Fig 2: 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. ß-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.

Fig 3: GM-CSF—a specific mediator of cellular transformation in a paracrine manner.A and B, wound healing and transwell migration assay of NIH3T3 cells incubated with conditioned media (CM)/GM-CSF depleted CM collected from RAW 264.7-PELP1 overexpression clones. The left panels represent percentage of wound closure and the number of migrated cells; the right panels show representative images of wound healing and transwell migration assay of NIH3T3 cells, respectively. C, soft agar colony formation assay; Left, the total number of transformed NIH3T3 cells/well when incubated with CM/GM-CSF–depleted CM collected from RAW 264.7-PELP1 overexpression clones for 1 to 2 weeks. Right, representative images captured by phase contrast microscope connected to a camera. D, quantitative RT–PCR analysis of CSF-2–specific receptor subunit alpha (CSFRa) expression on various cell lines. GM-CSF, granulocyte–macrophage colony-stimulating factor.

Fig 4: 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.

Fig 5: GM-CSF—a specific mediator of cellular transformation in a paracrine manner.A, wound healing assay of NIH3T3 cells incubated with conditioned media (CM) collected from RAW 264.7-PELP1 overexpression clones along with 0% serum media and GM-CSF pure protein as controls. The left panel depicts percentage of wound closure, and the right panel consists of representative images of wound closure. B, transwell migration assay of NIH3T3 (in the upper chamber) incubated with CM collected from RAW 264.7-PELP1 overexpression clones (in the bottom chamber). The left panel depicts the number of migrated cells, and the right panel shows representative images of migrated NIH3T3 cells captured by phase contrast microscope connected to a camera. C, ELISA to confirm increase in expression of GM-CSF along with increase in PELP1 expression in RAW 264.7 cells. Increasing concentration of PELP1 expression plasmid was transfected in RAW 264.7 cells, and the CM collected were concentrated to measure the CSF-2 concentration. Western blot showed the increased expression of PELP1 in RAW 264.7 cells transfected with pcDNA vector and increasing concentration of PELP1 expression plasmid. D, ELISA to confirm GM-CSF depletion in RAW 264.7-PELP1 clones. The depletion was achieved by incubating CM with anti GM-CSF antibody for 1 h at 37 °C followed by adding protein A/G beads for 2 to 4 h at 4 °C. GM-CSF, granulocyte–macrophage colony-stimulating factor; PELP1, proline, glutamic acid, and leucine-rich protein.