actual photo of tumors for each group was collected at the end of the experiment. (I) Hematoxylin and eosin staining of tumor tissues from nude mice. (J) Immunohistochemistry assays were performed to determine the impact of USP30-AS1 knockdown on Ki67 expression within xenograft tumor tissues.

Article Snippet: The cells were seeded into a 96-well plate (2 × 10 3 /well) and cultured for 0, 24, 48, 72, and 96 h. Cells were treated with 10% CCK-8 solution (MedChemExpress, # HY-K0301) and incubated for 2 h. OD 450 was determined using a microplate reader.

Techniques: Knockdown, In Vitro, In Vivo, shRNA, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Staining, Immunohistochemistry, Expressing

Journal: Translational Oncology

Article Title: Ferroptosis induction enhances anti-PD-1 efficacy in NSCLC via HIF-1α/PD-L1 modulation

doi: 10.1016/j.tranon.2026.102685

Figure Lengend Snippet: Ferroptosis inducers cause cell death and affect PD-L1 expression, which can be inhibited by iron chelators. (A, B) A549 and H1299 cells were treated with erastin or RSL3 at different concentration gradients for 24 h, and cell viability was detected using the CCK-8 kit. Subsequently, cells were co-treated with DFO (100 μM), ferrostatin-1 (2 μM), and the aforementioned ferroptosis inducers at different concentrations for another 24 h, with cell viability re-detected by CCK-8.(C) A549 and H1299 cells were treated with erastin (20 μM) and RSL3 (5 μM), respectively, for 0, 3, 6, 12, 18, and 24 h. PD-L1 expression was measured by qPCR.(D) After A549 and H1299 cells were treated with erastin (20 μM) for 24 h, they were further treated with DFO (100 μM) and ferrostatin-1 (2 μM) alone or in combination. PD-L1 expression in cells was then detected. (E) A549 and H1299 cells were treated with erastin (20 μM) for 24 h, and intracellular ROS was detected by flow cytometry. CCK-8, cell counting kit-8; DFO, deferoxamine; qPCR, quantitative polymerase chain reaction; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species. *** p < 0.001.

Article Snippet: Reagents and Antibodies Cell culture media and reagents: Ham's F-12 K, DMEM, RPMI-1640 (Servicebio), BCA protein assay kit, CCK-8 kit, RIPA lysis buffer (Servicebio), Lovastatin, Fluvastatin, RSL3, Erastin (MCE), deferoxamine mesylate, and Ferrostatin-1 (MCE).

Techniques: Expressing, Concentration Assay, CCK-8 Assay, Flow Cytometry, Cell Counting, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Bta-miR-30f promotes adipogenesis and unsaturated fatty acid accumulation by targeting RAD23B in buffalo intramuscular preadipocytes

doi: 10.1016/j.isci.2025.114578

Figure Lengend Snippet: Bta-miR-30f promoted proliferation and differentiation of buffalo intramuscular preadipocytes (A) Overexpression efficiency of bta-miR-30f mimics after 24 h ( n = 4 biologically independent experiments). (B–D) qRT-PCR was used to detect the adipogenesis genes PPARG , C/EBPα , FABP4 , (C) fatty acid uptake and triglyceride synthesis genes FAT/CD36 , GPAM , LIPN , XDH , and (D) Fatty acid desaturation and elongation genes SCD , FADS1 , FADS2 , and ELOVL5 ( n = 4 biologically independent experiments). (E) After transfection with MNC or bta-miR-30f mimics for 24 h, lipid accumulation was assessed by Oil Red O staining following an 8-day induction period ( n = 3 biologically independent experiments). Scale bars, 200 μm. (F) Histogram shows the quantitation of Oil Red O staining by spectrophotometry at 510 nm ( n = 3 biologically independent experiments). (G) After transfection with MNC or bta-miR-30f mimics for 24 h, triglyceride levels were measured following an 8-day induction period ( n = 4 biologically independent experiments). (H) The EdU assay was performed in intramuscular preadipocytes at 24 h after transfection with MNC or bta-miR-30f mimics. Cells were stained with EdU (red), and cell nuclei were stained with Hoechst (blue) ( n = 10 biologically independent experiments). Scale bars, 200 μm. (I) The percentages of EdU-positive cells ( n = 10 biologically independent experiments). (J) CCK-8 assays were performed to determine cell proliferation ( n = 10 biologically independent experiments). All data are presented as mean ± SE. (A, B, C, D, F, G, I, and J) Statistical analyses using unpaired t test were performed on GraphPad Prism 8.0, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Cell Counting Kit-8 (CCK-8) assay , Vazyme , cat#A311.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Staining, Quantitation Assay, Spectrophotometry, EdU Assay, CCK-8 Assay

Journal: iScience

Article Title: Bta-miR-30f promotes adipogenesis and unsaturated fatty acid accumulation by targeting RAD23B in buffalo intramuscular preadipocytes

doi: 10.1016/j.isci.2025.114578

Figure Lengend Snippet: Bta-miR-30f inhibitor suppressed the proliferation and differentiation of buffalo intramuscular preadipocytes (A) qRT-PCR analysis of bta-miR-30f transcript levels ( n = 4 biologically independent experiments). (B–D) bta-miR-30f knockdown reduced the mRNA levels of genes related to adipogenic ( PPARG , C/EBPα , FABP4 ), (C) triglyceride (TG) synthesis (FAT/ CD36 , LIPN , and XDH ), and (D) fatty acid desaturation and elongation ( SCD , FADS1 , FADS2 , ELOVL5 ) ( n = 4 biologically independent experiments), respectively. (E) After transfection with INC or Inhibitor for 24 h, lipid accumulation was assessed by Oil Red O staining following an 8-day induction period ( n = 3 biologically independent experiments). Scale bars, 200 μm. (F) Histogram shows the quantitation of Oil Red O staining by spectrophotometry at 510 nm ( n = 3 biologically independent experiments). (G) After transfection with INC or Inhibitor for 24 h, triglyceride levels were measured following an 8-day induction period ( n = 4 biologically independent experiments). (H) After transfection with the bta-miR-30f inhibitor or INC for 24 h, the EdU staining assay was performed ( n = 10 biologically independent experiments). Scale bars, 200 μm. (I) Quantification ratio of EdU cells/total cells ( n = 10 biologically independent experiments). (J) CCK-8 analysis ( n = 10 biologically independent experiments). All data are presented as the mean ± SE. (A, B, C, D, F, G, I, and J) Statistical analyses using the unpaired t test were performed on GraphPad Prism 8.0, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Cell Counting Kit-8 (CCK-8) assay , Vazyme , cat#A311.

Techniques: Quantitative RT-PCR, Knockdown, Transfection, Staining, Quantitation Assay, Spectrophotometry, CCK-8 Assay