mouse monoclonal α akt2 Search Results


mouse monoclonal α akt2  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc mouse monoclonal α akt2
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 <t>Akt2,</t> 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.
Mouse Monoclonal α Akt2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/Akt2+Mouse+mAb/bio_rxiv__2022__06__23__497315-264-54-81
Average 94 stars, based on 1 article reviews
mouse monoclonal α akt2 - by Bioz Stars, 2026-10
94/100 stars
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rabbit polyclonal α phospho akt2 ser474  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc rabbit polyclonal α phospho akt2 ser474
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 <t>Akt2,</t> 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.
Rabbit Polyclonal α Phospho Akt2 Ser474, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/Phospho-Akt2+(Ser474)+Rabbit+mAb/bio_rxiv__2022__06__23__497315-264-58-81
Average 95 stars, based on 1 article reviews
rabbit polyclonal α phospho akt2 ser474 - by Bioz Stars, 2026-10
95/100 stars
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goat α mouse starbright blue 700  (Bio-Rad)
96
Bio-Rad goat α mouse starbright blue 700
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 <t>Akt2,</t> 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.
Goat α Mouse Starbright Blue 700, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/StarBright+Blue+700+Goat+Anti-Mouse+IgG/bio_rxiv__2022__06__23__497315-264-160-167
Average 96 stars, based on 1 article reviews
goat α mouse starbright blue 700 - by Bioz Stars, 2026-10
96/100 stars
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hfab rhodamine anti gapdh igg  (Bio-Rad)
95
Bio-Rad hfab rhodamine anti gapdh igg
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 <t>Akt2,</t> 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.
Hfab Rhodamine Anti Gapdh Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/hFAB+Rhodamine+Anti-GAPDH+Primary+Antibody/bio_rxiv__2022__06__23__497315-264-155-167
Average 95 stars, based on 1 article reviews
hfab rhodamine anti gapdh igg - by Bioz Stars, 2026-10
95/100 stars
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fitc annexin v apoptosis detection kit i (556547)  (Becton Dickinson)
90
Becton Dickinson fitc annexin v apoptosis detection kit i (556547)
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 <t>Akt2,</t> 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.
Fitc Annexin V Apoptosis Detection Kit I (556547), supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/fitc+annexin+v+apoptosis+detection+kit/bio_rxiv__2022__06__23__497315-264-174-183
Average 90 stars, based on 1 article reviews
fitc annexin v apoptosis detection kit i (556547) - by Bioz Stars, 2026-10
90/100 stars
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rabbit polyclonal α h2ax  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit polyclonal α h2ax
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 <t>Akt2,</t> 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.
Rabbit Polyclonal α H2ax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/Histone+H2A%2EX+Antibody/bio_rxiv__2022__06__23__497315-264-76-81
Average 96 stars, based on 1 article reviews
rabbit polyclonal α h2ax - by Bioz Stars, 2026-10
96/100 stars
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rabbit polyclonal α phospho akt1 ser473  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit polyclonal α phospho akt1 ser473
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 <t>Akt1,</t> 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.
Rabbit Polyclonal α Phospho Akt1 Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+%CE%B1+akt2/Phospho-Akt1+(Ser473)+XP+Rabbit+mAb/bio_rxiv__2022__06__23__497315-264-67-81
Average 96 stars, based on 1 article reviews
rabbit polyclonal α phospho akt1 ser473 - by Bioz Stars, 2026-10
96/100 stars
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mouse α cd68  (Bio-Rad)
96
Bio-Rad mouse α cd68
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 <t>Akt1,</t> 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.
Mouse α Cd68, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse monoclonal p21  (Santa Cruz Biotechnology)
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Protein expression of <t>p21</t> in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).
Mouse Monoclonal P21, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Protein expression of <t>p21</t> in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).
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Protein expression of <t>p21</t> in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).
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Protein expression of <t>p21</t> in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).
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Image Search Results


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.

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

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.

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: 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.

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: Real-time Polymerase Chain Reaction, Amplification, Immunostaining, Immunoprecipitation, Control, Irradiation, Immunofluorescence, Imaging

a) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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: Activation Assay, Phospho-proteomics

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.

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

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.

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: 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.

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: Real-time Polymerase Chain Reaction, Amplification, Immunostaining, Immunoprecipitation, Control, Irradiation, Immunofluorescence, Imaging

a) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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: Activation Assay, Phospho-proteomics

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.

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

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.

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: 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.

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: Real-time Polymerase Chain Reaction, Amplification, Immunostaining, Immunoprecipitation, Control, Irradiation, Immunofluorescence, Imaging

a) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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) Scheme of cross-sectional area of uninjured (the comparable cross-sectional surface area of uninjured TA muscle as WT: 5.4 ± 1.2mm 2 and SCID: 5.5 ± 1.1mm 2 ), one-time injured TA at 5dpi and 29dpi, and 3 times injured (every 29 days) TA muscle at 89dpi from WT and SCID mice. Big cantaloupe circles represent TA muscle, orange circles represent individual fibers and green half circles represents SCs. The scheme, which is not at scale, aims to summarize the difference in SC number, fiber number, and size in tested conditions. b) Left panel: schematic representation of the activation of Myogenin by DNA-PKcs through phosphorylation of Akt2. The activation of other myogenic and differentiation factors (MyoD and MHC) by DNA-PKcs through Akt2 and/or Akt1 is also shown. The larger the arrow the stronger the effect. The minor role of PI3Ks is indicated with a white dotted arrow. In the absence of DNA-PKcs, PI3Ks in large part activates Myogenin (right panel).

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: Activation Assay, Phospho-proteomics

Protein expression of p21 in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).

Journal: The Journal of Physiology

Article Title: Thyroid hormone receptor function in maturing ovine cardiomyocytes

doi: 10.1113/JP276874

Figure Lengend Snippet: Protein expression of p21 in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).

Article Snippet: The antibodies used were: phospho‐p44/42 MAPK (ERK1/2) rabbit polyclonal antibody, which detects endogenous levels of p44 and p42 MAP kinase (ERK1 and ERK2) when phosphorylated either individually or dually at Thr202 and Tyr204 (Cell Signaling, Danvers, MA, USA); phospho‐Akt (Ser473) (D9E) XPTM rabbit monoclonal antibody, which detects endogenous levels of Akt only when phosphorylated at Ser473 (Cell Signaling); Akt rabbit polyclonal antibody, which detects endogenous levels of total Akt1, Akt2 and Akt3 proteins (Cell Signaling); rabbit monoclonal α‐tubulin, which detects endogenous levels of total α‐tubulin protein, and does not cross‐react with recombinant α‐tubulin (Cell Signaling); mouse monoclonal ERK2 antibody, which recognizes the C‐terminus of MAP kinase p42 (Santa Cruz Biotech, Inc.); mouse monoclonal TRα1/β1, which recognizes the ligand binding domain of the TRα1 and TRβ1 (Santa Cruz Biotech, Inc.); mouse monoclonal p21 (Santa Cruz Biotech, Inc.); and HRP linked secondary antibodies (goat anti‐rabbit IgG and horse anti‐mouse IgG) (Cell Signaling).

Techniques: Expressing, Cell Culture, Comparison