Article Title: Overturning the paradigm that IL6 signaling drives liver regrowth while shining light on a new therapeutic target for regenerative medicine
Figure Lengend Snippet: STAT-independent HyperIL6 activity inhibits APAP- or IL11-stimulated cell death through competitive binding to the shared gp130 co-receptor. ( A ) Representative fluorescent images and ( B ) quantification of DRAQ7 staining for cell death (scale bars, 200 µm) (n=3 independent experiments, 23 images per experiment) in APAP (20 mM) -treated hepatocytes in the presence of IgG (2 µg/ml), DMSO, anti-IL11RA (X209, 2 µg/ml), HyperIL6 (20 ng/ml), HyperIL6 supplemented with iSTAT3 (S3I-201, 20 µM), or anti-gp130 (2 µg/ml). ( C ) Western blots showing phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-JNK, JNK, NOX4, and GAPDH levels in APAP-treated hepatocytes in the presence of IgG, X209, HyperIL6, or anti-gp130. ( D ) Western blots of phosphorylated ERK, AKT and STAT3 protein and their respective total expression in hepatocytes in response to HyperIL6 stimulation. ( E ) GSH levels (n=4) in APAP-treated hepatocytes. ( F ) Representative fluorescent images of DCFDA (2’, 7’-dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100 µm) (n=4 independent experiments, 10 images per experiment) in APAP-treated hepatocytes. ( G ) Western blots showing ERK, STAT3 and JNK activation status, NOX4 protein expression in APAP-treated hepatocytes in the presence of DMSO, HyperIL6, or HyperIL6 supplemented with iSTAT3. ( H ) Proposed mechanism for competition of IL11 cis-signaling and IL6 trans-signaling by binding to gp130. ( I ) ALT secretion (n=4) and ( J ) Western blots showing ERK, STAT3, and JNK activation status, NOX4 protein expression by rhIL11 (10 ng/ml) -treated hepatocytes following a dose range stimulation of either HyperIL6 or sgp130 in the presence of iSTAT3. ( A-G, I-J ) Primary human hepatocytes; ( A-C, E-G, I-J ) 24 h stimulation. ( E, I ) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-max values (whiskers), one-way ANOVA with Dunnett’s correction.
Article Snippet: Recombinant proteinsCommercial recombinant proteins: rhIL11 (UniProtKB: P20809, Genscript), rmIL11 (UniProtKB: P47873, Genscript), rh-HyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R & D systems), rm-HyperIL6 (IL6R:IL6 fusion protein, 9038-SR, R & D systems), human soluble gp130 Fc (671-GP-100, R & D systems).
Techniques: Activity Assay, Binding Assay, Staining, Western Blot, Expressing, Activation Assay, Whisker Assay