Journal: Scientific Reports
Article Title: SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma
Figure Lengend Snippet: ATM inhibition stimulated SIRT3 activity in DLBCL. ( A ) Expression of SIRT3 targets in DLBCL cell lines inhibited for ATM expression compared to non-target controls. Expression of these targets in ABC DLBCL cell lines (HLY AND SUDHL2) and GCB cell line (SUDHL6) is depicted and quantitated normalized expression is provided below. Protein expression was quantitated using Image J software. Protein expression in shATM-DLBCL cell lines is expressed as relative percentage expression compared to non-target control. Protein abundance data shown here is a representative from triplicate experiments that were initiated from independent cell cultures. ( B ) Effects of SIRT3 on GDH acetylation in ATM-WT and ATM deficient DLBCL cell line HLY. Western blotting was performed using AcK and GDH antibodies on immunoprecipitated (IP) GDH. Protein expression was quantitated using Image J software. Percentage of GDH acetylation normalized to total GDH expression is depicted. VDAC was used as input control loading. The images are representative of two independent experiments. All western blots were run under same experimental conditions. (C) GDH activity assay in GM control cells and DLBCL cell lines expressing nt-shRNA and ATM-shRNA, experiments were repeated three times and data are expressed as mean + SD, asterisks define significant difference *p < 0.05. ( D ) Percentage acetylation of SOD2 as determined by Ack-68-SOD2 antibody in ATM CRISPR knock out (CKO) DLBCL cells compared to WT-ATM. The percentage decrease in acetylated SOD2 expression in ATM-CKO cells was estimated by normalizing the acetylated expression levels to total SOD2 levels in both wild type-ATM and ATM deficient DLBCL groups. The images are representative of two independent experiments. All western blots were run under same experimental conditions. ( E ) Representative density blots of FACs analysis showing percentage ROS accumulation in DLBCL transduced with GFP tagged lentiviral particles expressing nt-shRNA and ATM-shRNA. Experiment was repeated n = 3, data are expressed as mean + SD, asterisks define significant difference p < 0.05.
Article Snippet: Following antibodies were used in the current study: ATM (D2E2, Cell Signaling Technology), SIRT1 (SC-15404, Santa Cruz), SIRT3 (SC-365175, Santa Cruz), FOXO3A (75D8) (Cell Signaling Technology 2497), GAPDH (6C5) (abcam, ab8245), Tom20 (612278 BD Transduction Laboratories), VDAC (D73012, Cell Signaling Technology), Cytochrome C (EPR1327) (abcam, ab133504), H3 (Cell Signaling Technology, 9715), Acetylated lysine (9441, Cell Signaling Technology), MRPL10 (abcam, ab229097), SOD2 (E-10) SC-137254, Santa Cruz), SOD2 (acetyl K68) (EPVANR2, abcam), GDH (D9F7P, Cell signaling Technology), OPA1 (612606, BD Transduction Laboratories), NDUFA9 (abcam, ab14713), DRP1 (BD Transduction Laboratories, 611113), Acetyl CoA synthetase (abcam, EPR8500).
Techniques: Inhibition, Activity Assay, Expressing, Software, Western Blot, Immunoprecipitation, shRNA, CRISPR, Knock-Out, Transduction