Journal: bioRxiv

Article Title: Targeting CREB remodels the immune microenvironment to enhance immunotherapy responses in pancreatic cancer

doi: 10.64898/2025.12.04.691935

Figure Lengend Snippet: Cancer cell intrinsic CREB transcriptionally regulates LIF expression, driving macrophage-modulatory signaling in PDAC. (A) Experimental schematic demonstrating RNA sequencing (RNA-seq) analysis conducted in KPC mouse CREB WT and CREB KO tumor cells. (B) Bubble plot visualizing differentially regulated pathways (using molecular signature databases) in CREB KO transcriptomics compared with CREB WT tumor cells (n=3). (C) Ridge plot showing normalized enrichment scores for the hallmark JAK–STAT3 signaling pathway across different cellular constituents in the human HTAN pancreatic cancer dataset from treatment-naïve patients. (D) Western blot image depicting pSTAT3 activation, along with tSTAT3, vinculin and vimentin in mouse RAW macrophage 264.7 cell line treated with either recombinant (r) LIF alone (1 µg/mL) or in combination with EC359 (LIFR blockade, 0.2µM). (E) Chromatin Immunoprecipitation sequencing (ChIP-seq) peak signals visualized as heat maps which depict CREB binding sites across genomic regions in KPC mouse pancreatic tumor cells. The adjacent call out boxes with the heat maps showing essential genes regulated via CREB as its downstream mediators. (F) Integrative Genome Viewer (IGV) plot visualizing occupancy of CREB binding peaks in ChIP-seq data at the site of mouse Lif promoter gene regulatory sequences. (G-H) Violin dot plots showing downregulation of LIF expression via qPCR and ELISA in bulk tumor lysates of CREB KO KPC orthotopic PDAC tumors as compared to CREB WT with n=3-7 mice per group. (I) Representative photomicrographs of whole pancreas along with its corresponding quantification depicting significantly reduced LIF expression via IHC in age matched KPC C -/- as compared to KPC mice. Individual data points with mean ± SEM are shown and compared by two-tailed unpaired t test. *p<0.05; **p<0.01; ns non-significant (p>0.05).

Article Snippet: Under specific experimental conditions, bone marrow derived macrophages (BMDMs) were treated with murine recombinant LIF (Biotechne, 1 μg/mL), with or without pretreatment using an LIF receptor (LIFR) inhibitor (0.2 μM EC359, HY-120142, MedChem Express) at 37°C for 30 minutes.

Techniques: Expressing, RNA Sequencing, Western Blot, Activation Assay, Recombinant, ChIP-sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: Active gp130/STAT3 pathway in PANC-1 sphere cells. ( A ) Real-time qPCR analysis of IL-6 , LIF , IL-6R , LIFR , and gp130 in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. The results presented are normalized to values obtained for adherent cells (value = 1). Results are presented as means ± SD from three independent experiments. ( B ) FACS analysis of IL-6R, LIFR, and gp130 expression in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Controls are indicated by thin lines with gray color. ( C ) Cell surface levels of IL-6R, LIFR, and gp130 expression in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Mean fluorescence intensities (MFIs) relative to those of adherent cells are presented. Results are presented as means ± SD from three independent experiments. ( D ) Western blot analysis of p-gp130, gp130, p-STAT3, and STAT3 was performed in PANC-1 cells cultured in 2D (adherent) or 3D (sphere) conditions. Relative band intensity is provided. ** p < 0.01.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Cell Culture, Expressing, Fluorescence, Western Blot

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: The correlation of gp130/STAT3 pathway-related factor expression with overall survival and H19 expression correlated with gp130/STAT3 pathway-related factors in patients with PDAC. Each survival curve was presented according to the online database GEPIA. ( A ) IL-6 , ( B ) LIF , ( C ) IL-6R , ( D ) LIFR , ( E ) gp130 (IL-6ST) , ( F ) JAK1 , ( G ) STAT3 , ( H ) TGF β-RII (TGFBR2) , ( I ) MT1-MMP (MMP14) , and ( J ) H19 . ( K ) The correlation of H19 and gp130/STAT3 pathway-related factor expression in patients with PDAC was assessed using Spearman rank correlation analysis according to the online database GEPIA.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Expressing

Journal: Cancers

Article Title: Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression

doi: 10.3390/cancers14092055

Figure Lengend Snippet: Schematic representation of autocrine/paracrine IL-6 or the LIF/gp130/STAT3 pathway in PDAC sphere cells. In PDAC sphere cells in which CSCs are enriched (CSC-like cells), binding of autocrine/paracrine IL-6 or LIF to each receptor (IL-6R or LIFR, respectively) induces gp130 homodimerization or LIFR/gp130 complex formation to thereby activating JAKs followed by phosphorylation of gp130, ultimately leading to STAT3 activation. This pathway contributes to the maintenance of stemness features and expression of MT1-MMP and TGFβ-RII. Expression of TGFβ-RII via the gp130/STAT3 pathway affects TGF-β1/Smad signaling to promote EMT induction. Additionally, p-STAT3 can access the active promoter region of H19 and contribute to its transcription. Therefore, autocrine/paracrine IL-6 or the LIF/gp130/STAT3 pathway in PDAC CSC-like cells is believed to eventually lead to invasion and metastasis, both of which are hallmarks of malignancy.

Article Snippet: Cells were harvested, and dissociated single cells were incubated on ice for 30 min with PE-conjugated anti-human IL-6 receptor α (IL-6Rα) antibody (BioLegend, San Diego, CA), PE-conjugated anti-human LIF receptor (LIFR) antibody (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-human gp130 antibody (BioLegend), or PE-conjugated isotype control (Becton Dickinson) diluted in FACS buffer (0.5% ( w / v ) BSA and 0.1% ( w / v ) sodium azide in PBS).

Techniques: Binding Assay, Phospho-proteomics, Activation Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Leukemia Inhibitory Factor Induces Proopiomelanocortin via CRH/CRHR Pathway in Mouse Trophoblast

doi: 10.3389/fcell.2021.618947

Figure Lengend Snippet: CRH and LIF expression in mouse placenta during development. (A) Representative immunoblots and (B) quantification of CRH and LIF protein levels in mouse placenta at 13.5, 15.5, and 17.5 dpc. The housekeeping protein β-actin (42 kDa) was used as a loading control. Densitometry reveals a significant increase in CRH peptide and LIF at day 13.5 dpc in mouse placenta ( n = 4 of each group), with each point standardized to 17.5 dpc. All data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs. control (Student’s t -test). (C) Placental sections obtained from mice at 13.5, 15.5, and 17.5 dpc underwent immunofluorescence staining using a CRH-specific antibody ( left panel ) and LIFR-specific antibody ( right panel ). Nuclei were stained with Hoechst 33342 ( blue ). Scale bar 50 μm. Dec decidua, JC junctional zone, Lab labyrinth, Cont negative control at13.5 dpc.

Article Snippet: Sections were incubated overnight at 4°C with a rabbit anti-mouse CRF antibody (2 mg/mL; Bioss; RRID:AB_10885736 ) and a rabbit anti-mouse LIF receptor (LIFR) antibody (0.2 mg/mL; Santa Cruz Biotechnology) after washing with PBS.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, Negative Control