1. Volume 19, Issue 8, Pages 1512-1521 (May 2017)
Using hESCs to Probe the Interaction of the Diabetes-Associated Genes CDKAL1 and MT1E 
Min Guo, Tuo Zhang, Xue Dong, Jenny Zhaoying Xiang, Minxiang Lei, Todd Evans, Johannes Graumann, Shuibing Chen 
Cell Reports 
Volume 19, Issue 8, Pages (May 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 19, 1512-1521DOI: (10.1016/j.celrep.2017.04.070)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
CDKAL1−/− Causes the Downregulation of MT Genes, and Forced Expression of MT1E Rescues the Hypersensitivity of CDKAL1−/− Cells to Glucotoxicity and Lipotoxicity
(A) The relative fold change of the top 20 most downregulated genes in CDKAL1−/− insulin-GFP+ cells compared with WT insulin-GFP+ cells.
(B) qRT-PCR analysis of MT1E, MT1H, and MT1M gene expression in WT and CDKAL1−/− insulin-GFP+ cells.
(C and D) Immunocytochemistry analysis (C) and the quantification (D) of the percentage of PI+/insulin+ cells in WT, CDKAL1−/− insulin+ cells, or CDKAL1−/− insulin+ cells with forced expression of MT1E (MT1E), MT1H (MT1H), or MT1M (MT1M) when cultured in the presence of 2 mM d-glucose (ctrl-g), 35 mM d-glucose (glu), no palmitate (ctrl-p) or 1 mM palmitate (palm). PI+/insulin+ cells are highlighted by arrows. Scale bar, 50 μm. In (D), data are presented as mean ± SD.
(E and F) Flow cytometry analysis (E) and the quantification (F) of apoptotic rate of WT, CDKAL1−/− insulin+ cells, or CDKAL1−/− insulin+ cells expressing MT1E, MT1H, or MT1M. The data are presented as mean ± SD. n = 3 independent biological replicates for each condition. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t test were ∗,#p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figures S1 and S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

4. Figure 2
Forced Expression of MT1E Rescues the Functional Defects of CDKAL1−/− Cells In Vitro
(A–C) Human c-peptide (% of content) (A) and fold change of WT, CDKAL1−/−, CDKAL1−/−-MT1E cells at D30 with or without 50 μM IBMX (B) or 20 μM forskolin (Fosko), (C) stimulation in the presence of 2 mM d-glucose. n = 6 biological replicates.
(D and E) Human c-peptide (percentage of content, D) and fold change (E) of WT, CDKAL1−/−, CDKAL1−/−-MT1E cells at D30 with 2 or 20 mM d-glucose. n = 6 biological replicates. Forsk, forskolin; LG, 2 mM d-glucose; HG, 20 mM d-glucose. Human c-peptide secretion was calculated by dividing the secreted c-peptide by the total c-peptide of insulin-GFP+ cells or primary human beta cells. p values calculated by unpaired two-tailed Student’s t test were ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Data are presented as mean ± SD. See also Figure S3.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

5. Figure 3
Forced Expression of MT1E Rescues the Functional Defects and Cell Apoptosis of CDKAL1−/− Cells after Transplantation into STZ-Treated Immunodeficient Mice
(A) Human insulin GSIS at 2 weeks after transplantation of WT, CDKAL1−/−, and CDKAL1−/−-MT1E D30 cells.
(B) GSIS secretion of SCID-beige mice carrying human cells at 6 weeks after transplantation. p values calculated by one-way repeated-measures ANOVA.
(C and D) Intraperitoneal glucose tolerance test (IPGTT, C) and area under the curve (AUC, D) of STZ-treated mice 6 weeks after transplantation. In (C), data are presented as mean ± SD.
(E and F) Immunocytochemistry analysis (E) and quantification (F) of the percentage of TUNEL+/insulin+ (TUNEL+/INS+) cells in of WT, CDKAL1−/−, and CDKAL1−/−-MT1E D30 cells at 8 weeks after transplantation. Scale bar, 100 μm.
In (C)–(F), p values were calculated by two-way repeated-measures ANOVA with a Bonferroni test for multiple comparisons between WT and mutant cells. n = 8 mice for each condition. n.s. indicates a non-significant difference. p values were ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < The bottom and top of the box represent the first and third quartiles; the band inside the box represents the median. The ends of the whiskers represent the minimum and maximum of all the data. See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

6. Figure 4
Forced Expression of MT1E Rescues the ER Stress, Cell Death, and the Increased Production of Cellular ROS Induced by the Loss of CDKAL1
(A) Gene set enrichment analysis (GESA) of CDKAL1−/− and CDKAL1−/−-MT1E D30 insulin-GFP+ cells cultured in 35 mM d-glucose condition.
(B) qRT-PCR analysis of CHOP10, EDEM, and PDI expression in CDKAL1−/− and CDKAL1−/−-MT1E D30 insulin-GFP+ cells cultured in 35 mM d-glucose condition.
(C) Electron microscopy images of WT, CDKAL1−/−, and CDKAL1−/−-MT1E D30 insulin-GFP+ cells cultured in 35 mM d-glucose condition. Scale bar, 500 nm. ER was highlighted by red arrows.
(D) Flow cytometry analysis and the quantification of cellular reactive oxygen species (ROS) for CDKAL1−/− and CDKAL1−/−-MT1E D30 insulin-GFP+ cell when cultured in the presence of 2 mM d-glucose (ctrl-g) or 35 mM d-glucose (glu).
The data are presented as mean ± SD. See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

7. Figure 5
Chemical Chaperones Rescue Hypersensitivity to Glucotoxicity and Lipotoxicity Caused by the Loss of CDKAL
(A and B) Immunocytochemistry analysis (A) and the quantification (B) of the percentage of PI+/insulin+ cells in WT, CDKAL1−/−, and CDKAL1−/−-MT1E insulin-GFP+ cells treated with 1 mM 4PBA or 200 μM TUDCA when cultured in the presence of 2 mM d-glucose (ctrl-g), 35 mM d-glucose (glu), no palmitate (ctrl-p), or 1 mM palmitate (palm). PI+/insulin+ cells are highlighted by arrows.
(C and D) Flow cytometry analysis (C) and the quantification (D) of apoptotic rate for WT, CDKAL1−/−, and CDKAL1−/−-MT1E insulin-GFP+ cells treated with DMSO, 4PBA, or TUDCA.
n = 3 independent biological replicates for each condition. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t test were ∗,#p < Scale bar, 50 μm. Data are presented as mean ± SD. See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions