Journal: bioRxiv
Article Title: DNA-PKcs regulates myogenesis in an AKT-dependent manner independent of induced DNA damage
doi: 10.1101/2022.06.23.497315
Figure Lengend Snippet: a-g) Myogenic cells treated with inhibitor of kinases. C2C7 cells were grown until at 2.5 dps, following the scheme in Fig. S3d, and treated with inhibitors (alone or in combination, at the indicated doses) until 5 dps (panels a-c, e). SCs (panel d) were treated following the scheme in , i.e. treated at 5.5 dps and collected at 7.5dps. Control 1, untreated at 2.5 dps (or 5.5 dps, panel d); control 2, untreated at 5 dps (or 7.5 dps, panel d); vehicle, untreated (DMSO) at 5 dps (or 7.5 dps, panel d). a) mRNA fold changes of Myog expression (n=3-6) in C2C7 cells, mean ± SD normalised to TBP-housekeeping gene, black columns, left part of the histogram. In the righ part of the histogram, grey hatched columns indicate the same samples in the presence of (10 µg of insulin). Significance by Ordinary 1-way ANOVA (F=9.943, DFn=7, DFd=30, p<0.0001) with post-hoc Tukey’s multiple comparisons test, significant p-values are indicated on the histogram. b) Western blot (WB) of C2C7 cells treated with increasing doses of DNAPKi (NU7441), or 1 µM PI3Ki (ZSTK474), or 5 µM AKTi (MK2206); two bands of MyoD are present , and a band shift is observed at the highest doses of DNAPKi. c ) WB of Myogenin and p-Akts in C2C7 cells at increasing concentrations of DNAPKi. Two vehicle lanes (0.05% and 0.1%) correspond to the volume of DMSO used with 2.5 µM and 10 µM DNAPKi, respectively. d) WB of Myogenin, Akts, and p-Akts of SCs in culture until 5.5 dps before treatment with increasing concentrations of DNAPKi for two more days. e) WB of C2C7 cells treated with 10 µM DNAPKi, PI3Ki (LY294002), and 5µM AKTi, alone or in combination (left panels), and cultured in the presence or in the absence of inhibitor(s) until 5 dps; in panels b and e, vehicle contains 0.1% DMSO. For WBs, GAPDH was used as reference housekeeeping protein. f) C2C7 cell number and g) WB of Myogenin and Akt2, upon growth and differentiation in the presence and in the absence of various inhibitors and insulin (green columns). As expected, insulin did not improve proliferation of cells treated with PI3Ki, because PI3Ks are inhibited. In WB GAPDH was used as reference housekeeeping protein. Significance by 2-way ANOVA (F=4.66, DFn=18, DFd=69, p<0.0001), with post-hoc Tukey’s multiple comparisons test vs. corresponding vehicle conditions (vehicle or vehicle + insulin) when not specifically indicated (P values shown on the histogram). h-l) Analysis of Myogenic factors and Akt kinases upon shRNA-dependent silencing of Akt1, Akt2 or DNA-PKcs. h ) Scheme and readouts of the experiment: SCs tested at 2.5 dps and 5.5 dps (control 1 and control 2, respectively) in the absence of puromycine (selection) treatment; SCR (scramble RNA) or shAkt1, shDNA-PKcs (2 independent clones), and shAkt (2 independent clones) at 5.5 dps upon puromycine selection. i ) WB of myogenic factors (differentiation markers: MHC (MF20) and Myogenin; myoblast marker MyoD, and stem cell marker Pax7), kinases (Akt1, Akt2, phospho-Akt1, phosphor-Akt2, phospho-Akts, and DNA-PKcs), and the cell cycle marker Cyclin A2. MyoD levels remained relatively high in SCR, perhaps as a consequence of the lentiviral and selection procedure. Normalisation of protein levels with the reference protein GAPDH upon quantification of bands with Imagelab (BIORAD) for j ) myogenic factors, k ) individual Akt kinases and their phosphorylated forms, and global p-Akts, and l) DNA-PKcs. Uncropped gels are shown in Fig S5.
Article Snippet: The following antibodies have been used in this study: mouse monoclonal α-Pax7 (AB_528428), mouse monoclonal α−Myogenin (F5D, AB_2146602), mouse monoclonal α-Myosin Heavy chain, MHC (MF20, AB_2147781) and mouse monoclonal α-embryonic MyHC (F1.652, AB_528358) antibodies from Developmental Studies Hybridoma Bank; rabit polyclonal α-Myogenin (sc-576), rabbit polyclonal α-MyoD (sc-304), Rabbit polyclonal α-GAPDH (sc-25778) from Santa Cruz; mouse monoclonal α-Akt2 (5239), rabbit polyclonal α-phospho-Akt2 (Ser474) (8599), rabbit polyclonal α-Akt1 (C73H10), rabbit polyclonal α-phospho-Akt1 (Ser473) (9018), rabbit polyclonal α-phospho-Akt (9271), rabbit polyclonal α-H2AX (2595) from Cell Signaling technology; mouse monoclonal α-phospho-H2AX(Ser139) (05-636) from Millipore; rabbit polyclonal α-53BP1 (NB100-304) from Novusbio; chicken polyclonal-α-GFP (ab13970), rabbit polyclonal-α-DNAPKcs (ab70250) rabbit monoclonal α-Cyclin A2 (ab181591) from abcam; Rabbit α-Laminin (L9393), Propodium Iodide (P4864) from Sigma; goat α-rabbit fluor 555 (A21428), goat α-rabbit fluor 488 (A11034), goat α-mouse fluor 488 (A11029), goat α-mouse fluor 555 (A21425), goat α-mouse fluor cy5 (A10524), goat α-chicken fluor 488 (A11039), goat α-mouse-HRP (31430), goat α-rabbit-HRP (31460) from Invitrogen; hFAB Rhodamine Anti-GAPDH IgG (12004168), Goat α-mouse Starbright blue 700 (12004159) from Biorad; goat α-rabbit CF770 (10078-1) from Biotium; FITC Annexin V Apoptosis Detection kit I (556547) from BD Biosciences.
Techniques: Control, Expressing, Western Blot, Electrophoretic Mobility Shift Assay, Cell Culture, shRNA, Selection, Clone Assay, Marker
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![Quantitative PCR (histogram) analyses of several DNA fragments in the Myogenin promoter from ChIP assays with either (a) anti-MyoD or (b) anti-DNA-PKcs antibodies in differentiating cells Significance by Unpaired t-test (F=2.9, DFn=2, DFd=2). (c) Scheme not at scale of primer positions on the the Myog promoter wih the respective distances from the initiation of transcription site (+1), and summary of positive amplifications in light blue and statistically significant in dark blue (MyoD) or orange (DNA-PKcs) whereas crossed boxes in pink indicate no amplification, in the absence and in the presence of irradition: n=3 unless diffferently indicated. Immunostaining with (d) MyoD, (e) p300, and (f) DNA-PKcs of immunoprecipitation with DNA-PKcs and Akt1 (IgG rabbit, IgR), and Akt2 (IgG Mouse, IgM) pull-down. Input, empty IgG rabbit (IgGR, for control of DNA-PKcs and Akt1) and IgGM (IgG Mouse, IgM, for control of Akt2) in C2C7 cells at 5dps. g) Schematic representation of the interactions (dotted lines) observed in immuniprecipiatation experiments; dotted lines of the same colour indicate interactions detected with pulldown of the same protein. Protein sizes are not at scale. Histone ψH2AX immunostaining evaluated per cell in the absence [control, vehicle (0.2% DMSO)] and in the presence of 10 µM DNAPKi at different time points h) in non-irradiated cells and i ) irradiated C2C7 cells. Total ψH2AX immunofluorescence intensity was assessed with ImageJ of single cells after acquisition with Cell Voyager CV1000, confocal scanner box (Yokogawa). N= 40-80 cells from 3 independent experiments. Significance by 1-way ANOVA (Non-irradiated: F=16.37, DFn=11, DFd=588, p<0.0001. Irradiated: F=53.68, DFn=11, DFd=457, p<0.0001), with post-hoc Tukey’s multiple comparisons test. P-values are indicated on the histogram. j) WB of Myogenin, global histone H2AX, ψH2AX, and the housekeeping GAPDH protein in C2C7 cells at 4.5 dps. The ratio ψH2AX/H2AX is shown in the bottow line after superimposition of the (700 nm (Goat α-mouse Starbright blue 700) and 800nm (goat α-rabbit CF770) signal with the imagelab program of ChemiDoc MP imaging system (Biorad). The highest doses of DNAPKi results in slightly reduced levels of ψ-H2AX, compatibly with H2AX being also phosphorylated by DNA-PKcs. k ) Enumeration of Myogenin + and Myogenin - cells upon immunostaining per image in the presence and in the absence of inhibitors of DNA-PKcs and caspases (4 images/condition analysed, n=1). Uncropped gels are shown in Fig S5.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_15/10__1101_slash_2022__06__23__497315/10__1101_slash_2022__06__23__497315___F3.large.jpg)
