Journal: British Journal of Cancer

Article Title: A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity

doi: 10.1038/s41416-020-0889-4

Figure Lengend Snippet: Mutagenesis primers.

Article Snippet: Subsequently, MDA-MB-361-iCas9 cells were similarly transduced with lentiviral guide RNA constructs targeting AKT1 (Addgene, 75500) and AKT2 (Addgene, 77506), and stable expressing lines were subjected to puromycin selection (2 μg/mL).

Techniques: Mutagenesis

Journal: British Journal of Cancer

Article Title: A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity

doi: 10.1038/s41416-020-0889-4

Figure Lengend Snippet: a Allosteric AKT inhibitors have higher cell death-inducing activity compared to ATP-competitive inhibitors. A panel of cancer cell lines with the various PI3K/AKT-activating lesions (shown in the table on the left) was treated with three allosteric AKT inhibitors (MK-2206, miransertib and ARQ 751) and two ATP-competitive AKT inhibitors (GSK690693 and ipatasertib) at the indicated doses. The fraction of dead cells following 4 days of drug treatment is shown (cell death %). b Allosteric and ATP-competitive inhibitors have comparable effects on AKT kinase inhibition. The effects of AKT inhibitors on AKT and AKT substrate phosphorylation was assessed in MDA-MB-361 cells by immunoblot using the indicated antibodies following 18 h of treatment. c The cell death-inducing effects of the allosteric AKT inhibitor ARQ 751 and the ATP-competitive inhibitor ipatasertib were compared in washout experiments. Cells were treated with the indicated doses of drug for either 30 min or 1 h. The drug was then removed and cells were extensively washed before allowing them to grow for an additional 72 h. As a control, cells were also continuously exposed to drug for the duration of the experiment. The fold change in cell numbers (left) and the fraction of dead cells following 3 days of drug treatment (right) is shown. d The W80A mutation confers resistance to some but not all allosteric inhibitors in an isoform-selective manner. AKT1 W80A or AKT2 W80A was ectopically expressed in MDA-MB-361 cells. Cells were treated with MK-2206, miransertib or ARQ 751 at the indicated doses. The fold change in cell numbers (left) and the fraction of dead cells following 4 days of drug treatment (right) is shown. e Cells were also treated for 18 h and lysed. Lysates were analysed by immunoblot with the indicated antibodies. f HCT116 AKT1/2 DKO cells were stably transduced with wild-type or W80A variants of either AKT1 or AKT2. Cells were then treated with the indicated drugs and lysed. Lysates were analysed by immunoblot with the indicated antibodies. n.s., not significant. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.

Article Snippet: Subsequently, MDA-MB-361-iCas9 cells were similarly transduced with lentiviral guide RNA constructs targeting AKT1 (Addgene, 75500) and AKT2 (Addgene, 77506), and stable expressing lines were subjected to puromycin selection (2 μg/mL).

Techniques: Activity Assay, Inhibition, Phospho-proteomics, Western Blot, Control, Mutagenesis, Stable Transfection, Transduction

Journal: British Journal of Cancer

Article Title: A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity

doi: 10.1038/s41416-020-0889-4

Figure Lengend Snippet: a A docking model of MK-2206 bound to AKT1. b Crystal structure of AKT1 bound to miransertib. c A homology model of AKT2 bound to miransertib. d , e Effects of the Q79K mutation on the response to AKT inhibitors. d MDA-MB-361 cells were stably transduced with wild-type AKT1 or AKT1 Q79K. Cells were then treated with the indicated doses of MK-2206, ARQ 751, miransertib, GSK690693, ipatasertib or capivasertib. The fold change in cell numbers (top) and the fraction of dead cells (bottom) following 3 days of drug treatment is shown. e MDA-MB-361 cells expressing either AKT1 or the Q79K mutant were treated with increasing doses of MK-2206 or miransertib. The fold change in cell numbers (left) and the fraction of dead cells (right) following 3 days of drug treatment is shown. f The effects of the AKT1 Q79K mutation in response to MK-2206 and miransertib was evaluated in washout experiments. MDA-MB-361 cells stably expressing wild-type AKT1 or AKT1 Q79K were treated with the indicated doses of drug for either 1 h or continuously for 72 h. The fold change in cell numbers and the fraction of dead cells (bottom) at 72 h is shown. g HCT116 AKT1/2 DKO cells were stably transduced with wild-type AKT1 or AKT1 Q79K. Cells were subjected to western blot analysis, following o/n drug treatment, with the indicated antibodies. h A mutation that disrupts the kinase-domain/PH-domain interface confers resistance to all allosteric inhibitors. MDA-MB-361 cells were stably transduced with wild-type AKT1 or the mutant variants D323H, D292A (a kinase-deficient mutant), and the double mutant D323H-D292A. Cells were treated with allosteric AKT inhibitors as shown. The fold change in cell numbers and the fraction of dead cells following 4 days of drug treatment is shown (cell death %). i HCT116 AKT1/2 DKO cells were stably reconstituted with the indicated AKT variants and treated o/n with allosteric AKT inhibitors as shown. Cells were then lysed and analysed by immunoblot with the indicated antibodies. n.s., not significant. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.

Article Snippet: Subsequently, MDA-MB-361-iCas9 cells were similarly transduced with lentiviral guide RNA constructs targeting AKT1 (Addgene, 75500) and AKT2 (Addgene, 77506), and stable expressing lines were subjected to puromycin selection (2 μg/mL).

Techniques: Mutagenesis, Stable Transfection, Transduction, Expressing, Western Blot

Journal: British Journal of Cancer

Article Title: A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity

doi: 10.1038/s41416-020-0889-4

Figure Lengend Snippet: a MDA-MB-361 cells were stably transduced with wild-type AKT1 or AKT1 M227Q. Cells were then treated with the indicated doses of various AKT inhibitors as shown. The fold change in cell numbers (left) and the fraction of dead cells (right) following 4 days of drug treatment is shown. b HCT116 AKT1/2 DKO cells stably transduced with either wild-type AKT1 or AKT1 M227Q were treated with the indicated doses of various AKT inhibitors as shown. Cells were lysed following treatment and analysed by western blot with the indicated antibodies. c Crystal structure of AKT2 bound to GSK690693 (left) and a homology model of AKT1 bound to GSK690693 (right). The proteins in the wild-type models are depicted in magenta (left side, AKT2 crystal structure) and dark green (right side, AKT1 homology model), whereas GSK690693 is shown in blue. In the mutant models, the compound and mutant residue conformations are coloured yellow (left side) and rose and grey (right side, please note that two different M227Q side chain rotamers are included), respectively. d Alignment of the crystal structures of GSK690693 (magenta, PDB entry 3D0E, in complex with AKT2), ipatasertib (light blue, PDB entry 4EKL, in complex with AKT1) and capivasertib (dark blue, PDB entry 4GV1, in complex with AKT1). GSK690693 extends to an area between the gatekeeper M229 (orange sticks) and the K181–E200 salt bridge (orange sticks), which is not occupied by ipatasertib and capivasertib. We have highlighted the part of GSK690693 that extends into this area with dotted lines. The protein in all three complexes is depicted as grey cartoon. n.s., not significant. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.

Article Snippet: Subsequently, MDA-MB-361-iCas9 cells were similarly transduced with lentiviral guide RNA constructs targeting AKT1 (Addgene, 75500) and AKT2 (Addgene, 77506), and stable expressing lines were subjected to puromycin selection (2 μg/mL).

Techniques: Stable Transfection, Transduction, Western Blot, Mutagenesis, Residue

Journal: British Journal of Cancer

Article Title: A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity

doi: 10.1038/s41416-020-0889-4

Figure Lengend Snippet: a Conditioned media from HCT116 AKT1/2 DKO cells stably transduced with either wild-type (AKT2) or kinase-dead AKT2 (AKT2 K181M) was collected for acetate quantification. Acetate was quantified using a colorimetric assay (left). Expression of AKT2 was confirmed by western blot (right). b HCT116 AKT1/2 DKO cell stably transduced with wild-type (AKT2) or MK-2206-resistant AKT2 (AKT2 W80A) were cultured for 24 h in the presence or absence of MK-2206 as indicated. Culture media was collected following treatment and acetate levels were assessed with a colorimetric assay. c MDA-MB-361 and EBC1 cells were cultured for 24 h in the presence or absence of MK-2206 or GSK690693 as indicated. Culture media was collected following treatment and acetate levels were assessed with a colorimetric assay. d MDA-MB-361 cells were cultured for 24 h in the presence or absence of three allosteric (MK-2206, ARQ 751 and miransertib) or three ATP-competitive (GSK690693, ipatasertib and capivasertib) AKT inhibitors. Culture media was collected following treatment and acetate levels were assessed with a colourimetric assay. e MDA-MB-361 cells stably transduced with either wild-type AKT1 or AKT1 Q79K were cultured for 24 h in the presence or absence of MK-2206, ARQ 751, miransertib, GSK690693, or ipatasetrtib as indicated. Culture media was collected following treatment and acetate levels were assessed with a colourimetric assay. f Parental or MK-2206-resistant MDA-MB-361 were cultured for 24 h in the presence or absence of the indicated drugs. Culture media was collected and acetate levels were assessed with a colorimetric assay. n.s., not significant. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.

Article Snippet: Subsequently, MDA-MB-361-iCas9 cells were similarly transduced with lentiviral guide RNA constructs targeting AKT1 (Addgene, 75500) and AKT2 (Addgene, 77506), and stable expressing lines were subjected to puromycin selection (2 μg/mL).

Techniques: Stable Transfection, Transduction, Colorimetric Assay, Expressing, Western Blot, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Effect of Akt inhibition on neuronal insulin signaling and glucose uptake; and effect of insulin on Akt and its isoforms. A N2A cells were differentiated in 2% DMSO for 3 days and stimulated with or without 30 µM MK2206 and/or 100 nM insulin for 30 min as indicated. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt antibody. B Differentiated N2A cells were serum starved for 2 h, followed by 100 nM insulin for 30 min. Uptake of 2-NBDG was then measured. Bar represents relative change in uptake of 2-NBDG. C–E N2A cells were differentiated in 2% DMSO for 3 days and stimulated with or without 100 nM insulin for 30 min. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. C Bar represents relative change in pAkt1 (Ser-473) probed with anti-Akt1 antibody. D Bar represents relative change in pAkt2 (Ser-474) probed with anti-Akt2 antibody. E Post-insulin stimulation, lysates were subjected to immunoprecipitation using anti-Akt3 antibody. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt3 antibody. F–H HT22 cells were differentiated in neurobasal media containing 2 mmol/L glutamine and 1 × N2 supplement for 48 h and stimulated with or without 100 nM insulin for 30 min. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. F Bar represents relative change in pAkt1 (Ser-473) probed with anti-Akt1 antibody. G Bar represents relative change in pAkt2 (Ser-474) probed with anti-Akt2 antibody. H Post-insulin stimulation, lysates were subjected to immunoprecipitation using anti-Akt3 antibody. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt3 antibody. GAPDH has been used as a loading control (A, C, D, F, G). IgG band was used as loading control (E, H). Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01 compared to lane 1, ###P < 0.001, ##P < 0.01 compared to lane 2. IP Immunoprecipitation; IB Immunoblot; A.U Arbitrary Units

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Inhibition, Western Blot, Immunoprecipitation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Effect of silencing of one isoform, on expression and phosphorylation of other isoform in N2A cells. Three days post-proliferation, N2A cells were transfected with Akt1, Akt2 or Akt3 specific siRNA and then differentiated in 2% DMSO for 3 days. Cells were stimulated with or without 100 nM insulin for 30 min prior to cell lysis. Lysate was subjected to western blotting, followed by probing with relevant primary antibodies. A, D, G Bar represents relative change in pAkt1 (Ser-473) when probed with anti-Akt1 antibody. B, E, H Bar represents relative change in pAkt2 (Ser-474) when probed with anti-Akt2 antibody. C, F, I Bar represents relative change in pAkt (Ser-473) when probed with anti-Akt3 antibody. GAPDH has been used as a loading control (A, B, D, E, G, H). IgG band was used as loading control (C, F, I). Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01 as compared to lane 1; ###P < 0.001, ##P < 0.01 as compared to lane 2. IP Immunoprecipitation; IB Immunoblot

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Expressing, Phospho-proteomics, Transfection, Lysis, Western Blot, Control, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Mechanism underlying Akt isoform specificity in neuronal cells. A N2A cells were differentiated in 2% DMSO for 3 days. Three days post-differentiation, membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change in Akt1, Akt2 or Akt3 when probed with anti-Akt isoform specific antibody. B HT22 cells were differentiated in neurobasal media containing 2 mmol/L glutamine and 1 × N2 supplement for 48 h. Three days post-differentiation, membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change in Akt1, Akt2 or Akt3 when probed with anti-Akt isoform specific antibody. C, D Bar represents relative change in AS160 when probed with anti-AS160 antibody (N2A/HT22 as indicated). Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody (N2A/HT22 as indicated). GAPDH has been used as a loading control. Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1 or as indicated. IB Immunoblot

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Membrane, Isolation, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Mechanism underlying Akt isoforms specificity in mice whole brain tissue. A Subcellular translocation of Akt isoforms in mice whole brain tissue. Mice whole brain was lysed, membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change in Akt1, Akt2 or Akt3 when probed with anti-Akt isoform specific antibody. Experiments were executed with three independent animals and a representative result is shown. B Subcellular redistribution of AS160 post-insulin stimulation. Bar represents relative change in AS160 when probed with anti-AS160 antibody. Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody. GAPDH has been used as a loading control. Experiments were executed with three independent animals and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1 or as indicated. (ND Normal Diet; HFD High-Fat-Diet). IB Immunoblot

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Translocation Assay, Membrane, Isolation, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Expression and activation of Akt1, Akt2, and Akt3, glucose uptake and subcellular translocation under insulin-resistant condition in neuronal cells. (A–C) N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change when probed with anti-Akt isoform specific antibody. A Bar represents relative change in pAkt1 (Ser-473) probed with anti-Akt1 antibody. B Bar represents relative change in pAkt2 (Ser-474) probed with anti-Akt2 antibody. C Post-insulin stimulation, lysates were subjected to immunoprecipitation using anti-Akt3 antibody. Bar represents relative change in pAkt (Ser-473) probed with anti-Akt3 antibody. D Three days post-proliferation, N2A cells were transfected with double (Akt−2–3, Akt−1–3 or Akt−1–2) specific siRNA and then differentiated under MF MFI condition for 3 days. Differentiated N2A cells were serum starved for 2 h, followed by 100 nM insulin for 30 min. Uptake of 2-NBDG was then measured. E Subcellular translocation of Akt isoforms post-insulin stimulation. N2A cells were differentiated under MF MFI condition for 3 days and stimulated with or without 100 nM insulin for 30 min as indicated. Cells were lysed, and membrane and cytosol fraction were isolated and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change in Akt1, Akt2 or Akt3 when probed with anti-Akt isoform specific antibody. F Subcellular translocation of AS160 post-insulin stimulation. Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody. GAPDH has been used as a loading control. Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1 or as indicated; ###P < 0.001, ##P < 0.01, #P < 0.05 compared to lane 2. IP Immunoprecipitation; IB Immunoblot; A.U Arbitrary Units

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Expressing, Activation Assay, Translocation Assay, Western Blot, Immunoprecipitation, Transfection, Membrane, Isolation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Effect of Akt1, Akt2, and Akt3 over-expression on expression and activation of AS160, and neuronal glucose uptake under insulin-resistant condition in neuronal cells. A–F Three days post-proliferation, Akt1, Akt2 or Akt3 were over-expressed using isoform specific plasmids. N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. Cells were lysed and subjected to western blotting, followed by probing with relevant primary antibodies. Bar represents relative change when probed with anti-Akt isoform specific antibody. A, C, E Bar represents relative change in Akt1/Akt2/Akt3 probed with anti-Akt isoform specific antibody. GAPDH was used as loading control B, D, F Bar represents relative change in pAS160 (Thr-642) when probed with anti-AS160 antibody. G–I Three days post-proliferation, Akt1, Akt2 or Akt3 were over-expressed using isoform specific plasmids. N2A cells were differentiated in serum-free medium in the absence of (MF) or chronic presence of 100 nM insulin (MFI) for 3 days. N2A cells were serum starved for 2 h, followed by 100 nM insulin for 30 min. Uptake of 2-NBDG was then measured. Experiments were executed three times and a representative result is shown. Data expressed are mean ± SE. ***P < 0.001, **P < 0.01, *P < 0.05 compared to lane 1 or as indicated, ###P < 0.001, ##P < 0.01, #P < 0.05 compared to lane 2. IB Immunoblot; A.U Arbitrary Units

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Over Expression, Expressing, Activation Assay, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Role of Akt isoforms in neuronal insulin signaling and resistance

doi: 10.1007/s00018-021-03993-6

Figure Lengend Snippet: Schematic diagram depicting role of Akt isoforms in regulating neuronal insulin signaling. A In an insulin-sensitive, under unstimulated condition, Akt (Akt1, Akt2, and Akt3) are present in the cytoplasm. AS160 binds to GSVs (GLUT4 Storage Vesicles) and tethers GLUT4. This does not allow GLUT4 exocytosis under basal conditions. B In an insulin-sensitive, insulin stimulated condition, Akt translocates to plasma membrane in an isoform specific insulin-dependent way (1), getting phosphorylated there (2). In neuronal system, all Akt isoforms translocate in the order Akt2 > Akt3 > Akt1. An activated Akt phosphorylates AS160, hence inactivating it. Phosphorylated and thus inactivated AS160 translocated to cytoplasm (3), promoting GLUT4 dissociation from GSVs and allowing glucose uptake (4). C In an insulin-resistant, unstimulated condition, hyperinsulinemia occurs due to defects in insulin signaling. D In an insulin-resistant, insulin stimulated condition, hyperinsulinemia triggers insulin receptor down-regulation, not allowing Akt isoform specific translocation to the plasma membrane (1). This leads to inadequate phosphorylation (2), leading to Akt’s inability to phosphorylate AS160. Unphosphorylated, thus, active AS160 continues tethering GLUT4 to GSVs, not allowing GLUT4 exocytosis, hence affecting neuronal glucose uptake. (Created with BioRender.com)

Article Snippet: Plasmid DNA transfection using Akt isoform specific plasmids Akt isoform specific constructs were purchased from Addgene [Akt1 (#86631) and Akt2 (#86593)] and Origene [Akt3 (NM_RC221051)].

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Phospho-proteomics

Journal: The FASEB Journal

Article Title: Post‐translational regulation of PGC‐1α modulates fibrotic repair

doi: 10.1096/fj.202100339R

Figure Lengend Snippet: Pulmonary fibrosis does not require Akt2. Akt2 fl/fl or Akt2 −/− Lyz2‐cre mice were exposed to saline or bleomycin (Bleo). BAL cells were isolated 21 days later. A, TUNEL staining ( n = 5), B, TUNEL quantification ( n = 4‐5), and C, caspase‐3 activity ( n = 5). Scale bar represent 50 μm. mRNA analysis of D, Ppargc1a ( n = 5‐6), E, Tfam ( n = 5‐6), F, Cs ( n = 5‐6), and G, Cox4i1 expression ( n = 5‐6) in BAL cells. H, Histology of lung sections with Masson’s trichrome staining ( n = 5) and I, hydroxyproline analysis of homogenized lung ( n = 5‐6). *** P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized

Article Snippet: The pcDNA‐MKK6(Glu), pCMV‐Flag‐p38 DN (generous gifts from Dr. Roger Davis, University of Massachusetts), and pUSE‐Akt1 CA plasmids have been previously described., Constitutively active Akt2 (Akt2 CA ) plasmid was a gift from Dr. William Sellers, purchased from Addgene #9016.

Techniques: Saline, Isolation, TUNEL Assay, Staining, Activity Assay, Expressing, Comparison

Journal: The FASEB Journal

Article Title: Post‐translational regulation of PGC‐1α modulates fibrotic repair

doi: 10.1096/fj.202100339R

Figure Lengend Snippet: Akt1‐mediated mtROS activates PGC‐1α via phosphorylation of p38 MAPK. A, Nuclear immunoblot analysis for p‐PGC‐1α (S570) and PGC‐1α in transfected macrophages. B, PPARGC1A promoter activity in transfected macrophages ( n = 8). Inset, p‐Akt1 and p‐Akt2 immunoblot. C, Nuclear immunoblot analysis in transfected macrophages. D, Schematic of Akt2 phosphorylation and mutation sites on PGC‐1α. E, NFE2L2 mRNA analysis in macrophages expressing empty (emp), Akt1 C(A) or Akt2 CA together with PGC‐1α WT (WT) or PGC‐1α S570A (S570A) ( n = 3). Macrophages were transfected with empty or Akt1 CA together with MKK6(Glu) or p38 DN . F, PPARGC1A mRNA expression ( n = 3), G, nuclear immunoblot analysis, H, quantification of PGC‐1α expression ( n = 3); inset, Akt1 immunoblot analysis, I, NRF1 mRNA expression ( n = 3). J, Schematic of p38MAPK phosphorylation and mutation sites on PGC‐1α. K, NFE2L2 mRNA analysis of macrophages expressing empty or Akt1 CA together with PGC‐1α WT (WT), PGC‐1α T262A (T262A), PGC‐1α S265A (S265A), PGC‐1α T298A (T298A) ( n = 3). L, Immunoblot analysis of BAL cells from saline or bleomycin‐exposed Akt1 fl/fl and Akt1 −/− Lyz2‐cre mice. Macrophages were transfected with scramble or PGC‐1α siRNA and empty or Akt1 CA . M, Mitochondrial DNA content ( n = 3) and N, Tfam mRNA expression ( n = 3). Inset, PGC‐1α mRNA expression. O, Nuclear immunoblot analysis in macrophages expressing empty or Akt1 CA and treated with vehicle or mitoTEMPO (10 µM, overnight). ** P < .001; ***, P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized. CPD = Cdc4 phosphodegron motifs

Article Snippet: The pcDNA‐MKK6(Glu), pCMV‐Flag‐p38 DN (generous gifts from Dr. Roger Davis, University of Massachusetts), and pUSE‐Akt1 CA plasmids have been previously described., Constitutively active Akt2 (Akt2 CA ) plasmid was a gift from Dr. William Sellers, purchased from Addgene #9016.

Techniques: Phospho-proteomics, Western Blot, Transfection, Activity Assay, Mutagenesis, Expressing, Saline, Comparison