pbabe zeo kras g12d construct  (Addgene inc)

Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: A The scheme for generating the HPNE-based in vitro stepwise transformation model system. B Diagram illustrating the retroviral vector that expresses Doxycycline (Dox)-inducible human PR55α. HPNE cells were transduced with Dox-inducible PR55α retroviral vector (pRevTRE-PR55α) or control empty vector and selected with 200 μg/ml Hygromycin for stably transduced cells. The PR55α-transduced cells were induced by 1 μg/ml Dox for 3 days and analyzed for the protein levels of PR55α and GAPDH by Western blotting. C HPNE/Control and HPNE/PR55α cells were transduced with a retroviral vector expressing V5-tagged human p53 R175H mutant or control vector. The stably transduced clones were selected for Blasticidin (4 μg/mL) and verified for the expression of ectopic V5-p53 R175H by Western blot analysis with an anti-V5 antibody. D The indicated HPNE isogenic cell lines were transduced with a retroviral vector expressing the KRAS G12D mutant, the most frequently detected KRAS mutant in human pancreatic cancer (PC), and selected by 800 μg/ml Zeocin for stably transduced cells. The expression of KRAS G12D in the resulting cells was validated by Western blot analysis using a specific antibody.

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Transformation Assay, In Vitro, Retroviral, Plasmid Preparation, Transduction, Control, Stable Transfection, Western Blot, Expressing, Mutagenesis, Clone Assay

Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: A The indicated HPNE isogenic cell lines were subcutaneously implanted into the flanks of athymic mice and monitored for tumor growth over a 10-week period. Tumor size was measured weekly using a digital caliper. For mice implanted with cells expressing Dox-inducible PR55α, Dox (1 mg/ml) was administered in the drinking water and refreshed every 48 h. As a positive control, CD18/HPAF pancreatic cancer cells were implanted into athymic nude mice and monitored for tumor growth in parallel. The table summarizes the tumor formation rates for each cell line. In a separate cohort, CA3 treatment (1 mg/kg) was initiated 10 days after implantation of HPNE/PR55α + KRAS G12D cells and administered via intraperitoneal injection three times per week for three weeks, as described previously [ 41 ]. Following treatment, mice remained in remission for an additional six weeks. B Representative images of tumor-bearing mice and excised tumor samples at the end of the experiment. C Line graph depicts the number of mice with tumors over time. Statistical significance ( p -value) was assessed using Student’s t-test with SigmaPlot software. D Line graph shows average tumor volume measured weekly for 8 weeks following implantation. E At the study endpoint, tumor xenografts from mice implanted with PK and BK cells were excised and weighed. F Tumor xenograft and adjacent normal tissues were analyzed by IHC analysis for expression of Ki67 (a proliferation biomarker), PR55α, and YAP, as described in Materials and Methods . Histological analysis was also performed using H&E staining. Scale bar = 50 μm.

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Expressing, Positive Control, Injection, Software, Biomarker Discovery, Staining

Journal: Oncogene

Article Title: PR55α subunit of protein phosphatase 2A supports KRAS G12D -driven tumorigenesis that requires YAP activation

doi: 10.1038/s41388-025-03477-y

Figure Lengend Snippet: Oncogenic mutations in the KRAS oncogene cause KRAS activation, which is present in 90–95% of PC and serves as the driver of PC. The Raf/MEK/ERK oncogenic pathway is the major downstream effector of KRAS [ 45 ]. PR55α/PP2A was shown to facilitate the activation of the Raf/MEK/ERK signaling pathway by dephosphorylating the inhibitory sites in Raf and KSR1 [ 16 , 17 ]. Raf1 activation can block MST activity in activating LATS1/2. Conversely, the activation of LATS1/2 can suppress Raf1 [ 36 – 38 ]. YAP oncogene activation is required for KRAS-driven PC progression [ 26 – 28 ]. Our studies show that PR55α-associated PP2A promotes YAP activation by inhibiting the LATS/MOB1 auto-activation loop and dephosphorylating YAP, thus stabilizing YAP protein [ 19 ]. Our previous studies also identified a positive feedback regulation of the MST/LATS cascade by PR55α expression [ 19 ]. However, this PR55α-stimulated MST/LATS signaling was abolished by oncogenic KRAS G12D .

Article Snippet: The pBABE-zeo/KRAS G12D construct was generated by cloning human KRAS G12D cDNA into the pBABE-zeo retroviral vector (Addgene #1766).

Techniques: Activation Assay, Blocking Assay, Activity Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: Sprouty2, PTEN, and PP2A interact to regulate prostate cancer progression

doi: 10.1172/jci63672

Figure Lengend Snippet: Figure 11 Impact of SPRY2, PTEN, and PP2A status in clinical PC. (A) TMA of clinical PC and BPH cohorts was analyzed for expression of SPRY2 and nuclear and cytoplasmic PTEN. Box and whisker plots show median (lines within boxes), interquartile range (bounds of boxes), and upper and lower range (whiskers). Data were analyzed by ANOVA using Dunnett’s multiple comparison test. (B) Representative IHC images in clinical BPH samples. Scale bars: 100 μm. (C) Kaplan-Meier survival plot for PC patients with reduced SPRY2 expression (below median histoscore); analysis was according to the levels of nuclear PTEN. (D) Heat map of alterations in SPRY2, PTEN, and PPP2CB (PP2A catalytic subunit) generated from metastatic tumors (27 cases) using MSKCC Prostate Oncogenome Project data set from cBio genomic portal. (E) Schematic of PC progression. SPRY2 loss leads to tumor suppression by inducing growth arrest via PP2A-mediated nuclear accumulation of PTEN. Subsequent inactivation of PTEN or PP2A as observed in clinical PC may drive tumor progression.

Article Snippet: Retroviral PP2A-A expression construct pMIG-Aα WT (10884), empty backbone pMIG (9044), and PP2AC expression construct pBABE PPP2CA WT (10689) were obtained from Addgene (William Hahn, Dana Farber Cancer Institute).

Techniques: Expressing, Whisker Assay, Comparison, Generated