Journal: The Journal of Biological Chemistry

Article Title: Inflammation-induced PELP1 expression promotes tumorigenesis by activating GM-CSF paracrine secretion in the tumor microenvironment

doi: 10.1016/j.jbc.2021.101406

Figure Lengend Snippet: GM-CSF—a new molecular target of PELP1. A , the schematic represents coculturing of Caco2 cells with MD-CRL9850 (with/without LPS) in a transwell migration apparatus. B , left , scattergram represents the number of migrated Caco2 cells when cocultured with 0% media, 0% media with LPS, LPS (1 μg/ml) treated/untreated MD-CRL 9850 cells. Right , representative phase contrast images of migrated Caco2 cells. C , left , specific activity of alkaline phosphatase (mU/mg of protein) in undifferentiated and differentiated Caco2 cells. Right , representative phase contrast images of Caco2 cells. D , quantitative RT (qRT)–PCR analysis of PELP1 in LPS-treated and untreated MD-CRL9850. E , heat map showing the expression of 84 key regulatory genes in mouse IL-6/STAT3 inflammatory signaling pathway determined by using PELP1 knocked down RAW 264.7 lysate. Green and red color backgrounds indicate the downregulation and upregulation of specific gene expressions, respectively. The position, gene symbol, and fold change of genes downregulated in PELP1 knockdown condition were represented below the heat map. F , Western blot and qRT–PCR images that confirmed PELP1 KD when RAW 264.7 cells were transfected with PELP1 siRNA. G , Western blot that confirmed PELP1 KD by shRNA lentiviral particles in RAW 264.7. H – I , validation of GM-CSF and IL18R1 expression by qRT–PCR using RNA from RAW 264.7 PELP1 KD clone with and without LPS induction. J , cell culture supernatants from PELP1 siRNA-transfected RAW 264.7 cells were collected after 48 h of transfection and assessed for GM-CSF concentration (pg/ml) using ELISA. GM-CSF, granulocyte–macrophage colony-stimulating factor; IL, interleukin; LPS, lipopolysaccharide; PELP1, proline, glutamic acid, and leucine-rich protein 1; STAT3, signal transducer and activator of transcription 3.

Article Snippet: IFN-γ from Calbiochem, TNF-α from Peprotech, Ptx from MP Biomedicals, mouse recombinant GM-CSF protein (catalog no.: PMC2014) from Thermo Fisher Scientific, and STAT3 inhibitor S31-201 (catalog no.: sc-204304) from Santa Cruz Biotechnology were procured.

Techniques: Migration, Activity Assay, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Transfection, shRNA, Biomarker Discovery, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Inflammation-induced PELP1 expression promotes tumorigenesis by activating GM-CSF paracrine secretion in the tumor microenvironment

doi: 10.1016/j.jbc.2021.101406

Figure Lengend Snippet: Feedback regulation of PELP1 expression in cancer. A and B , Western blot and quantitative RT–PCR analysis of PELP1 expression in HC11 cells incubated with CM collected from RAW 264.7-PELP1 clones for 6 h. β-Actin was used as control. C , Western blot analysis of PELP1 expression in HC11 cells when incubated with CM/GM-CSF–depleted CM collected from RAW 264.7-PELP1 clones for 6 and 12 h time intervals. D , Western blot analysis of various cytokine-mediated signaling pathway proteins along with PELP1 in HC11 cells incubated with CM collected from RAW 264.7-PELP1 clones. E , Western blot analysis of HC11 cells pretreated with S31-201 inhibitor (86 μM) followed by CM treatment collected from RAW 264.7-PELP1 clones. Actin and total STAT3 as loading controls. CM, conditioned media; GM-CSF, granulocyte–macrophage colony-stimulating factor; PELP1, proline, glutamic acid, and leucine-rich protein 1.

Article Snippet: IFN-γ from Calbiochem, TNF-α from Peprotech, Ptx from MP Biomedicals, mouse recombinant GM-CSF protein (catalog no.: PMC2014) from Thermo Fisher Scientific, and STAT3 inhibitor S31-201 (catalog no.: sc-204304) from Santa Cruz Biotechnology were procured.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Incubation, Clone Assay, Control

Journal: Stem Cell Reports

Article Title: Aberrant α-Adrenergic Hypertrophic Response in Cardiomyocytes from Human Induced Pluripotent Cells

doi: 10.1016/j.stemcr.2014.09.002

Figure Lengend Snippet: Phosphokinase Proteome and Kinase Inhibitor Analyses of PE-Induced Signaling in hESC-CMs and hiPSC-CMs (A) Relative changes in phosphoprotein levels in response to PE (10 μM, 48 hr) in hESC-CMs (H7) and hiPSC-CMs (iCell). (B) Schematic diagram of Ingenuity Pathway Analysis-mapped mechanistic interactions of active epidermal growth factor receptor kinase, STAT family members, and GSK3β/ β-catenin and Src kinase pathways. Arrows between nodes represent direct (solid lines) and indirect (dashed lines) interactions between molecules as supported by information in the Ingenuity Pathway Knowledge Base. (C) Assessment of kinase inhibitors on hypertrophy. Human ESC-CMs (H7) and hiPSC-CMs (iCell and ReproCell) were treated with PE in the presence of kinase inhibitors. Bar graphs show changes in response to selected kinase inhibitors on cell area by automated high-content microscopy. Robust Z score was computed and visualized. (D–F) Representative image and quantitation of nuclear translocation of STAT3 (D and E) and cell area (F) in hiPSC-CMs treated with PE in the presence of interleukin-6 (100 ng/ml) in hiPSC-CMs. White scale bar represents 20 μm. (G) Cell area in hiPSC-CMs treated with PE in the presence of combined inhibition of GSK3β/EGFRK/CAMKII/src/PDGFRK (1 μM each); mean ± SEM; four biological replicates; one-way ANOVA with Tukey’s post hoc test. See also and .

Article Snippet: However, STAT3 inhibitor protein had no effect on hiPSC-CMs (iCell, from CDI) ( A and S4B).

Techniques: Microscopy, Quantitation Assay, Translocation Assay, Inhibition