Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Immunostaining of GIT1 and ERα in one ER(+) and one ER(−) patient breast tumour section ( a ) and the quantitative analysis of GIT1 immunofluorescence relative to DAPI ( b ; ER(+) ( n = 45) versus ER(−) ( n = 30), P = 0.0006, t test). Scale bar, 10 μm. c Mass spectrometry data from CPTAC of the GIT1 protein levels in ER(+) and ER(−) patients (ER(+) ( n = 51) versus ER(−) ( n = 11), P = 0.024, t test). d , e , Western blots of GIT1 in various human breast cancer cells ( d ) and the quantitative analysis ( e ; n = 7 independent biological replicates, 184A1 versus BT474, P = 0.0004; MCF7, P = 0.0003; MDA-MB-134-VI, P = 0.015; MDA-MB-361, P < 0.0001; HCC1954, P = 0.017; MDA-MB-157, P = 0.023; MDA-MB-231, P = 0.0067; t tests). ER, oestrogen receptor. PR, progesterone receptor. HER2, human epidermal growth factor receptor 2. f Kaplan–Meier plot of relapse-free survival of the breast cancer patients from the TCGA database stratified by GIT1 expression ( n = 292, P = 0.0084, log-rank test). g – i Kaplan–Meier plots of relapse-free survival of all breast cancer (BC) patients ( g ; n = 3,310, P < 0.0001, log-rank test), ER(+) patients ( h ; n = 2527, P = 0.032, log-rank test), and ER(−) patients ( i ; n = 779, P = 0.0007, log-rank test) from KM-plotter stratified by GIT1 expression. Hazard ratios (HRs) and their 95% confidence intervals (95%CI) are indicated. j , Forest plot of HRs for survival analysis of all BC, ER(+), and ER(−) patients stratified by GIT1 expression (ER(+) ( n = 2527) versus ER(−) ( n = 779), P = 0.047, one-sided unpaired t test). Error bars show 95%CI. All data are shown as the mean ± s.e.m. For the box plots, the centre line shows the median, the plus sign shows the mean, the upper and lower boundaries of the box show the upper and lower quartiles, and the whiskers show the minimum and maximum values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-sided unpaired t tests. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Immunostaining, Immunofluorescence, Mass Spectrometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a Immunostaining of GIT1 and Notch1 ICD (N1ICD) in one ER(+) and one ER(−) breast cancer sample. Nuclei were detected using DAPI. Images from representative micrographs; the experiment was repeated n = 6 times for ER(+) samples and n = 4 times for ER(−) samples with similar results. Scale bar, 10 μm. b – d Luciferase reporter assays of 12xCSL-Luc ( b ; n = 5, Ctrl-siRNA versus DAPT, P < 0.0001; Ctrl-siRNA versus GIT1-siRNA1, P = 0.0038; one-way ANOVA, F 2,12 = 64.17, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P < 0.0001; t tests) and western blots of Hey1 ( c ) and the quantitative analysis ( d ; n = 6, Ctrl-shRNA versus GIT1-shRNA2, P = 0.030; mRFP versus GIT1-mRFP; P = 0.0002; t tests) in MDA-MB-231 cells treated as indicated. e , f Volcano plot of differentially expressed genes for high and low GIT1 e ; n = 2509 patients) and a correlation analysis between GIT1 and ALDH1A1 mRNA expression ( f ; n = 2509 patients, Spearman ρ = −0.45, P < 0.0001) in breast cancer samples from the METABRIC database . Breast cancer stemness genes are indicated. g , h Flow cytometric analysis of Aldefluor-assayed MDA-MB-231 cells treated as indicated ( g ) and the quantitative analysis ( h ; n = 5, vehicle versus DAPT, P = 0.0014, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.031; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.040; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.44; one-way ANOVA, F 3,16 = 7.216, P = 0.0028 (shaded area); mRFP versus GIT1-mRFP, P = 0.025, t test). SSC, side scatter. i Clonogenic assay of MDA-MB-231 cells treated as indicated ( n = 6, vehicle versus DAPT, P < 0.0001, t test; Ctrl-shRNA versus GIT1-shRNA2, P < 0.0001; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT ( n = 3) versus GIT1-shRNA2 + DAPT ( n = 3), P = 1.00; one-way ANOVA, F 3,14 = 57.84, P < 0.0001 (shaded area); mRFP ( n = 5) versus GIT1-mRFP ( n = 6), P = 0.010, t test). j , k Quantitative analyses of Cyclin A ( j ; n = 3, vehicle versus DAPT, P = 0.036, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0032; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P < 0.0001; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.84; one-way ANOVA, F 3,8 = 32.06, P < 0.0001 (shaded area); mRFP versus GIT1-mRFP, P = 0.028, t test) and Cyclin B1 ( k ; n = 3, vehicle versus DAPT, P = 0.024, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.049; GIT1-shRNA2 versus GIT1-shRNA2 + DAPT, P = 0.0022; Ctrl-shRNA + DAPT versus GIT1-shRNA2 + DAPT, P = 0.94; one-way ANOVA, F 3,8 = 15.92, P = 0.0010 (shaded area); mRFP versus GIT1-mRFP, P = 0.043, t test) from western blots of MDA-MB-231 cells treated as indicated. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Immunostaining, Luciferase, Western Blot, shRNA, Expressing, Clonogenic Assay

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a , b Co-IP of GIT1 with Notch1 from lysates of the indicated mouse organs ( a ) and human breast cell lines ( b ). c Co-IP of the Notch1-2 ICDs with GIT1 from lysates of the MDA-MB-231 cells treated as indicated. d , Schematic diagrams of recombinant fusion protein fragments. e , f GIT1 pulled down by GST Notch fragments from MDA-MB-231 cell lysates. g Pulldown of purified GIT1-His by purified GST-N1ICD/N. CB, Coomassie Blue. Images from representative blots; the experiment was repeated n = 3 times with similar results. *, indicates recombinant GST-fused peptides. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Co-Immunoprecipitation Assay, Recombinant, Purification

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c Quantitative analyses of Notch1 ( a ; n = 4, vehicle versus DAPT, P = 0.032; Ctrl-shRNA versus GIT1-shRNA2, P = 0.26; mRFP versus GIT1-mRFP, P = 0.40; t tests) and the Notch1 ICD (N1ICD) ( b ; n = 4, vehicle versus DAPT, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.67; mRFP versus GIT1-mRFP, P = 0.46; t tests) western blots ( c ) from lysates of MDA-MB-231 cells treated as indicated. d – f Quantitative analyses of cytosolic ( d ; n = 7, vehicle versus DAPT, P = 0.013; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0045; mRFP versus GIT1-mRFP, P = 0.035; t tests) and nuclear ( e ; n = 7, vehicle versus DAPT, P = 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0042; mRFP versus GIT1-mRFP, P = 0.0025; t tests) subcellular fractionation assays with western blots ( f ) from MDA-MB-231 cells treated as indicated. g , h Confocal images ( g ) and quantitative analysis of EGFP intensities in the cytoplasm and nucleus ( h ; Notch1ΔE−EGFP ( n = 150 cells) versus: Notch1ΔE−EGFP + DAPT ( n = 79 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP ( n = 180 cells), P < 0.0001; Notch1ΔE-mutNLS1+2−EGFP + DAPT ( n = 63 cells), P < 0.0001; one-way ANOVA, F 3,468 = 42.82, P < 0.0001) of MDA-MB-231 cells treated as indicated. Nuclei were detected using DAPI. i , j Luciferase reporter assays of Notch1 ICD (UAS-Luc) in MDA-MB-231 cells with knockdown or overexpression of GIT1 ( i ; n = 4, vehicle versus DAPT, P = 0.0019; Ctrl-siRNA versus GIT1-siRNA1, P = 0.020; mRFP versus GIT1-mRFP, P = 0.027; t tests) or mutated NLS1 and NLS2 ( j ; n = 7, Notch1ΔE-GVP versus: Notch1ΔE-GVP + DAPT, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP, P < 0.0001; Notch1ΔE-mutNLS1+2-GVP + DAPT, P < 0.0001; one-way ANOVA, F 3,24 = 1828, P < 0.0001). Scale bar, 10 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t tests or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: shRNA, Western Blot, Fractionation, Luciferase, Over Expression

Journal: Nature Communications

Article Title: GIT1 protects against breast cancer growth through negative regulation of Notch

doi: 10.1038/s41467-022-28631-y

Figure Lengend Snippet: a – c , k Kaplan–Meier plots showing the percentage of tumour-free mice out of ten mice transplanted with MDA-MB-231 ( a , k ) or HCC1395 cells ( b , c ) stably expressing the indicated plasmids. All injection sites were assessed independently, and a tumour was defined as >100 mm 3 (MDA-MB-231) and >500 mm 3 (HCC1395). Comparisons of Kaplan–Meier curves, MDA-MB-231 implants: Ctrl-shRNA versus GIT1-mRFP, P < 0.0001; Ctrl-shRNA versus GIT1-shRNA2, P = 0.12 ( a ); HCC1395 implants: LV-mRFP versus LV-GIT1-mRFP, P = 0.012 ( b ); LV-Ctrl-shRNA versus LV-GIT1-shRNA3, P = 0.025 ( c ); MDA-MB-231 implants: Ctrl-shRNA versus DNMM1, P = 0.0038; Ctrl-shRNA versus GIT1-shRNA2 + DNMM1, P = 0.030; DNMM1 versus GIT1-shRNA2 + DNMM1, P = 0.58 ( k ); log-rank tests. d Scatter plot of tumour volume over time in the mice transplanted with HCC1395 cells expressing LV-GIT1-shRNA3 and LV-GIT1-mRFP. Time is days from the tumour volume >100 mm 3 . Solid line, exponential regression. Shaded area, 95% confidence bands. Comparison of growth rate constants k (Methods) for LV-GIT1-shRNA3 ( n = 290 tumour measurements, k = 0.057) and LV-GIT1-mRFP ( n = 440 tumour measurements, k = 0.023), P < 0.0001; F -test. e , f Tumour doubling time ( e ) and volume change ( f ) for LV-GIT1-shRNA3 versus LV-Ctrl-shRNA and LV-GIT1-mRFP versus LV-mRFP. Solid line, exponential regression. Shaded area, 95% confidence interval. g , j Flow cytometric analysis of ALDH1A1+ cells ( g ; n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.0090, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.019; Ctrl-shRNA versus DNMM1, P = 0.018; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 7.877, P = 0.011 (shaded area)) and Hey1+ cells ( j , n = 4, Ctrl-shRNA versus GIT1-shRNA2, P = 0.012, t test; Ctrl-shRNA versus GIT1-shRNA2, P = 0.0063; Ctrl-shRNA versus DNMM1, P = 0.0059; GIT1-shRNA2 + DNMM1 versus DNMM1, P = 1.00; one-way ANOVA, F 2,9 = 11.61, P = 0.0032 (shaded area)) in MDA-MB-231 xenograft tumours. h , i Confocal images of the Notch1 ICD (N1ICD)-immunolabelled MDA-MB-231 xenograft tumours ( h ) and quantitative analysis ( i ; n = 8 tumours, P = 0.0004, t test). Nuclei were detected using DAPI. Scale bar, 5 μm. All data are shown as the mean ± s.e.m. n denotes the number of biologically independent replicates, unless stated otherwise. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant by two-sided unpaired t test, F -test, or one-way ANOVA with Tukey’s post hoc comparison. Source data are provided as a Source Data file.

Article Snippet: Lentiviral transduction was performed with control shRNA (LV-Ctrl-shRNA, Cat. SHC002V, Sigma-Aldrich) and GIT1 shRNAs (LV-GIT1-shRNA3, Cat. TRCN0000008401; LV-GIT1-shRNA4, Cat. TRCN0000008403; Sigma-Aldrich) for one day. mRFP and mRFP-GIT1 lentiviral transduction particles were produced using the lentiviral package system: pCDF1-MC2_EF1Puro (Cat. CD110B-SBI, BioCat GmbH, Heidelberg, Germany), p.MD2.G (Cat. 12259, AddGene, Watertown, MA, USA), and ps.PAX2 (Cat. 12260, AddGene).

Techniques: Stable Transfection, Expressing, Injection, shRNA

Journal: The Journal of Biological Chemistry

Article Title: α-Actinin 4 Potentiates Nuclear Factor κ-Light-chain-enhancer of Activated B-cell (NF-κB) Activity in Podocytes Independent of Its Cytoplasmic Actin Binding Function *

doi: 10.1074/jbc.M114.597260

Figure Lengend Snippet: IκBα blocks ACTN4 from interacting with p65 in the cytosol. A, purified, immobilized GST-p65 was incubated with cell extracts expressing HA-ACTN4 or HA-p50. Pulldown fractions were subject to Western blotting with anti-HA antibodies. B, purified, immobilized GST-ACTN4 was incubated with cell extracts expressing HA-p65 (full-length), HA-p65 (1–313), or HA-p65 (314–551) followed by Western blotting with anti-HA antibodies. C, knockdown of IκBα results in an association between ACTN4 and p65. HPCs were transiently transfected with a control or IκBα shRNA, and nuclear (N) and cytoplasmic (C) fractions were prepared, immunoprecipitated (IP) with anti-p65 antibodies, and Western blotted with the indicated antibodies.

Article Snippet: shRNAs against human ACTN4 (TRCN0000055783, TRCN0000055784, TRCN0000055785, TRCN0000055786, and TRCN0000055787), shRNA against human IκBα (TRCN0000004539, TRCN0000004540, TRCN0000004541, and TRCN0000004542), and shRNA against human HDAC7 (TRCN0000195442) were purchased from Sigma-Aldrich.

Techniques: Purification, Incubation, Expressing, Western Blot, Transfection, shRNA, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: We evaluated the Gli1 mRNA expression in ( A ) BT549 shCtrl and shNRP2 cells ( n = 3), ( B ) 4T1-RR cells that had been treated with either IgG or aNRP2-10 for 24 hours ( n = 3), and ( C ) BT549 cells given a combined treatment of radiation (0, 5, and 10 Gy) with antibody for 24 hours ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D ) NOS2 mRNA expression was quantified in BT549 cells that had been treated with either DMSO or GANT61 (10 μM) for 24 hours ( n = 3). *** P < 0.001. ( E ) Gli1 and NOS2 mRNA expression was quantified in BT549 shCtrl and shGli1 cells ( n = 3). *** P < 0.001; **** P < 0.0001. ( F ) NOS2 mRNA expression in BT549 shNRP2 cells that had been transfected with either empty vector or a Gli1 -HA construct ( n = 3). The immunoblot shows the protein expression of NOS2, Gli1, and GAPDH in the same cells. *** P < 0.001; **** P < 0.0001. ( G ) Binding of Gli1 on the NOS2 promoter was analyzed using ChIP-qPCR in BT549 cells ( n = 2, representative image). ** P < 0.01. ( H ) NOS2 expression of CRISPR-generated mutations of the Gli1-binding site (Gli1-bind KO1 and KO2) compared with control ( n = 3). ( I ) Clonogenic assay of control (sgCtrl), Gli1-bind KO1, and Gli1-bind KO2 cells that had been irradiated (0–6 Gy; n = 2, representative image). * P < 0.05 Data are presented as means ± SD ( A – G , and I ). Statistical analysis was performed using 2-tailed Student’s t test ( B , D , and F ), 1-way ANOVA multiple comparisons ( A , C , and E ), or 2-way ANOVA multiple comparisons ( I ).

Article Snippet: The following lentiviral shRNAs were obtained from our core facility: human NRP2 (TRCN0000063309, TRCN0000063312), mouse NRP2 (TRCN0000063309 and TRCN0000063310), NOS2 (TRCN0000003764, TRCN0000003765), Gli1 (TRCN0000020484, TRCN0000020488), and KEAP1 (TRCN0000155340, TRCN0000158081). shCtrl vectors were pLKO scramble shRNAs (Addgene, 1864).

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot, Binding Assay, ChIP-qPCR, CRISPR, Generated, Control, Clonogenic Assay, Irradiation

Journal: Scientific Reports

Article Title: Early Cellular Responses of Prostate Carcinoma Cells to Sepantronium Bromide (YM155) Involve Suppression of mTORC1 by AMPK

doi: 10.1038/s41598-019-47573-y

Figure Lengend Snippet: Role of AMPK in cellular responses to YM155. YM155 rapidly activates AMPK in PC-3 cells, and the AMPK inhibitor, Compound C, partially reverses the suppression of P-S6K1 by YM155. ( A ) PC-3 cells were treated with 100 nM YM155 for various early times (from 10 min to 4 h), and changes in the expression of P-AMPKα (T172), AMPKα, P-ULK1(S555), ULK1, P-Raptor (S792), and Raptor were assessed by Western blot. ( B ) PC-3 cells were pre-treated (30 min) with the AMPK kinase inhibitor Compound C or vehicle, followed by treatment with vehicle followed by 4 h treatment with YM155 or vehicle at the indicated drug concentrations, and P-S6K1(T389) and S6K were analyzed by Western blotting. ( C – F ) AMPKα1 and AMPKα2 were stably silenced in PC-3 cells by lentiviral-mediated shRNA transduction for 5 to 7 days as described in Materials and Methods. Stably transduced cells were treated for 4 h with 100 nM YM155 or vehicle for 4 h, and the resulting cell lysates were subjected to Western blot for expression of the various protein shown, such as Survivin, Mcl-1, S6K1, PS6K1(T389) P-rS6 (S240) and P-4E-BP1(S65). Knockdown of AMPKα1 or AMPKα2 by lentiviral-mediated shRNA transduction each led to loss of reduced P-S6K1(T3489) changes in P-rS6 (S240) relative to rS6 ( E ) and cell growth/viability by MTT assay ( F ). Relative changes in cell growth/viability of transduced cells by MTT assay as described in Material and Methods. Unless indicated on the figure, each group of blots represents data obtained from a single gel and experiment. Results shown are representative of 2 to 3 independent experiments.

Article Snippet: Mission pLKO.1 lentiviral shRNA constructs (TRCN0000000861, TRCN0000002170, TRCN0000001271 were obtained from Sigma to silence human AMPKα1 and α2 genes.

Techniques: Expressing, Western Blot, Stable Transfection, shRNA, Transduction, MTT Assay

Journal: Scientific Reports

Article Title: Early Cellular Responses of Prostate Carcinoma Cells to Sepantronium Bromide (YM155) Involve Suppression of mTORC1 by AMPK

doi: 10.1038/s41598-019-47573-y

Figure Lengend Snippet: Summary of our model on the molecular action of YM155 in prostate cancer cells. Panel A represents the overall molecular steps in which our model supports that YM155 rapidly promotes the activation of AMPK and subsequent inactivation of mTORC1 and suppression of cap-dependent translation of proteins involved in cell cycle and survival. This model also illustrates that YM155 additionally promotes the loss of AMPKα1 or AMPKα2, and that such loss counter-intuitively promotes growth arrest and cell death. Panel B is a simplified version of panel A.

Article Snippet: Mission pLKO.1 lentiviral shRNA constructs (TRCN0000000861, TRCN0000002170, TRCN0000001271 were obtained from Sigma to silence human AMPKα1 and α2 genes.

Techniques: Activation Assay