1 Volume 19, Issue 2, Pages 281-294 (April 2017)An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation  Jonna Saarimäki-Vire, Diego Balboa, Mark A. Russell, Juha Saarikettu, Matias Kinnunen, Salla Keskitalo, Amrinder Malhi, Cristina Valensisi, Colin Andrus, Solja Eurola, Heli Grym, Jarkko Ustinov, Kirmo Wartiovaara, R. David Hawkins, Olli Silvennoinen, Markku Varjosalo, Noel G. Morgan, Timo Otonkoski  Cell Reports  Volume 19, Issue 2, Pages (April 2017) DOI: /j.celrep Copyright © 2017 The Author(s) Terms and Conditions

2 Figure 1 Correction of STAT3K392R Mutation with CRISPR/Cas9 Rescues the Premature Differentiation Phenotype (A) Seventeen-days differentiation protocol (see Experimental Procedures for details). Abbreviations: ACTA, activin A; B27, B-27 supplement; CHIR, CHIR-99021, GSK-3 inhibitor; CYC, cyclopamine; F10, fibroblast growth factor 10; LDN, LDN Alk inhibitor; RA, retinoid acid; RPMI, RPMI-1640 medium. No SB was used during stages 2 and 3 in this protocol. (B) Schematic presentation of the targeting strategy for CRISPR/Cas9-mediated mutation correction. See also Figure S3. (C) DNA sequences of correction template, STAT3K392R, and CORRECTED cell lines. See also Figure S6. (D) Relative gene expression of pancreatic genes in CTRL (n = 8–15/stage independent experiments; two different cell lines), STAT3K392R (n = 7–12/stage independent experiments; three different cell lines), and CORRECTED (n = 3; three different CORRECTED clones) cells. Data represent the mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Significant differences between STAT3K392R (STAT3) cells relative to control (CTRL) and CORRECTED cells are shown as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S4. (E) Double immunocytochemistry for NEUROG3 (NGN3) plus SOX9 and INSULIN plus CHROMOGRANIN A in CTRL, STAT3K392R, and two corrected lines after 17 days differentiation. The scale bar represents 100 μm. See also Figure S5. (F) Quantification of NEUROG3+ cells in STAT3K392R (n = 3; three different clones) and CORRECTED cells (n = 3; three different corrected clones) at day 17 of differentiation. Data represent the mean ± SEM. Statistical analysis was performed with Student’s t test. ∗p < 0.05. (G) Quantification of CHGA+ and INS+ cells in STAT3K392R (n = 3; three different clones) and CORRECTED cells (n = 3; three different corrected clones) at D17 of differentiation. Data represent the mean ± SEM. Statistical analysis was performed with Student’s t test. ∗∗p < 0.01. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

3 Figure 2 Extended Differentiation into Islet-like Aggregates(A) Protocol for extended differentiation (for abbreviations, see Supplemental Experimental Procedures). (B) Gene expression levels analyzed by qRT-PCR of pancreatic genes in CTRL (n = 4; pooled data from two healthy-donor control lines and two CORRECTED clones) and STAT3K392R (four independent experiments) cells. Data represent the mean ± SEM. ∗p < 0.05; Student’s t test. Note the logarithmic scale for INS and GCG. (C) Immunocytochemistry analysis for PDX1, NKX6.1, SOX9, NEUROG3, CHGA, and INS after D10 differentiation in monolayer, in CTRL (healthy donor) STAT3K392R cells. The scale bars represent 100 μm. (D) Quantification of NEUROG3+ cells in CTRL (healthy-donor; n = 3; three different cell lines), STAT3K392R (n = 4; HEL72D), and CORRECTED cells (n = 4; two different corrected clones) at day 17 of differentiation. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. Significant differences between STAT3K392R (STAT3) cells and CTRL or CORRECTED cells are shown as ∗p < 0.05 and ∗∗p < 0.01. (E) Immunohistochemistry analysis for PDX1, NKX6.1, CHGA, INS, and GCG on sections of islet-like clusters at day 30. The scale bars represent 100 μm. (F) Flow cytometry analysis of dissociated islet-like clusters with NKX6.1 and INS antibodies. Data represent the mean ± SEM. (G) Flow cytometry analysis of dissociated islet-like clusters with GCG and INS antibodies. Results are presented separately for single- and double-hormone-positive cells. Data represent the mean ± SEM. Student’s t test; ∗p < 0.05. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

4 Figure 3 Differentially Expressed Genes between STAT3K392R and Mutation-Corrected Clones Reveal a NEUROG3-Regulated Network (A) The most up- and downregulated genes related with endocrine differentiation in STAT3K392R cells. NEUROG3 levels are marked in red. See also Figure S6. (B) Interaction network of genes regulated by NEUROG3 differentially expressed in STAT3K392R cells. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

5 Figure 4 Premature Differentiation Is Not Caused by Increased STAT3-Y705 Phosphorylation (A) Relative gene expression analyzed by qRT-PCR of pancreatic progenitor genes (PDX1 and NKX6.1) and differentiation markers NEUROG3 (NGN3), INS, and GCG in CTRL and STAT3K392R cells with and without IL-6 or LIF stimulation during stages 2 and 3 of differentiation to test whether activation of STAT3 signaling pathway is enough to mimic effect of STAT3 mutation. Data represent the mean ± SEM of two to five independent experiments. Statistical analysis with one-way ANOVA did not reveal significant differences between treatment groups. (B) Western blot analysis of total STAT3 and pSTAT3-Y705 in pancreatic progenitors representing either STAT3K392R (two different clones; HEL72.1 and HEL72D) or controls (two corrected lines and one control line) at 11 days of differentiation. Exposure time for STAT3 blots was for 1 s and for pSTAT3 blots 10 min. Quantitative analysis was performed by normalizing values against ACTIN expression, and the data are expressed relative to CTRL. Data represent the mean ± SEM. See also Figure S7. (C) The same cells as in (B) after IL-6 stimulation for 30 min. Exposure time for STAT3 was 1 s and 22 s for pSTAT3. Quantitative analysis was performed by normalizing values against ACTIN expression, and the data are expressed relative to CTRL. Data represent the mean ± SEM. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

6 Figure 5 K392R Mutation Does Not Alter the Intrinsic DNA-Binding Ability of STAT3 (A) COS7 cells were transfected with an empty vector or expression vector for STAT3 (S3 WT) or STAT3K392R mutant (S3 KR). Cells were stimulated, where indicated, with pervanadate for 20 min to induce STAT3 phosphorylation and nuclear extract used in the binding reaction with an infrared-dye-labeled STAT3 consensus sequence. The complexes were resolved on a native polyacrylamide gel and visualized with LI-COR Odyssey instrument. (B) Oligonucleotide pull-down with the indicated oligonucleotides using whole-cell extracts of COS-7 cells transfected as in (A). Oligonucleotide-bound proteins were resolved by SDS-PAGE, blotted and detected with a STAT3 antibody. (C) Oligonucleotide pull-down using whole-cell extracts of HEK293T cells transfected as in (A) and detected by STAT3 immunoblotting. (D) NEUROG3 promoter activity in PANC1 cells after transfection of cells with empty vector (E/V), normal STAT3 (STAT3 WT), STATK392R, or STAT3Y640F (highly activating STAT3 mutation) in unstimulated (−IL-6) and stimulated (+IL-6) conditions. Data represent the mean ± SEM of three independent experiments. Statistical significance against empty vector ∗p < 0.05; ∗∗∗p < 0.001; statistical significance against K392R a = p < 0.05; one-way ANOVA followed by Tukey’s test. (E) STAT3-regulated SIE (sis-inducible element) promoter activity in PANC1 cells in unstimulated (−IL-6) and stimulated (+IL-6) conditions. Statistics as in (D). Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

7 Figure 6 K392R Mutation Increases STAT3 Nuclear Localization(A) Biotin proximity assay identifies interactions between STAT3 and proteins involved in transcriptional regulation (STAT1 [known STAT3 partner], GSE1, TLE3, MTA1, BCOR, and DIDO1), chromatin remodeling (SMARCC2, YEATS2, SMARCA4, WDR5, and MYSM1), and nuclear transport (NUP50, RANBP3, NUP62, IMA1, NU153, and NXF1). Compared to the STAT3 WT, STATK392R in general shows significantly increased interactions with nuclear proteins and notably transport proteins involved in nuclear import, suggesting an increased nuclear translocation. ∗, significantly different interacting proteins; p < 0.05; Student’s t test; n = 3. ∗∗, detected proteins interacting only with STAT3 K392R or WT. (B) Nuclear localization of STAT3 determined by manual blinded scoring of HEK293 overexpressing STAT3 WT, STAT3 K392R, or STAT3 Y640F, a mutant with increased nuclear localization. n = 16–30 individually scored cells. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparison test. Differences between the conditions are marked in the figure. ∗∗p < 0.01; ∗∗∗p < (C) Nuclear localization of STAT3 determined by measuring nuclear to cytoplasmic ratio of STAT3 versions overexpressed in HEK293 cells as in (B). n = 26–30 individually measured cells. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparison test. Differences between the conditions are marked in the figure. ∗p < 0.05; ∗∗∗p < (D) Nuclear localization of STAT3 determined by quantification of nuclear intensity of stained STAT3 WT and STAT3 K392R-overexpressing HEK293 cells, treated or not with IL-6. Data represent mean ± SEM; n = 3 biological replicates. Analysis was performed on five random fields imaged per well, with an average of 63 nuclei/image, 315 nuclei/well, and 945 nuclei/condition. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparison test. Stars mark the differences between measured condition and WT STAT3. ∗p < 0.05. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

8 Figure 7 Prematurely Differentiated STATK392R Cells Form GCG-Positive Cells after Transplantation (A) Hematoxylene-Eosin (HE) staining of pancreatic grafts derived from CTRL (healthy donor) and STAT3K392R iPSCs grafts removed at 3 months after transplantation under the kidney capsule of NSG mice (borderline between kidney and graft tissue marked by a dashed line). Four grafts were produced using the 30-day, seven-stage protocol for islet-like aggregates (Figure 2; two derived from healthy-control iPSCs and two from mutant iPSCs) and four grafts using the 17-day, four-stage pancreatic progenitor protocol (Figure 1; two derived from healthy-control iPSCs and two from mutant iPSCs), totally eight different grafts, four per genotype. The scale bars represent 200 μm. (B) Double immunohistochemistry of graft sections for INS + CHGA (top), INS+ GCG (middle), and PDX1 + C-PEPTIDE (CPEP) (bottom). The scale bars represent 100 μm. (C) Quantification of relative proportion of INS+, GCG+, and double-positive cells in CTRL and STAT3K392R grafts (n = 4 individual grafts per genotype, totaling eight different grafts). Statistical analysis was performed with Student’s t test; ∗∗p < 0.01. Cell Reports  , DOI: ( /j.celrep ) Copyright © 2017 The Author(s) Terms and Conditions

9 Cell Reports 2017 19, 281-294DOI: (10.1016/j.celrep.2017.03.055)Copyright © 2017 The Author(s) Terms and Conditions