EMBO/soda-nel huggingface dataset:
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 6

Figure 6. BIC1 and BZR1/PIF4 associate with the promoters of common target genes interdependently.

A Diagram of the promoter structures of SAUR-AC and IAA19. Arrows indicate the primer positions for ChIP-qPCR. E-box, CACATG. G-box, CACGTG.

B ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of BZR1/PIF4 target genes. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays.

C ChIP-qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1/PIF4 target genes is dependent on PIFs. The 6-d-old 35S:BIC1-Flag and 35S:BIC1-Flag/pifQ seedlings were used for ChIP assays.

D ChIP-qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10-d-old 35S:BZR1-MYC and 35S:BZR1-MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays.

E ChIP-qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs. The 6-d-old 35S:PIF4-MYC and 35S:PIF4-MYC/bic1bic2 seedlings were used for ChIP assays.

Data information: For (B to E), chromatin of each sample was immunoprecipitated (IP) using an anti-Flag, anti-MYC or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n=3).
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 7

Figure 7. BIC1 activates PIF4 expression and interacts with BZR1 in bread wheat.

A RT-qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4. The Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 plants were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3). Different letters indicate significant differences (Fisher's LSD, P< 0.05).

B Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical-inducible construct pERGW-BIC1. 8-d-old pERGW-BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT-qPCR analysis. The PP2A gene served as internal control. Data are means ± SD (n=3).

C ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of PIF4. The top panel shows the diagram of the promoter structure of PIF4. Arrows indicate the primer positions for ChIP-qPCR. G-box, CACGTG. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays. The chromatin of each sample was immunoprecipitated (IP) using an anti-Flag or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n=3). *p < 0.05, as determined by Student's t-test.

D RT-qPCR analysis showing the transcript levels of endogenous PIF4 in the 5-d-old Col-0, 35S-PIF4 and PIF4-HA seedlings. The PP2A gene served as internal control. Data are means ± SD (n=3).

E Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1/PIF4 synergistically activate PIF4 promoter. The ProPIF4:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratios represent the ProPIF4:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3). *p < 0.05, as determined by Student's t-test.

F LCI assays showing that TaBIC1 interacts with TaBZR1. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-TaBIC1 or nLUC and cLUC-TaBZR1 or cLUC as indicated.

G In vitro pull-down assays showing that TaBIC1 directly interacts with TaBZR1. Purified GST-TaBIC1 proteins were incubated with MBP or MBP-TaBZR1 for the MBP pull-down assay. Arrowhead indicates specific band.

H BiFC assays showing the interaction of TaBIC1 and TaBZR1 in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-TaBIC1 and cYFP-TaBZR1. The yellow dots represent interaction signals. BF, bright field. Scale bars, 20 μm.

I A propose working model for a transcriptional regulatory module BIC1-BZR1-PIF4. BIC1 acts as a transcriptional coactivator and interacts with the transcription factors BZR1 and PIF4 to activate PIF4 and other target genes, consequently promoting cell elongation.
Endothelial-immune crosstalk contributes to vasculopathy in non-alcoholic fatty liver disease (2022)
Figure 1

Figure 1. Generation and characterization of blood outgrowth endothelial cells from healthy and NAFLD subjects.

(a) Workflow schematic of BOEC generation from PBMC samples isolated from NAFLD patients and healthy donors.

(b) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm).

(c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test).

(d) Left panel: Flow cytometry characterization of BOECs for endothelial, leukocyte and progenitor cell markers (grey - isotype control; red/blue/green/purple - cell lineage marker staining). HUVEC and monocytic THP-1 cells were used as positive controls for endothelial and leukocytic markers respectively. Right panel: Percentages of positively expressing cells. Sample sizes are n = 4-6 healthy, n = 2-7 NAFLD. Results are indicated by mean ± SD. *p<0.05; **p<0.01; ****p<0.0001; ns, non-significant (one-way ANOVA).

(e) Representative images of immunostaining for endothelial markers, PECAM1 and CDH5 (scale bar, 100 µm).

(f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead. ns, not significant (t-test). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers).
Endothelial-immune crosstalk contributes to vasculopathy in non-alcoholic fatty liver disease (2022)
Figure 4

Figure 4. Immunoprofiling of NAFLD patients and healthy subjects by single-cell transcriptomics.

(a) Upper panel: Single-cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects; n = 10,786 cells. We identified 15 distinct cell types by DICE annotation. Lower panel: Expression patterns of CXCR3, CXCR4, ACKR3, CX3CR1, CCR6 across different cell types in NAFLD and healthy PBMC samples.

(b) Chemokine ligand-receptor mapping to identify potential immune cell interactors based on NAFLD endothelial chemokine signatures.

(c) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single-cell analysis.

(d) Upper panel: Representative flow cytometry dot plots, characterizing the isolated CD8+ T cells and CD8- immune cell fraction (mainly CD4+ T cells, NK cells and monocytes) from PBMCs of healthy and NAFLD subjects. Lower panel: Flow cytometry quantification of % of positively expressing CXCR4+ and CX3CR1+ cells within each immune subset. Sample sizes are n = 14 NAFLD; n = 6 healthy. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test).

(e) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD (n = 3) and healthy (n = 3) BOECs, with 3 biological replicates per donor.

(f) Upper panel: Representative flow cytometry histograms of HLA-A/B/C, HLA-DM and IFN-γR1 expressions in NAFLD (red) and healthy (blue) BOECs. Grey, isotype control. Lower panel: Box-whisker plots reflect mean fluorescence intensity (a.u., arbituary unit), normalized to isotype control, as a readout for epitope density. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). n = 4 donors; *p<0.05; ns, not significant (Mann-Whitney test for HLA-A/B/C and IFN-γR1; t-test for HLA-DM).
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf1

(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined. (c) U2OS cells expressing GFP–FYVE were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. GFP-positive puncta per cell were calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment. (d) U2OS cells expressing GFP–FOXO3A were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf2

(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound’s donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3)Kα active site. The displacement of the PI(3)Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf3

(a) H4 cells expressing mCherry–GFP–LC3 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged by automated acquisition. Representative images of an overlay of GFP (green), mCherry (red) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP- and mCherry-positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment. (b) HeLa cells expressing GFP–Parkin were treated with PIK-III for 12 h followed by the addition of CCCP for 12 h, fixed, stained for endogenous Tom20 and imaged. Representative images of an overlay of Tom20 (red) and Hoechst (blue) are shown with a scale bar equivalent to 50 μm. Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment. (c) H4 cells expressing GFP–p62 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged. Representative images of an overlay of GFP (green) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP-positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment. (d) Steady-state levels of autophagy markers visualized by western blotting in H4 GFP–p62 cells treated overnight with 0.5 μM AZD8055 or 5 μM PIK-III. (e) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in PSN-1 cells treated for 48 h with the indicated doses of PIK-III. (f) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in Panc10.05 cells treated for 48 h with the indicated doses of PIK-III. (g) RKO cells expressing inducible HA–ATG4B-C74A were treated with doxycycline (DOX induction) for five days and then incubated in DOX-free medium for 24 h (DOX washout). Bafilomycin A1 was added with or without PIK-III at the beginning of the washout period (lanes 5 and 6). Steady-state levels of ATG4B, LC3 and GAPDH were visualized by western blotting. Uncropped images of blots are shown in Supplementary Fig. 7.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf4

(a) Schematic of experimental workflow. DLD1 cells were converted to SILAC medium, incubated overnight in the presence or absence of 5 μM PIK-III, lysed, and ubiquitin-modified peptides enriched with anti-diglycyl antibody and quantified as described in the Methods. (b) Proteins represented by unique ubiquitylation sites with >1.5 log2 (fold change) in both replicates after diglycyl-peptide enrichment were searched for >1.5 log2 (fold change) at the proteome level in both replicates. The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow-through replicate analyses. Two independent biological replicates are shown in crossplots. (c) Steady-state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK-III for 20 h. (d) The levels of NCOA4 and p62 in ATG7 wild-type (+/+), heterozygous (+/−) or homozygous knockout (−/−) DLD1 clones 1 and 2 were determined. (e) DLD1 cells cultured in rich medium (Fed) were deprived of serum and amino acids (Starve) for the indicated times (hours, h) and levels of NCOA4, p62, LC3 and GAPDH were determined by western blotting. (f) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4, LC3, LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK-III. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full panels on left, and zoomed panels on right correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf5

(a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates. (b) Steady-state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated (hours, h). (c) DLD1 cells were cultured in the presence or absence of bafilomycin A1 overnight, fixed and processed for confocal immunofluorescence to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively. (d) DLD1 parental or NCOA4−/− cells (clones 1 and 2; KO-1, KO-2) were starved for 6 h and the levels of NCOA4, FTH1, FTL and GAPDH determined. (e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1 overnight, fixed and processed for confocal immunofluorescence to determine the cellular distribution of endogenous ferritin (FTH1–FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf6

(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only. (b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot. (c) NCOA4−/− cells were transfected with control or SBP–NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors, lysed and the occurrence of the faster-migrating FTH1 band was determined. (d) Endogenous NCOA4 immunoprecipitations from DLD1 parental, FTH1−/− or FTL−/− (clones 1 and 2 for each genotype) protein extracts. The presence of FTH1 and FTL was detected by western blot. Uncropped images of blots are shown in Supplementary Fig. 7.
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo (2014)
figf7

(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting. (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined. (c) Ncoa4−/− mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting. Note the complete absence of the lower Fth1 band consistent with earlier findings. (d) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4−/− mice is shown and brown pigmented regions apparent in Ncoa4−/− sections are indicated (white arrows). (e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls’ Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.
Identification of novel genes and networks governing hematopoietic stem cell development (2016)
Figure 1

Figure 1. General outline of the experimental strategy.

A: Developmental stages of ESC differentiation to HSPCs, showing the populations isolated on days 6 and 20.

B: Schematic overview of the experimental strategy.

C: FACS plots from a representative experiment showing gates for purification of the cell populations. Upper left: GFP+ cells in the population of ESCs infected with pGIPZ-shRNA library lentiviruses. Upper right: Isolation of mesodermal (SSEA1–Flk1+Cxcr4–) and endodermal (SSEA1–Flkl–Cxcr4+) cells. Lower panels: Isolation of D20LS (Lin–Sca1+c-Kit–), D20LK (Lin–Sca1–c-Kit+), and D20LSK (Lin–Sca1+c-Kit+) cells.
Identification of novel genes and networks governing hematopoietic stem cell development (2016)
Figure 2

Figure 2. Confirmation of the identity of the sorted cell populations.

A: mRNA analysis of expression of endothelial (Tie2, CD31, Cdh5), epithelial (Cdh1), and early hematopoietic (Scl, CD41) marker genes in the indicated sorted cell populations. Error bars represent standard deviation (SD), n=3. Student’s t-test. *p<0.05.

B: Confirmation of the purity of sorted populations by qRT-PCR: c-Kit is highly expressed only in the c-Kit+ (D20LSK and D20LK) populations, and Sca1 is highly expressed only in the in Sca1+ (D20LSK and D20LS) populations. Error bars represent standard deviation (SD), n=3. Student’s t-test. *p<0.05.

C: qRT-PCR analysis of Gata1, Gata2, Lyz2, and Scl expression in the sorted cell populations on day 20. Error bars represent standard deviation (SD), n=3. Student’s t-test. *p<0.05.

D: FACS analysis of ESC-derived cells confirming that D20LS, D20LK, and D20LSK cell populations express the early hematopoietic marker CD41 and the pan-hematopoietic marker CD45.
Identification of novel genes and networks governing hematopoietic stem cell development (2016)
Figure 4

Figure 4. Functional validation of candidate genes in groups IV, X, XI, and XII required for differentiation of ESCs towards HSPCs.

A: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Esrra, S100a8, or Hcfc2 and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). The percentages of Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were decreased on day 6 with shRNA knockdown of Group XII genes Esrra, S100a8, and Hcfc2 compared to control as quantified by flow cytometric analysis.

B: Knockdown of Esrra, S100a8, and Hcfc2 showed that the ratios of percentage of Mesoderm-Endothelium population vs Endoderm population were decreased as measured by Flk1+/Cxcr4- vs. Flk1-/Cxcr4+ on day 6. The data were generated based on the quantification of flow cytometry analyses. Error bars represent standard deviation (SD), n=2. Student’s t-test, *p<0.05.

C: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Atp5g3, Bub1b, Eci1, or Dis3l and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). Flow cytometric analysis showed that Endoderm cells (Cxcr4+/Flk1-) were deceased while Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were slightly increased in the cells with shRNA knockdown of Group XI genes Atp5g3, Bub1b, Eci1, and Dis3l compared to control.

D: Knockdown of Atp5g3, Bub1b, Eci1, or Dis3l led to increase of Mesoderm-Endothelium/Endoderm population ratio as quantified based on the flow cytometry data. Error bars represent standard deviation (SD), n=3. Student’s t-test, *p<0.05.

E: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Wbp5, Rbm26, Gdpd4, and Nrxn1 and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Silencing of group IV genes including Wbp5, Rbm26, Gdpd4, and Nrxn1 decreased overall LK, LS, and LSK population on day 20.

F: Quantification of LK+LSK+LS cell fractions with Wbp5, Rbm26, Gdpd4, and Nrxn1 silencing based on flow cytometric analyses. Error bars represent standard deviation (SD), n=2. Student’s t-test, *p<0.05.

G: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1, Mettl22, Lrsam1, or Hal and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Flow cytometric analysis showed notable reduction of LSK population on day 20 with Group X genes Ap2a1, Mettl22, Lrsam1, or Hal knockdown.

H: Quantification of flow cytometric analyses showed knockdown of Ap2a1, Mettl22, Lrsam1, and Hal significantly impaired generation of LSK population on day 20 in vitro compared to control. Error bars represent standard deviation (SD), n=3. Student’s t-test, *p<0.05.