Journal: Advanced Science

Article Title: S100A8/A9‐High Macrophages Activate Intestinal Fibroblasts via mCCL6/hCCL15‐CCR1 Axis to Drive Intestinal Fibrosis in Crohn's Disease

doi: 10.1002/advs.76353

Figure Lengend Snippet: S100A8/A9 hi macrophages mediate CCL6 expression through activation of the transcription factor STAT3. (A) Venn diagram integrating multiple bioinformatic databases to identify potential upstream transcription factors regulating Ccl6 expression. (B) Protein‐protein interaction network between the nine transcription factors and S100A9. (C–F) BMDMs were stimulated with recombinant S100A8‐S100A9 protein (1 µg/mL) with or without pretreated with FPS‐ZM1 (1 µ m ), TAK‐242 (1 µ m ) or DMSO vehicle for 1 h. p‐STAT3 and total STAT3 were assessed by Western blot at 2 h (C and D), and CCL6 levels in culture supernatants were measured by ELISA at 24 h (E). The colocalization of S100A8/A9 with TLR4 in BMDMs was assessed by immunofluorescence staining (F); Scale bar, 20 µm. (G,H) Following pretreatment with or without Stattic (5 µ m ) for 1 h, BMDMs were stimulated with recombinant S100A8‐S100A9 protein (1 µg/mL). p‐STAT3 and total STAT3 levels were assessed by Western blot at 2 h (G), and CCL6 in culture supernatants was quantified by ELISA at 24 h (H). (I,J) BMDMs were transfected with siNC or si S100a9 , followed by treatment with Colivelin TFA (50 µg/mL) for 4 h to activate STAT3. Protein levels of S100A9, p‐STAT3/STAT3, and CCL6 were measured by Western blot (I), and p‐STAT3 expression was visualized by immunofluorescence (J; Scale bar: 500 µm). (K) Western blot analysis of p‐STAT3 levels in nuclear and cytoplasmic fractions of BMDMs transfected with siNC or siS100a9 . (L) Schematic representation of putative STAT3 binding sites within the Ccl6 promoter. (M) Dual‐luciferase reporter assays were performed in HEK 293T cells co‐transfected with a control vector (NC) or a Stat3 expression plasmid, together with reporter vectors pGL1‐Control, pGL1‐Ccl6 wild‐type (WT), or pGL1‐Ccl6 mutant (mut). Promoter activity was measured and normalized. N, O) BMDMs were treated with recombinant S100A8‐S100A9 protein (1 µg/mL) for 2 h. STAT3 recruitment to the Ccl6 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. The enrichment of p‐STAT3 at the promoter region was quantified by RT‐qPCR and expressed as a percentage of the total input (N). Representative agarose gel images confirmed the specificity of the PCR amplification (O). All values are expressed as mean ± SD. ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To investigate the role of the STAT3 pathway in S100A8/A9‐mediated CCL6 production, BMDMs were stimulated with recombinant S100A8‐S100A9 heterodimer (1 μg/mL; HY‐P71076; MCE, USA) for 2 h. To identify the functional receptors mediating S100A8/A9‐induced STAT3 activation, BMDMs were pretreated with the TLR4 inhibitor TAK‐242 (1 μM; HY‐11109; MCE, USA) or the RAGE inhibitor FPS‐ZM1 (1 μM; HY‐19370; MCE, USA) for 1 h. For STAT3 pathway blockade, BMDMs were pretreated with the STAT3 inhibitor Stattic (5 μM; HY‐13818; MCE, USA) for 1 h before stimulation.

Techniques: Expressing, Activation Assay, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Transfection, Binding Assay, Luciferase, Control, Plasmid Preparation, Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Amplification

Journal: Redox Report : Communications in Free Radical Research

Article Title: Macrophage metabolic reprogramming via HIF-1α–glycolysis drives osteoblast ferroptosis and bone loss through an IL-6–STAT3–dependent redox axis

doi: 10.1080/13510002.2026.2667673

Figure Lengend Snippet: Proposed pathophysiological framework of ovariectomy (OVX)-induced osteoporosis and the protective effects of valproic acid (VPA). (Top) OVX disrupts bone homoeostasis, characterised by sustained osteoclast overactivation and compromised osteoblast survival. Concurrently, the accumulation of M1-polarised macrophages is associated with inflammatory infiltration, contributing to exacerbated bone loss. (Bottom) The M1 macrophage secretome, enriched in pro-inflammatory cytokines (e.g. IL-6), creates a microenvironment that supports osteoclastogenesis and contributes to ferroptosis in osteoblasts. VPA counteracts these pathological shifts by targeting HIF-1α to restrict glycolysis-driven M1 polarisation in macrophages. This attenuation of M1 polarisation correlates with reduced IL-6 secretion and the subsequent downregulation of the p -STAT3/HIF-1α/TFRC signalling axis, ultimately alleviating iron overload-associated oxidative stress.

Article Snippet: For targeted osteoblast interventions, MC3T3-E1 cells were treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1; 10 μM, MedChemExpress, #HY-100579), an IL-6 neutralising antibody (1 μg/mL; Invitrogen, #16-7061-81), or the STAT3 specific inhibitor Stattic (5 μM; MedChemExpress, #HY-13818).

Techniques:

Journal: Redox Report : Communications in Free Radical Research

Article Title: Macrophage metabolic reprogramming via HIF-1α–glycolysis drives osteoblast ferroptosis and bone loss through an IL-6–STAT3–dependent redox axis

doi: 10.1080/13510002.2026.2667673

Figure Lengend Snippet: VPA-reprogrammed macrophage secretome rescues osteoblasts from ferroptosis via the IL-6/STAT3 axis. (A) CCK-8 assay assessing the viability of MC3T3-E1 cells treated with the indicated conditioned media or targeted inhibitors. (B) Viability of MC3T3-E1 cells treated with 100 ng/mL LPS for 24 h, to rule out direct endotoxin cytotoxicity. (C, D) Representative merged fluorescence images of JC-1 staining and corresponding quantitative analysis of the red/green fluorescence intensity ratio for evaluating mitochondrial membrane potential.(E) Representative merged fluorescence images of intracellular lipid peroxidation detected using the BODIPY 581/591 C11 probe.(F) Representative fluorescence images of the intracellular labile iron pool detected using the Phen Green SK probe.(G, H) Flow cytometry analysis of oxidised BODIPY 581/591 C11 (FITC channel), with representative stacked histograms and corresponding quantification of mean fluorescence intensity. (I) Intracellular GSH/GSSG ratio measured via biochemical assay. All quantitative data are presented as mean ± SD ( n = 3 independent biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the LPS-CM group.

Article Snippet: For targeted osteoblast interventions, MC3T3-E1 cells were treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1; 10 μM, MedChemExpress, #HY-100579), an IL-6 neutralising antibody (1 μg/mL; Invitrogen, #16-7061-81), or the STAT3 specific inhibitor Stattic (5 μM; MedChemExpress, #HY-13818).

Techniques: CCK-8 Assay, Fluorescence, Staining, Membrane, Flow Cytometry

Journal: Redox Report : Communications in Free Radical Research

Article Title: Macrophage metabolic reprogramming via HIF-1α–glycolysis drives osteoblast ferroptosis and bone loss through an IL-6–STAT3–dependent redox axis

doi: 10.1080/13510002.2026.2667673

Figure Lengend Snippet: Ultrastructural and mechanistic validation of the IL-6/STAT3-driven ferroptosis axis in osteoblasts. (A) Representative transmission electron microscopy (TEM) images of MC3T3-E1 osteoblasts exposed to the indicated treatments. Red arrows indicate typical ferroptotic mitochondria, characterised by reduced volume, increased membrane electron density, and disrupted cristae. (B, C) Representative Western blot images and corresponding quantitative densitometric analysis of p -STAT3 and total STAT3, demonstrating that IL-6 neutralising antibody (anti-IL-6) blocks upstream STAT3 activation. (D–H) Representative Western blot images and corresponding quantifications of ferroptosis-related proteins (TFRC, HIF-1α, GPX4, and ACSL4), revealing that the STAT3 inhibitor Stattic effectively reverses the molecular signature of ferroptosis. All quantitative data are presented as mean ± SD ( n = 3 independent biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the LPS-CM group.

Article Snippet: For targeted osteoblast interventions, MC3T3-E1 cells were treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1; 10 μM, MedChemExpress, #HY-100579), an IL-6 neutralising antibody (1 μg/mL; Invitrogen, #16-7061-81), or the STAT3 specific inhibitor Stattic (5 μM; MedChemExpress, #HY-13818).

Techniques: Biomarker Discovery, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Activation Assay

Journal: International Journal of Oncology

Article Title: Smoking promotes colorectal cancer via the CKAP2L/AREG axis

doi: 10.3892/ijo.2026.5872

Figure Lengend Snippet: CKAP2L promotes proliferation and migration of colorectal cancer cells through the STAT3/AREG/EGFR axis. (A) Expression levels of STAT3 and p-STAT3 proteins measured by western blotting. (B) Calculation of IC 50 in HCT116 cells treated with Stattic for 24 h. (C) Levels of AREG were measured by reverse transcription-quantitative PCR and enzyme-linked immunosorbent assay. (D) Migration of cells was detected by Transwell assay (×100 magnification). (E) Proliferation of cells was measured by Cell Counting Kit 8. (F) Expression levels of proteins measured by western blotting. (G) Binding peak of STAT3 on the promoter region of AREG was detected by Cistrome Data Browser, the binding motif was predicted using the JASPAR database, and the binding of STAT3 to the AREG promoter was evaluated by chromatin immunoprecipitation assay. Data were analyzed using (A and G) Unpaired Student's t-test, and (C-F) two-way ANOVA followed by Tukey test. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. AREG, amphiregulin; CKAP2L, cytoskeleton-associated protein 2-like; EGFR, epidermal growth factor receptor; NC, negative control; p-, phosphorylated; sh, short hairpin; STAT3, signal transducer and activator of transcription 3; TSS, transcription start site.

Article Snippet: The signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitor Stattic (cat. no. HY-13818; MedChemExpress) was dissolved in DMSO for cell culture.

Techniques: Migration, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transwell Assay, Cell Counting, Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: International Journal of Oncology

Article Title: Smoking promotes colorectal cancer via the CKAP2L/AREG axis

doi: 10.3892/ijo.2026.5872

Figure Lengend Snippet: Smoking may promote colorectal cancer progression through the CKAP2L/STAT3/AREG/EGFR axis. This figure was drawn by Figdraw ( https://www.figdraw.com/ ). AREG, amphiregulin; CKAP2L, cytoskeleton-associated protein 2-like; EGFR, epidermal growth factor receptor; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitor Stattic (cat. no. HY-13818; MedChemExpress) was dissolved in DMSO for cell culture.

Techniques:

Journal: Nature Metabolism

Article Title: Tissue-specific fibroblast lipid cues impose the rate of epithelial cancer invasion

doi: 10.1038/s42255-026-01514-y

Figure Lengend Snippet: a , SM metabolic pathway and inhibitors targeting enzymes. b – d , Spheroid invasion of oSCC with control media (grey), 5 µM SM (blue) or 5 µM SM and 20 µM imipramine (yellow) ( b ), oral FB secretomes (grey), with 20 µM imipramine (blue) or 20 µM imipramine and 2 µM S1P (yellow) ( c ) and oral FB secretomes (grey), with 2 µM PF543 (blue) or 2 µM PF543 and 2 µM S1P ( d ) (two-sided Mann–Whitney U- test, n = 6 replicates, two cell lines, two independent experiments). e , S1P immunofluorescence quantification of FADU control (grey), dermal (yellow) and oral (blue) FB secretomes (two-sided Mann–Whitney U- test, n = 6 replicates, three independent FB secretomes). f , Immunofluorescence images of FADU + dermal (left) or oral (right) FB secretomes; blue, Hoechst; orange, phalloidin; green, S1P; scale bar, 20 µm (representative images from n = 6 biologically independent wells). g , Spheroid invasion of oSCC cells treated with oral FB secretomes (grey) with 5 µM S1PR1 inhibitor (S1PR1i, ponesimod, blue) or 10 µM S1PR3 inhibitor (S1PR3i, TY-52156, yellow; Kruskal–Wallis, Dunn’s multiple comparisons, n = 5 replicates). h , Spheroid invasion of oSCC treated with oral FB secretomes (grey) with 20 µM STAT3 inhibitor (STAT3i, STX-0119, blue), 10 µM NF-κB inhibitor (NF-κBi; JSH-23, yellow) or 2 µM AKT inhibitor (AKTi; MK-2206, red; Kruskal–Wallis, Dunn’s multiple comparisons, n = 5 replicates). i , Western blot of STAT3, phospho-STAT3 Tyr705 , B-actin in oSCC (FADU) + dermal or oral FB secretomes, ±PF543, S1P rescue, S1PR1 inhibitor (ponesimod) or STAT3 inhibitor (STX-0119); numbers represent normalized pSTAT3 to total STAT3 fold change relative to oral FB secretomes, run on separate blots with independent loading controls under identical conditions (western blots representative of two independent experiments). j , oSCC basal respiration in oSCC cells treated with oral FB secretomes ± lipid stripping (Cleanascite, blue, 24 h; OCR, oxygen consumption rate; n = 24 replicates in two cell lines treated with three FB secretomes, two independent experiments, two-sided Mann–Whitney U- test). k , Mitochondrial activity in oSCC cell lines treated with oral FB secretomes (blue) measured by membrane potential-dependent immunofluorescence (two-sided Mann–Whitney U- test, n = 8 replicates, two independent cell lines). l , Mitochondrial abundance in oSCC cell lines treated with oral FB secretomes (blue) measured by immunofluorescence (two-sided Mann–Whitney U- test, n = 8 replicates, two independent cell lines). m , Basal respiration in oSCC cells treated with combinations of oral FB secretomes, 20 µM imipramine, 2 µM S1P or 20 µM STAT3i (STX-0119; n = 5 independent replicates, two-sided Mann–Whitney U- test). n , Subcutaneous tumour growth rate in NSG mice injected with oSCC UMSCC01 (grey), UMSCC01 + dermal FBs (oSCC + dermal FB, yellow) or UMSCC01 + oral FBs (oSCC + oral FB, blue; two-sided Mann–Whitney U- test, n = 7 per group). o , Ki67 proliferation quantification in oSCC (grey), oSCC + dermal FB (yellow) and oSCC + oral FBs (blue) tumours (two-sided Mann–Whitney U- test, n = 14, two measurements per tumour). p , Kaplan–Meier survival curve of oSCC (black) and oSCC + oral FBs (blue; n = 7 per group, two-sided Mantel–Cox test). Box plots show minimum to maximum values (error bars), the box indicates 25th and 75th percentiles, and the line denotes the median.

Article Snippet: STAT3 inhibitor STX-0119 (MedChemExpress, HY-103692-1ml), AKT inhibitor MK-2206 (MedChemExpress, HY-10358-1ml) and NF-κB inhibitor JSH-23 (MedChemExpress, HY-13982-1ml) were supplied at 10 mM in DMSO and diluted to 20 μM, 2 μM and 10 μM, respectively, in secretomes.

Techniques: Control, MANN-WHITNEY, Immunofluorescence, Western Blot, Stripping Membranes, Activity Assay, Membrane, Injection

Journal: Nature Metabolism

Article Title: Tissue-specific fibroblast lipid cues impose the rate of epithelial cancer invasion

doi: 10.1038/s42255-026-01514-y

Figure Lengend Snippet: a , oSCC spheroid invasion with oral fibroblast secretome treated with (blue) or without (grey) 5 µM ARC39 (two-sided Mann Whitney U, n = 6 replicates, two cell lines, two independent experiments). b , oSCC (FADU) spheroid invasion with oral fibroblast secretome (grey) with 5 µM (blue) and 10 µM (yellow) carmofur (Kruskal-Wallis, Dunn’s multiple comparisons, Oral FB, 10 µM: n = 8 replicates, 5 µM: n = 7). c , oSCC spheroid invasion +/- oral fibroblast secretomes control (grey), 20 µM SPHK2 inhibitor ABC294640 (blue) (two-sided Mann Whitney U, ns: non-significant, n = 5 replicates, two cell lines, two independent experiments). d , Spheroid invasion of oSCC with control media (grey) or sphingsine-1-phosphate (S1P, 1 µM yellow, 2 µM blue), (two-sided Mann Whitney U, n = 6 replicates, two cell lines, two independent experiments). e , Proliferation (relative confluence) of oSCC treated with 20 µM sphingomyelin (SM) and with 20 µM imipramine or 2 µM PF543 for 24 h (two-sided Mann Whitney U, n = 5 replicates, bars represent mean, error bars SEM). f , Replicate western blot of STAT3, phospho-STAT3Tyr705, B-actin in oral SCC (FADU) + dermal or oral fibroblast secretome, +/- PF-543, S1P rescue, S1PR1 inhibitor (ponesimod), or STAT3 inhibitor (STX-0119), run on separate blots with independent loading controls under identical conditions. g , Fold change in pSTAT3 levels relative to oral fibroblast secretome (Oral sec) in FADU western blots (n = 2 independent western blots). h , Relative expression (RE) of EMT genes VIM, FN1, and SNAI2 in oral SCC cells treated with oral fibroblast secretome (grey) and 20 µM STAT3 inhibitor (STAT3i, blue) STX-0119 (two-sided Mann Whitney U, n = 9 replicates across three independent samples). i , Oxygen consumption rate (OCR) plots of Seahorse assay of oSCC treated with fibroblast secretome ± Cleanascite (control: n = 8 biological replicates, oral FB: n = 24 replicates in two cell lines treated with three fibroblast secretomes). j , ATP production in oSCC cells treated with oral fibroblast secretomes +/- lipid stripping (Cleanascite: blue) (OCR: oxygen consumption rate, n = 24 replicates in two cell lines treated with three fibroblast secretomes, two independent experiments, two-sided Mann Whitney U). k , Basal respiration and ATP production ( l ) in oSCC (FADU) treated with sphingomyelin (SM), sphingosine-1-phosphate (S1P) or oral fibroblast secretome (24 h, two-sided Mann Whitney U, n = 5 replicates). m , Oxygen consumption rate (OCR) plots of Seahorse assay of FADU treated with sphingomyelin (SM), sphigosine-1-phosphate (S1P) or oral fibroblast secretome (n = 5 biological replicates). n , Representative immunofluorescence images of oSCC mitochondria stained with MitoView 633 (red), before and after exposure to oral fibroblast secretome, scale = 25 µm. o , Basal respiration of FADU cells treated with fibroblast secretome, Cleanascite treated secretome and rescue with addition of 2 µM S1P (Kruskal-Wallis, Dunn’s multiple comparisons, n = 5 replicates). Boxplots: minimum to maximum values (error bars), box: 25th and 75th percentiles; line: median. Bar plots display mean ± standard error of the mean.

Article Snippet: STAT3 inhibitor STX-0119 (MedChemExpress, HY-103692-1ml), AKT inhibitor MK-2206 (MedChemExpress, HY-10358-1ml) and NF-κB inhibitor JSH-23 (MedChemExpress, HY-13982-1ml) were supplied at 10 mM in DMSO and diluted to 20 μM, 2 μM and 10 μM, respectively, in secretomes.

Techniques: MANN-WHITNEY, Control, Western Blot, Expressing, Stripping Membranes, Immunofluorescence, Staining