cp 466722  (MedChemExpress)

Journal: Nature Communications

Article Title: ATR/Chk1 signaling induces autophagy through sumoylated RhoB-mediated lysosomal translocation of TSC2 after DNA damage

doi: 10.1038/s41467-018-06556-9

Figure Lengend Snippet: ATR/Chk1 activity is required for UV or MMS-induced autophagy. a and b ATR but not ATM activity is required for UV or MMS-induced LC3 aggregation. HeLa cells with stable expression of mRFP-LC3 pretreated 0.5 h with or without ATM inhibitor CP-466722 (10 μM) or ATR inhibitor VE-821 (1 μM) were subjected to fluorescence microscopy 4 h after UV (80 Jm −2 ) or 6 h after MMS (0.5 mM) treatment ( a ). The numbers of red puncta per cell were quantified using Imaris x64 image analysis software. Five random areas were counted for each experiment and data are presented as mean ± SD of three individual experiments. One-way ANOVA (F (8,126) = 165.07) followed by LSD post hoc test for multiple comparisons ( b ). c and d Chk1 but not Chk2 activity is required for UV or MMS-induced autophagy. HeLa cells with stable expression of mRFP-LC3 pretreated 0.5 h with or without Chk1 inhibitor (0.2 μM) or Chk2 inhibitor-II (4 μM) were treated and subjected to fluorescence microscopy as in panel a . Quantification of red puncta per cell was as in panel b . One-way ANOVA (F (8,126) = 229.17) followed by LSD post hoc test for multiple comparisons. e Treatment with Chk1 inhibitor attenuates UV or MMS-induced augment of LC3-II. HeLa cells pretreated 0.5 h with or without Chk1 inhibitor (0.2 μM) were subjected to immunoblotting assay 4 h after treated with UV (80 Jm −2 ) or 6 h after treated with MMS (0.5 mM). f Knockdown of Chk1 decreases the autophagic flux promoted by UV or MMS treatment. HeLa cells with stable expression of mRFP-GFP-LC3 and control shRNA (sh-Con) or shRNA against Chk1 (sh-Chk1-1 or sh-Chk1-2) were subjected to fluorescence microscopy 8 h after treated with UV (80 Jm −2 ) or 10 h after treated with MMS (0.5 mM). Scale bar, 10 μm

Article Snippet: Mouse anti-actin (1:2000, sc-47778), anti-Myc (1:2000, sc-40), anti-RhoB (1:1000, sc-8048), anti-GST (1:2000, sc-138), anti-UBC9 (1:2000, sc-271057), anti-LAMP1 (1:200, sc-20011), anti-tuberin (1:1000, sc-271314), rabbit anti-Chk1 (1:1000, sc-7898), and rabbit anti-RhoB (1:1000, sc-180) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse anti-HSP60 (1:200, H3524) and anti-FLAG (M2) (1:2000, F1804) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA); rabbit anti-TSC2 (1:1000, #4308), anti-ULK1 (1:1000, #8054), anti-AMPKα (1:1000, #2532), anti-PIAS1 (1:1000, #3550), anti-p70S6 kinase (1:1000, #2708), anti-phospho-p70S6 kinase (1:1000, #9205), anti-phospho-ULK1 (1:1000, Ser757#14202), anti-phospho-AMPKα (1:1000, Thr172#2531), anti-HSP60 (1:1000, #4870), anti-phospho-Threonine (1:1000, #9381), anti-Chk2 (1:1000, #2662), anti-Phospho-Chk1 (1:1000, ser345, #2348), and anti-Phospho-Chk2 (1:1000, Thr68, #2661) antibodies were purchased from Cell Signaling Technology; mouse anti-SQSTM1/P62 (1:1000, ab56416) antibody was purchased from Abcam; rat anti-HA (1:2000, #11867431001) monoclonal antibody was purchased from Roche (Mannheim, Germany); rabbit anti-LC3B/MAP1LC3B (1:1000, NB100-2220) was purchased from Novus; mouse anti-phosphoserine (1:1000, #05-1000), mouse anti-phospho-Histone H2A.X (Ser139)(1:200, #05-636) were purchased from Millipore; rabbit anti-RhoB (1:1000, 14326-1-AP) and rabbit anti-ATR (1:1000, 19787-1-AP) were purchased from Proteintech; inhibitors for Chk1 (#681637) and Chk2 (#220485) were purchased from Calbiochem; inhibitors for ATM CP-466722(#11002) and ATR VE-821(#14731) were purchased from MedChemExpress; chloroquine, methyl methanesulphonate, guanidine hydrochloride, and urea were purchased from Sigma-Aldrich (St Louis, MO, USA); Doxorubicin (HY-15142) and Campathecin (HY-16560) were purchased from MedChemExpress.

Techniques: Activity Assay, Expressing, Fluorescence, Microscopy, Software, Western Blot, Knockdown, Control, shRNA