pbabe-pten wt  (Addgene inc)

Journal: STAR Protocols

Article Title: Using the single-cell imaging system and orthotropic footpad injection to establish mouse models for experimental and spontaneous melanoma metastasis

doi: 10.1016/j.xpro.2023.102349

Figure Lengend Snippet:

Article Snippet: pBabe-PTEN WT , Addgene , #10785.

Techniques: Recombinant, Software, Inverted Microscopy, Injection, Sterility

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) (Left) Western blots showing PTEN and phospho-AKT expression in PTEN-deficient H1650 cancer cell line stably expressing PTEN WT or empty vector (EV). (Right) List of genes significantly upregulated (fold change > 2, multiple t-test * p < 0.05, FDR adjustedp-value or q < 0.2) in PTEN-deficient H1650-GFP cells compared to H1650 cells engineered to re-express PTEN stably by microarray analysis. Highlighted in red boxes are the top upregulated energy metabolism genes, including PDHK1 , in H1650-GFP cells. (B) Western blots showing PTEN, phospho-AKT and PDHK1 expression in PTEN-deficient cancer cell lines stably expressing PTEN WT or empty vector. (C-D) Same as (B) in PTEN-proficient cancer (C) or non-cancer (normal) (D) cell lines with stable PTEN knockdown. shPTEN, shRNA to PTEN and SC, scrambled control shRNA. See also - and -S2.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Microarray, shRNA

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Quantitative real-time PCR analysis of PDHK1 mRNA expression in PTEN-deficient H1650 cancer cell line stably expressing PTEN WT or in PTEN-proficient HCC827 cancer cell line with stable PTEN knockdown. Data are shown as mean ± SEM (n = 3 replicates). *** p < 0.001 compared to ‘empty vector (EV) expressing PTEN-deficient cells’ or * p < 0.05 compared to ‘scrambled control shRNA expressing PTEN-proficient cells’ by two-tailed unpaired t test with Welch’s correction. (B) Effects of adenovirus mediated transient PTEN re-expression in PTEN-deficient H1650, U87MG and A2058 cancer cell lines on phospho-AKT and PDHK1-4 expression by immunoblot analysis are shown. (C) Relative mRNA expression of PDHK1 by microarray analysis in lung epithelial cells with conditional PTEN deletion in vivo (GSE47520). Data are shown as mean ± SEM (n = 3 replicates). * p < 0.05 compared to ‘PTEN expressing normal lung epithelial control cells’ by two-tailed unpaired t test with welch’s correction. PDHK1 probes: 1435836_at, 1423748_at, 1434974_at and 1423747_at.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Plasmid Preparation, shRNA, Two Tailed Test, Western Blot, Microarray, In Vivo

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Effects of stable PDHK1 knockdown in various PTEN-deficient (A) or -proficient cancer cell lines (B) on cell growth by crystal violet staining assay (left) and apoptosis induction as measured by cleaved PARP levels by immunoblot analysis (right) are shown. shPDHK1#1 and shPDHK1#2, shRNAs to PDHK1 and shScramble, scrambled control shRNA. (C) Effects of pharmacologic inhibition of PDHK1 in PTEN-deficient or -proficient cancer or normal cell lines with DCA (dose response: 0, 5, 10, 20 and 50 mM) treatment or 72 hours on cell viability by CellTiter-Glo luminescent assay are shown. Data are shown as mean ± SD. (D) Western blots showing phospho-PDHA1 expression in PTEN-deficient cancer cell lines in response to PDHK1 inhibition by DCA (dose response: 0, 5, 10, 20, 50 mM) treatment. (E) Effects of chemotherapeutic agents used at indicated concentrations on cell growth of PTEN-deficient cancer cell lines stably expressing PTEN WT or empty vector (EV) by crystal violet staining assay are shown. (F) Effects of pharmacologic inhibition of PDHK1 with 25 mM DCA in PTEN-deficient cancer cell lines with or without stable PTEN re-expression on pyruvate dehydrogenase complex (PDC) activation and apoptosis induction as measured by phospho-PDHA1 and cleaved PARP levels, respectively, by western blot are shown.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Staining, Western Blot, shRNA, Inhibition, Luminescence Assay, Expressing, Stable Transfection, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Quantitative real-time PCR analysis of PDHK1 mRNA expression in PTEN-deficient A2058 cancer cell line stably expressing PTEN WT or PTEN G129E or PTEN Y138L or GFP. Data are shown as mean ± SD (n = 2 replicates). **** p < 0.0001; n.s, not significant compared to ‘GFP expressing PTEN-deficient cells’ by Tukey’s multiple comparisons one-way ANOVA test. (B) Western blots showing phospho-AKT and PDHK1 expression in PTEN-deficient cancer cell lines in response to 1 μM BKM-120 (PI3-kinase inhibitor) or vehicle treatment for 24 hrs. (C) Western blots showing phospho-AKT, phospho-S6 (mTOR effector) and PDHK1 expression in PTEN-proficient cancer cell lines expressing empty vector (EV) or myristoylated-AKT (Myr-AKT) to constitutively activate AKT signaling. (D) Effects of pharmacologic inhibition of PDHK1 with DCA (dose response: 5, 10 and 20 mM) in PTEN-deficient stable cancer cell lines expressing PTEN WT or PTEN G129E or PTEN Y138L or GFP on cell growth by crystal violet staining assay are shown, with quantification of cell viability in 20 mM DCA treatment relative to vehicle (water) treatment reported as rescue score (Methods). See also .

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Western Blot, Plasmid Preparation, Inhibition, Staining

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Western blots showing PTEN, PDHK1 and phospho-AKT expression in PTEN-deficient cancer cell lines stably expressing PTEN WT or PTEN G129E or PTEN C124S or empty vector (EV). (B) Effects of PI3K inhibition in BKM-120 or vehicle treated PTEN-deficient 786-O cancer cells stably expressing PTEN WT or empty vector (EV) for 24 hours on PDHK1, phospho-AKT and HK2 expression by immunoblotting analysis are shown. (C) Effects of PI3K or AKT inhibition in PTEN-deficient cancer cell lines treated for 24 hours with BKM-120 or MK-2206 on phospho-AKT, HK2 and PDHK1 expression by immunoblotting analysis are shown. (D) Effects of PI3K inhibition in PTEN-deficient cancer cell lines treated for 4-24 hours with BKM120 on phospho-AKT, phospho-GSK3, phospho-S6 and PDHK1 expression by immunoblotting analysis are shown. V: vehicle (DMSO) treatment. (E) Effects of PI3K inhibition in PTEN-deficient cancer cell lines treated with GDC-0941 (0, 0.5, 1 μM) for 24 hours on phospho-AKT, total AKT, phospho-S6 and PDHK1-4 expression by immunoblotting analysis are shown. PI3K or AKT inhibitors were used at 1 μM final concentration for the indicated times in B-D.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) (Top) Identification of NFκB consensus binding site in the promoter of PDHK1 gene located between nucleotide positions 173420479 and 173420488 in chromosome 2, ~300 bp upstream of the transcription start site (TSS, red arrow) at nucleotide position 173420779. (Bottom) NFκB (RELA) recruitment at PDHK1 promoter in PTEN-deficient cancer cells by ChIP assay. Primers (forward and reverse arrows) used to amplify a 118 bp region (spanning nucleotide position 173420380) ~40 bp upstream of the NFκB binding site in the PDHK1 promoter are shown. Fold enrichment [RELA ChIP DNA (pg) to IgG DNA (pg)] data are shown as mean ± SEM (n = 3 replicates). ** p < 0.01; * p < 0.05 compared to ‘IgG control’ by two-tailed unpaired t test with Welch’s correction. See also . (B) Western blots showing NFκB (RELA), PDHK1 and phospho-PDHA1 expression in PTEN-deficient cancer cell lines with or without stable RELA knockdown. shRELA, shRNA to RELA. (C) Effects of PTEN WT or PTEN G129E or PTEN Y138L or empty vector (EV) expression in PTEN-deficient cancer cell lines on NFκB activity by luciferase reporter assays (Methods) are shown. Data are shown as mean ± SD (n = 2 replicates). *** p < 0.001, ** p < 0.01 compared to ‘empty vector control expressing PTEN-deficient cells’ by Tukey’s multiple comparisons one-way ANOVA test. (D) Effects of PTEN WT or PTEN G129E or PTEN Y138L or GFP expression in PTEN-deficient cancer cell lines on NFκB (RELA) subcellular localization by nuclear-cytoplasmic fractionation and immunoblotting are shown. Western blots were also probed with anti-LaminB1 and anti-actinβ antibodies as nuclear and cytoplasmic markers, respectively. (E) Western blots showing PTEN, phospho-RELA, PDHK1 and phospho-AKT expression in PTEN-deficient stable cancer cell lines expressing PTEN WT , or PTEN G129E , or PTEN 138L , or GFP. See also and .

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Binding Assay, Two Tailed Test, Western Blot, Expressing, shRNA, Plasmid Preparation, Activity Assay, Luciferase, Fractionation

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) (Left) Workflow of the unbiased, global phospho-proteomic profiling used in PTEN-deficient cancer cell lines stably expressing PTEN WT or PTEN G129E or PTEN Y138L or GFP to identify the phospho-peptides (and corresponding proteins) affected specifically by the protein or lipid phosphatase (PP or LP) activity of PTEN. (Right) Venn-diagram showing the number of proteins with phospho-sites specifically affected by the protein phosphatase activity of PTEN in H1650 (n = 169) and A2058 (n = 248) cells, with the phospho-proteins (including NFκB activating protein, NKAP highlighted in white) affected in both cell lines shown in the overlap (n = 42). (B) Schematic representation of NKAP phospho-sites at serines 9 and 149 affected by the protein phosphatase activity of PTEN. (C) Phosphate released (μM) from a phospho-S9-NKAP or phospho-S149-NKAP or phospho-S72-Rab7 (control) peptide after incubation without or with recombinant WT PTEN or mutant Y138L PTEN enzyme in an in-vitro Malachite green based colorimetric assay. Data are shown as mean ± SD (n=2 replicates). * p < 0.05; ** p < 0.01; **** p < 0.0001 compared to ‘no PTEN’ control by two-tailed unpaired t test with Welch’s correction. (D) Detection of phospho-NKAP and its de-phosphorylated species (indicated by the red dotted inset) in PTEN-deficient cancer cell lines stably expressing either PTEN WT or PTEN G129E or PTEN Y138L or GFP by Phos-tag PAGE and immunoblotting. De-phosphorylation score indicates the extent to which expression of each PTEN mutant in PTEN-deficient cells suppresses NKAP de-phosphorylation relative to WT PTEN (set at 1). A lower de-phosphorylation score indicates less de-phosphorylation of NKAP. (E) Co-immunoprecipitation of NKAP (indicated by the red dotted inset) with PTEN upon over-expression of both NKAP-V5 and FLAG-PTEN and not NKAP-V5 over-expression alone or no over-expression in 293T cells followed by IP-FLAG is shown. Arrowhead denotes NKAP-V5; while the dark band below is background due to secondary antibody cross-reactivity to the immunoglobulin heavy chain (IgH) used in IP. See also .

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Stable Transfection, Expressing, Activity Assay, Incubation, Recombinant, Mutagenesis, In Vitro, Colorimetric Assay, Two Tailed Test, Western Blot, De-Phosphorylation Assay, Immunoprecipitation, Over Expression

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A) Phospho-proteins (n=42, listed alphabetically), including NKAP (highlighted in red box), regulated specifically by the PTEN protein-phosphatase in both H1650 and A2058 PTEN-deficient cancer cell lines stably expressing either PTEN WT or PTEN G129E or PTEN Y138L or GFP identified by an unbiased global phospho-proteomic profiling analysis (Methods) are shown. Venn diagram representation of the phospho-proteins regulated specifically by the PTEN protein phosphatase in either H1650 (n = 169) or A2058 (n = 248) or both (n = 42) cell lines. (B) Co-immunoprecipitation of NHERF2 with PTEN upon over-expression of both GST-NHERF2 and FLAG-PTEN in 293T cells followed by IP-FLAG is shown.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Over Expression

Journal: bioRxiv

Article Title: Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

doi: 10.1101/441295

Figure Lengend Snippet: (A-B) Effects of stable NKAP knockdown in PTEN-deficient cancer cell lines on NFκB activity (A) or PDHK1 promoter activation (B) by luciferase reporter assays (Methods) are shown. shA and shB, shRNAs to NKAP and SC, scrambled control shRNA. Data are shown as mean ± SEM (n = 3 replicates). **** p < 0.0001; *** p < 0.001; ** p < 0.01 compared to ‘scrambled control shRNA expressing PTEN-deficient cells’ by Tukey’s multiple comparisons one-way ANOVA test. (C) Western blots showing phospho-RELA, PDHK1 and phospho-AKT expression in PTEN-deficient cancer cell lines with or without stable NKAP knockdown. shA and shB, shRNAs to NKAP and SC, scrambled control shRNA. (D-E) Effects of stable NKAP knockdown in PTEN-deficient cancer cell lines without (D) or with stable PTEN WT or PTEN G129E or PTEN Y138L or GFP expression (E) on cell growth by crystal violet staining assay are shown, with quantification of cell viability under each condition relative to cells expressing the scrambled control shRNA. shA and shB, shRNAs to NKAP and SC, scrambled control shRNA. (F) Model of cellular survival and energy metabolism regulation specifically by the protein-phosphatase activity of PTEN via a NKAP-NFκB-PDHK1-driven signaling axis. Loss of the PTEN protein-phosphatase activity promotes NKAP phosphorylation, NFκB activation, and PDHK1 upregulation, thereby enhancing aerobic glycolysis and rendering PTEN protein-phosphatase deficient cells dependent on NKAP and PDHK1 for survival.

Article Snippet: Mammalian retroviral expression plasmids pBABE-PTEN WT (Cat# 10785), pBABE-PTEN C124S (Cat# 10931), pBABE-PTEN G129E (Cat# 10771), pBABE-Puro (Cat# 1764) and pBABE-GFP (Cat# 10668) were purchased from Addgene (Cambridge, MA). pBABE-PTEN Y138L expression plasmid was engineered using standard molecular biology techniques.

Techniques: Activity Assay, Activation Assay, Luciferase, shRNA, Expressing, Western Blot, Staining