Fig 2: IL11 induces pancreatic stellate cell activation and invasion. (a) t-distributed stochastic neighbour embedding (tSNE) plots showing scRNA-seq expression of Il11ra1, Il6ra and Il-6st (gp130) in mouse pancreatic tissue. Cell clusters were identified using the Tabula Muris web tool (https://tabula-muris.ds.czbiohub.org/ (accessed on 21 February 2022)). [20]. Black arrows indicate pancreatic stellate cells (PSCs). (b) Representative immunostaining images of gp130, IL11RA, IL6RA in PSCs. Cells were counterstained with DAPI. Scale bars: 50 µm. (c) Western blot analysis of phosphorylated and total ERK and STAT3 and aSMA in lysates from PSC treated with IL11 (10 ng/mL) or IL 6 (10 ng/mL) across the indicated time-points (0 to 24 h). GAPDH serves as a loading control. (d) ELISA-based quantification of secreted MMP2 levels in PSC supernatants. (e,f) Representative immunofluorescence images and quantification of aSMA+ve cells, Collagen I intensity/area and EdU+ve cells at baseline and after 24 h treatment with either recombinant human TGFß1 (5 ng/mL), IL11 (5 ng/mL), bFGF (10 ng/mL), CTGF (50 ng/mL), PDGF (200 ng/mL) or EDN1 (250 ng/mL). Cells were counterstained with DAPI. Scale bar: 200 µm. (g) Matrigel invasion capacity of PSCs was determined at baseline and after 24 h treatment with PDGF (20 ng/mL) or with increasing concentrations of IL11 (5–20 ng/mL). Scale bars: 150 µm. AU: Arbitrary Unit. Data are represented as mean ± SD in panel (d) and median and whiskers extending from minimum to maximum values in panels (f,g). p values were determined by one-way ANOVA with Dunnet’s correction. BL: baseline.

Fig 3: Cells were cultured in 5 mmol/L (control) or 25 mmol/L (high glucose; HG) glucose throughout the completion of each cellular activity assay.In all graphs, values indicate mean ± SEM. (A) Growth of abEPCs was assessed as cell viability over a 12 day period in high serum media (EBM2/20% FBS). A spike in activity occurred in the hyperglycemic group at day 6 (*p<0.05 compared to cells in control media). However, abEPCs showed no significant differences between control and HG-treated groups over long-term culture. (B) Caspase-3 activity level was measured in abEPCs and HDMECs exposed to control or HG media (in EBM-2/1% FBS) for 24 hours [*p<0.05 compared to respective control media; †p<0.05 compared to HDMECs in HG media; n = 3]. (C) A 24-hour migration assay was performed on abEPCs, assessed as their ability to migrate through an 8-µm pore with bFGF as the chemoattractant. HG had no effect on the migratory abilities of abEPCs, cbEPCs, and HDMECs. (D) Growth of bmMPCs was assessed as cell viability over a 12 day period in high serum media (DMEM/20% FBS). HG-bmMPCs showed significantly less growth at day 1 as compared to control (*p<0.05). However, growth over long-term culture appeared to be unaffected by HG. (E) Migration of bmMPCs was assessed in the presence of 10% FBS as the chemoattractant. Exposure of bmMPCs to HG significantly reduced the migratory ability towards FBS (*p<0.05).

Fig 4: Autocrine IL11 activity causes mesenchymal transition of human renal TECs and paracrine IL11 activity from TECs stimulates myofibroblasts.a Representative IF images of aSMA+ve and SNAI1+ve cells and Collagen 1 immunostaining (scale bars: 100 µm, representative dataset from n = 3/group) and b Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3ß, GSK3ß, SNAI1, ZEB, E-Cadherin, PCNA, Cyclin D1, aSMA, and GAPDH (representative dataset from n = 4/group) from TECs stimulated with either IL11 or TGFß in the presence of IgG/X203. c Heatmaps showing quantification of aSMA+ve and SNAI1+ve cells and Collagen 1 intensity/area (n = 14/group; values are shown in Supplementary Fig. 8c) and d Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3ß, GSK3ß, SNAI1, ZEB, E-Cadherin, PCNA, Cyclin D1, aSMA, and GAPDH for TECs stimulated with various pro-fibrotic factors in the presence of IgG/X203 (representative dataset from n = 4/group). e Quantification of aSMA+ve and SNAI1+ve cells and Collagen 1 intensity/area from TECs stimulated with TGFß or IL11 subjected to siRNA knockdown for SNAI1 (NT: non-targeting siRNA control) (24 h; n = 14/group; Bsl, baseline). f Schematic showing mechanism and signaling pathway by which IL11 induces repression of SNAI1 transcription. g Schematic and h quantification of aSMA+ve cells and Collagen 1 immunostaining of HKFs (n = 14/group) for media transfer experiments in which conditioned media from control or TGFß-stimulated TECs (24 h) were used to treat primary human kidney fibroblasts (HKFs) in the presence of anti-TGFß (1D11) alone or with either IgG or X203. a–h AngII (100 nM), bFGF (10 ng/ml), Endothelin 1 (ET-1, 250 ng/ml), H2O2 (0.2 mM), IL11 (5 ng/ml), PDGF (20 ng/ml), TGFß1 (5 ng/ml), IgG (2 µg/ml), X203 (2 µg/ml), anti-TGFß (clone 1D11, 2 µg/ml), siNT/siSNAI1 (25 nM); 24-h stimulation. e, h Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers); one-way ANOVA with Tukey’s correction (except for H, aSMA+ve which was analyzed by Kruskal–Wallis with Dunn’s correction). Bsl: Baseline; I/A: Intensity per area. Source data are provided as a Source data file.

Fig 5: Representative photomicrographs obtained from culture wells before (day 0) and 7 days after incubation with the tri-lineage differentiation media. The overall morphological features of gonocytes (black arrows), embryoid body-like colonies (EBLCs) (dotted circles), and somatic cell monolayer (white arrowheads) did not differ between control groups provided with regular media (DMEM + 15% FBS + 10 ng/mL bFGF) and treatment groups incubated with ectoderm, mesoderm, or endoderm differentiation media. However, the diameter of EBLCs decreased in the three-differentiation media compared with those in the control group (0.001 < p ≤ 0.002). Also, the EBLCs in differentiation media possessed a narrow marginal area close to the periphery (black arrowheads) and more central areas (*). Insets show higher magnification. (Scale bar = 150 µm). “A” and “B” denote significant differences and p < 0.05 was considered as the significance level.