Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: BMC genomics

Article Title: Autophagy is involved in the toxicity of the biocontrol agent GC16 against Tetranychus pueraricola (Acari: Tetranychidae) based on transcriptomic and proteomic analyses.

doi: 10.1186/s12864-025-11312-7

Figure Lengend Snippet: Fig. 6 GC16 induced autophagy in Sf9 cells. (A) Representative fluorescence staining images of the LC3 protein in Sf9 cells treated with GC16 or water (for control, designated as CK) for 24 h (scale bar = 20 μm). The region of the LC3 protein that displays positive staining is observed to be red in color. The staining of the nucleus is visible in blue with DAPI. Autophagic vacuoles were observed to be red in color. (B) Average fluorescence intensity of the LC3 protein in Fig. 6A. Data are represented as mean ± SE of three replicates (*P < 0.05, **P < 0.01, and *** P < 0.001 with compared to control). (C) Western blotting analysis for expression of autophagy marker LC3 in Sf9 cells treated with GC16 for 24 h. β-Actin was used as a loading control. The blots were cropped and in supplementary file the full-length blots/gels are presented. (D) A quantitative analysis of western blot was shown by LC3-II/LC3-I ratios in the down panel. All data are represented as mean ± SE of three experiments in triplicate (*P < 0.05, **P < 0.01, and *** P < 0.001 with compared to control)

Article Snippet: Sf9 cells were cultured on coverslips and treated with water (control) or 2.1 mg/mL GC16 for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 at 25 °C for 5 min, and then blocked (AR1009; BOSTER Biological Technology Co., Ltd., Wuhan, China) at 25 °C for 30 min. LC3 expression was detected by incubating the samples with primary antibodies against LC3 (AP0762, Bioworld Technology, Inc., USA) at 4 °C overnight and then with corresponding secondary antibodies (BA1032, BOSTER Biological Technology Co. Ltd, Wuhan, China) at 37 °C for 1 h. Subsequently, the samples were stained with DAPI (C1002, Beyotime Biotechnology, Shanghai, China) for 5 min, and covered with a sealing agent to prevent fluorescence quenching (0100- 01, Southern Biotechnology Associates, Inc., USA).

Techniques: Fluorescence, Staining, Control, Western Blot, Expressing, Marker