EMBO/soda-nel huggingface dataset:
Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. (2015)
Figure 7

Inhibition of mTOR signaling increases survival of Phb2pko mice

A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.

B Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm).

C Kaplan–Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).

D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.
Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement (2019)
Figure 2

Figure 2. Binding of MSL2 to High Affinity Sites is established in early embryonic stages.

A) Example genomic region of MSL2 MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. DNA binding is defined as sub-nucleosomal fragments (10 - 130 bp), while chromatin binding as mono-nucleosomal fragments (130 - 220 bp). Called peaks/regions (Dataset EV1) are represented as boxes above the tracks and HAS below. Late-appearing regions are marked by a plus (+) sign. Graph represents pooled IP/Input signal of 3 biological replicates.

B) Average composite plots of sub-nucleosomal MSL2 MNase-ChIP-seq (DNA) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos. Total number of PionX = 56 and HAS = 247. Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.

C) Average composite plots of mono-nucleosomal MSL2 ChIP-seq (Chromatin) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos.
Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement (2019)
Figure 3

Figure 3. MSL2 binds broad chromatin regions in early embryonic stages.

A) Venn diagram showing the overlap of sub-nucleosomal MSL2 DNA peaks and mono-nucleosomal MSL2 chromatin regions.

B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.

C) Average composite plots of MSL2 [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across regions. Total number of early = 212 and late = 87.

D) Average composite plots of MOF [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right).
Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement (2019)
Figure 4

Figure 4. MOF-dependent H4K16ac accumulates at X-chromosomal gene bodies during embryonic development.

A) Example genomic region of DNA binding by MSL2 (DNA), and chromatin binding of MSL2 (Chrom.), MOF and H4K16ac MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. Called peaks/regions (Dataset EV1-2) are represented as boxes above the tracks. Graph represents pooled IP/Input signal of 3 biological replicates.

B) Size distribution of X-chromosomal MOF and H4K16ac regions, which are present in both early and late embryos. P-value (Wilcoxon rank sum test) is indicated. Total number of regions are 459, 443, 854 and 400, respectively.

C) Average composite plots of MOF MNase-ChIP-seq centered at Transcription Start Site (TSS) or Transcription Termination Site (TTS) in early or late embryos for X-chromosomal genes (total=1515). Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.

D) Average composite plots of H4K16ac MNase-ChIP-seq centered at TSS or TTS in early or late embryos for X-chromosomal genes (total=1515).
Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement (2019)
Figure 5

Figure 5. H4K16ac levels distinguish early from late compensated genes.

A) Scatterplot comparing X-chromosomal genic H4K16ac MNase-ChIP-seq levels in early (stage 5-8) and late (stage 13-15) mixed-sex embryos. Genes are grouped by k-means clustering (k=3). The numbers of genes are cluster 1 = 979, cluster 2 = 364 and cluster 3 = 172.

B) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos at genes grouped as in A (cluster 1-3).

C) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak grouped as in A (cluster 1-3). Red line indicates median distance to the nearest peak.

D) Comparison of cluster 1 (left) or cluster 3 (right) genes defined by H4K16ac MNase-ChIP-seq levels with constitutive and developmental genes as defined in Figure 1C.

Expanded View Figure Legends
Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors (2022)
Figure 1

Figure 1. Establishment of Fah knockout NSI mice for the humanized liver mouse model. (A) Schematic outline of the pronuclear microinjection procedure. In brief, female NSI mice were superovulated with PMSG and hCG and then mated with male NSI mice. Fertilized eggs were collected from NSI mice with a vaginal plug. TALEN mRNA pairs were injected into the cytoplasm of pronuclear-stage mouse embryos. The injected embryos were then transferred into pseudopregnant surrogate mothers. The mouse pups were genotyped. The mouse was drawn by the authors (Z. J. and P. Li) using Adobe Photoshop; (B) DNA-binding sequences and the spacer region (in blue) of TALENs targeting the exon 3 of Fah; (C) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype (left). Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting (right). Cropped blots are shown; (D) Histological staining of livers from Fah-deficient (NSIF) with and without NTBC treatment and Fah intact (NSI) mice without NTBC treatment. Scale bars, 50 μm; (E) (Left) Kaplan-Meier survival curves of PHH-infused NSIF mice (in blue, n=15) and NSIF mice without PHHs (in red, n=9) after NTBC withdrawal and NTBC-treated NSIF mice (in green, n=10); (Right) Serum ALT and AST levels in NSIF mice (+NTBC, n=5), moribund NSIF mice (-NTBC, n=5) and PHH-engrafted (8-week-old) mice (-NTBC, n=5) at indicated time points post-NTBC withdrawal; data are presented as the mean ± SD, and statistical significance was determined by two-way ANOVA with Sidak's multiple comparisons test and defined as ***P≤0.001; (F) Human ALB levels in the sera of PHH-engrafted NSIF mice (n=5) were measured by ELISA at indicated time points post-NTBC withdrawal, data are presented as the mean ± SD. N-numbers refer to biological replicates; (G) (Left) Quantification of the repopulation efficiencies of PHHs in livers estimated by hALB-positive staining at indicated time points post-transplantation. Data are presented as the mean ± SD. N-numbers refer to biological replicates (n=5); (Right) Representative immunohistochemical staining of hALB in the livers of NSIF mice at indicated time points post-PHH transplantation. Scale bars, 1000 μm; (H) Engraftment of PHHs into NSIF mouse livers was detected immunostaining for human ALB, CK8/18, and FAH. Scale bars, 50 μm.
Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors (2022)
Figure 3

Figure 3. Screening of oncogenes involved in the transformation of PHHs into HCC cells. (A) Schematic representation showing the process for induction of in situ HCC after lentiviral transduction of PHHs with a cocktail of 10 OC lentiviruses; (B) Bioluminescence imaging of three recipient NSIF mice (n=3) that had been transplanted with OC-transduced PHHs four months ago. The colors from blue to red indicate intrahepatic bioluminescence signal strength 4 months post-transplantation of OC transduced PHHs; (C) ALB and AFP levels in sera from NSIF mice that were infused with OC-transduced PHHs (OC-PHHs) or EGFP-transduced PHHs (mock-PHHs) were measured by ELISA at indicated time points. (n=5, the data are presented as the mean ± SD values. Two out of five mice died before 120 days post-transplantation in the OC-PHHs group. The levels of ALB and AFP in the rest three mice were measured.); (D) Bright and EGFP imaging of an explanted liver of a mouse from the OC group revealed an advanced intrahepatic tumor, Scale bars, 0.5 cm; (E) Anti-GFP immunofluorescence (left), H&E staining (center), anti-HLA-I IHC staining (right) images of tumor dissection from the OC group. Scale bars, 50 μm; (F) Representative PCR amplification of MYC, TP53R249S, KRASG12D, NRASG12D, CTNNB1S45F, BRAFV600E, AXIN1G652S, IL6, PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC-PHHs (Tumor) and their corresponding overexpression vector plasmids as positive controls (OCs); (G, H) Genomic DNA (gDNA) and cDNA of tumor samples were obtained for PCR amplification using forward primers that recognize TP53R249S or KRASG12D and reverse primers specific for EGFP. DNA sequencing confirmed mutations of TP53 and KRAS in the genomic DNA (G) and mRNA (H) of dissected primary tumors harvested from tumor-bearing mice. Red squares indicate mutant codes in TP53R249S and KRASG12D; (I) Expression of MYC, p53, and KRASG12D was detected in the iHCC samples (n=3, biological replicates) by Western blotting. Cropped blots are shown; (J) Immunofluorescence analysis of MYC, p53, and KRAS expression in iHCC samples derived from OC-PHHs and normal humanized livers that were reconstituted with mock-PHHs. Scale bars, 20 μm.
Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors (2022)
Figure 4

Figure 4. Generation of genetically defined in situ humanized liver cancers. (A) Representative images of in situ liver carcinomas derived from PHHs transduced with a combination of MYC, TP53R249S, and KRASG12D (MTK, n=22) or a combination of MYC and TP53R249S (MT, n=12) in NSIF mice. No tumors were detected in any of the mice injected with PHHs that were transduced with TP53R249 and KRASG12D (TK, n=9) or PHHs that were transduced with MYC and KRASG12D (MK, n=9). Scale bar, 1 cm; (B) Genomic DNA of colonies that were derived from single cells of iHCC1-1 and iHCC2-1 tumor samples were extracted for PCR amplification of MYC, TP53R249S, and KRASG12D; (C) Immunofluorescence analysis of the human cell marker HLA, the ductal/CC markers KRT19 and EpCAM, and the HCC markers AFP and GPC3 in a iHCC sample from NSIF mice transplanted with MTK-transduced PHHs from the donor PHH1 (iHCC1-1) and a clinical patient HCC sample (pHCC1). Nuclei were counterstained with DAPI. Scale bars, 20 μm; (D) Growth curves of PHHs after transduction with various combinations of MYC (M), TP53R249S (T) and KRASG12D (K) in culturing medium supplemented with hHGF (10 ng/ml), hEGF (5 ng/ml), and WNT3a (5 ng/ml), P=0.0038 for MTK- vs MT-transduced PHH by two-way ANOVA with Tukey's multiple comparison test; **P=0.0038 for MTK- vs MT-transduced PHH by two-way ANOVA with Tukey's multiple comparison test; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3); (E) Cell cycle distribution analysis of mock-PHHs, MTK-transduced PHHs, and MT-transduced PHHs on day 10. **P=0.0034 for MT-transduced PHHs vs. mock-PHHs and ***P=0.0009 for MTK-transduced PHHs vs. mock-PHHs by two-way ANOVA with Tukey's multiple comparison test; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3); (F) Morphology of PHHs transduced with various combinations of MYC (M), TP53R249S (T), and KRASG12D (K) at day 10. EGFP positivity indicates transduction of oncogenic candidates: scale bars, 100 μm.
Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors (2022)
Figure 5

Figure 5. Genetically defined iHCCs recapitulate the transcriptional profiles of human HCC. (A) PHHs from five donors (PHH1 (AKB, female, 39 years old), PHH2 (XSM, female, 59 years old), PHH3 (ANG, male, 3 months old), PHH4 (HVN, male, 33 years old), and PHH5 (QBU, male, 50 years old)) and their corresponding iHCC cells were used for RNA-seq analysis. Four iHCC samples (iHCC1-1~iHCC1-4) from the donor PHH1, three iHCC samples (iHCC2-1~iHCC2-3) from the donor PHH2, three iHCC samples (iHCC3-1~iHCC3-3) from the donor PHH3, one iHCC sample (iHCC4-1) from the donor PHH4, and an iHCC sample (iHCC5-1) from the donor PHH5 were used. The PCA plot shows samples plotted in two dimensions using their projections onto the first two principal components (PC1 and PC2). Each data point represents one sample (blue, PHHs; red, iHCC). PC1 was correlated with the type of sample (iHCCs vs. PHHs); (B) Volcano plot of RNA-seq data showing genes with differential expression (1,591 upregulated and 2,243 downregulated genes, fold change≥2 and P value≤0.05) in iHCC cells compared to PHHs. TP53, KRAS, MYC, MUC1, and AFP were highly expressed in iHCC cells, whereas hepatocyte signature markers, including FOXA2, FOXA3, ATF5, HNF4A, and ALB were highly expressed in PHHs; (C) Gene ontology (GO) analysis of the pathways enriched with DEGs (fold change≥2 and P value≤0.05) in iHCC cells and PHHs. Genes associated with telomere maintenance and the cell cycle were highly enriched in iHCC cells (top), while genes associated with biomacromolecule metabolisms were highly enriched in PHHs (bottom); (D) Heat map analysis of the log2(RPKM) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells. These genes were classified into three categories based on their functions; (E) UMAP visualization of iHCC and clinical HCC samples from the TCGA-LIHC database based on their gene expression profiles; (F) UMAP visualization of iHCC and three subtypes of clinical HCC samples from the TCGA-LIHC database (iClust 1-3, TCGA-LIHC) based on their gene expression profiles; (G) Unsupervised clustering analysis of iHCC and clinical HCC samples in relative to normal PHHs. The distribution of molecular attributes (TP53 and KRAS mutation, MYC amplification) of iHCC, clinical HCC samples, and PHHs were displayed.
Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors (2022)
Figure 6

Figure 6. Genetically defined iHCC as a platform for validation of potential therapeutic targets. (A) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs. Within these 20 genes, seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA-HCC (TCGA-LIHC) analysis; (B) Representative cytotoxicity CARFAP-T and CARMUC1-T cells assessed by coincubation with iHCC2-1 cells. P=0.0038 (CARFAP-T vs. mock T), and P<0.0478 (CARMUC1-T vs. mock-T). P values were determined by paired t-test, **P≤0.05. Cells were seeded at E: T ratios ranging from 4:1 to 1:4; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3); (C) Growth of iHCC in xenografts (Left) and tumor weights of iHCC dissected from xenografts (Right) (n = 5), the xenografts were treated with mock, CARFAP-, or CARMUC1-T cells when tumors reach 50 mm3 volume. ***P≤0.001 (tumor volume of CARFAP-/CARMUC1-T vs. mock T) with two-way ANOVA with Tukey's multiple comparison test. **P≤0.01 (tumor weight of CARFAP-/CARMUC1-T vs. mock T) with one-way ANOVA with multiple comparison test. Data are presented as the mean ± SD. N-numbers refer to biological replicates; (D) Clonogenic assay of iHCC1-1 cells treated with a MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates; (E) Luciferase and EGFP marked iHCC1-1 cells (1×106) were intrasplenically injected into NSI mice on day 0. iHCC-bearing mice were treated with DMSO (mock group, n=4), or trametinib (3 mg/kg body weight) plus palbociclib (150 mg/kg body weight, n=5) for two weeks (Daily oral gavage of drugs from Day 3 to Day 17). Top, on days 0, 7, 14, and 21, bioluminescence imaging was conducted. Bottom, quantification of the total flux analyzed by in vivo bioluminescence imaging of luciferase activity. P=0.0057 on day 21 by two-way ANOVA with Sidak's multiple comparison test, **P≤0.01. Data are presented as the mean ± SD. N-numbers refer to biological replicates; (F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of iHCC in xenografts (n=5) treated with PBS or XL413 at 50 mm3 tumor volume. P=0.0486 (XL413 vs. PBS group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.

EV figure legends
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 1

Figure 1. Expression and distribution of Pdia4 in pancreatic islets and/or other tissues.

(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies.

(B) Confocal analysis of Pdia4 and insulin (Ins) or glucagon (Gcg) in mouse islets. The tissues were stained with the indicated antibodies and analyzed using a confocal microscope. Scale bar = 100 μm.

(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies.

(D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis.

(E) The islets of WT BKS and diabetic Leprdb/db mice underwent immunoblotting analysis.

(F) IHC analysis of the islets from Leprdb/db and Lepob/ob mice at the indicated ages using anti-Pdia4 antibody and hematoxylin. Scale bar = 100 μm. The dash circles indicate islet regions.

(G) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit.

(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies.

Data information: Data from 3 experiments (A-E, F and H) and more (G) are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 2

Figure 2. Pdia4 ablation increases survival rate and longevity and reduces islet atrophy, ROS production, and islet cell death, elevates serum insulin, and potentiates β-cell GSIS in Pdia4-/-Leprdb/db mice.

(A) Survival rate and life span of WT, Pdia4-/-(KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks (Appendix Figures S2A and S6A). (B) Pancreata of 18-week-old WT and Pdia4-/- (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions.

(C) Serum ROS of the 18-week-old mice (B) were determined.

(D) The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions and the black arrowheads indicate TUNEL+ cells.

(E, F) ELISA kits were used to quantify serum insulin (E) of the same mice as in (B) and their supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) were used in GSIS assays.

Data information: The number of mice (n) is indicated in parentheses. Data from over 3 experiments are presented as the mean ± SD. Log rank (A) and one-way ANOVA test (B-F) were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 3

Figure 3. Pdia4 overexpression increases islet atrophy and ROS production, decreases serum insulin, and impairs β-cell GSIS in Pdia4tg/tgLeprdb/db mice.

(A) Serum insulin of WT and Pdia4tg/tg (TG) mice on BKS or Leprdb/db background (Appendix Figure S6A), at the indicated ages, was measured using an ELISA kit.

(B) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions.

(C) Serum ROS of the mice from (A) were determined.

(D) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays.

(E) Pancreatic islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice, which received an intraperitoneal dose of glucose (1 g/kg body weight) for 0.5 h, were isolated and divided into 2 aliquots. One aliquot of the islets was stained with anti-insulin antibody and measured for their basal insulin content using flow cytometry analysis (0 h). The other aliquot of the islets were grown in a complete medium for 24 h and their insulin content was measured (24 h).

Data information: Data from 3 experiments (E) and more (A-D) are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses.