EMBO/soda-nel huggingface dataset:
Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells (2022)
Figure 2

Figure 2. Automated circuit design and miRNA validation

A) Summary of computationally identified circuit using a constraint of maximum three inputs. Shown are input miRNAs and predicted circuit output levels. Expression levels of identified miRNAs inputs are given as fold change over the pre-set input abundance threshold (t) of the total miRNA pool (where t = 1%) (left). Calculated circuit output levels are given as mol / cell (middle) and logic connectivity of the identified miRNA is depicted (right). EB: embryoid bodies, MPC: mesenchymal progenitor cells, NPC: neural progenitor cells, R-NSC: neural rosettes. miRNA expression data and nomenclature in Data Set EV1, raw output data and constraint files of the algorithm in Source data file. See also Figure EV1.

B) Illustration of bi-directional miRNA sensor system.

C) Bar chart showing relative DsRed expression of sensors containing the indicated miRNA target sites as four fully complementary repeats (discrete miRNA sensing). The control vector (n.t.) does not contain any target sites. Two-tailed unpaired t-tests were performed to compare the definitive endoderm derived from H1 (H1 DE) to H1 and the control vectors. For comparison to H1, data were normalized with the control vector (nt) before performing the t-test. *Pvalue = 0.011 and ** P- value = 0.006 are both considered very significant. Representative scatterplots of HES-2 are shown on top.

D) Bar chart showing relative DsRed expression of sensors containing different mutated target sequence of miR-17 as listed in Appendix Table S1. Additionally, a sensor containing no target (n.t.) and two wildtype (WT) T17 target sites with 1x and 4x repeats were tested. Two-tailed unpaired t-tests were performed to compare H1 and HES-2 for each sensor. *P-values < 0.01, were considered very significant (**), < 0.02 significant (*) and > 0.5 not significant.. Representative scatterplots of HES-2 are shown on top.

Data information: Each bar in C and D corresponds to mean +/- s.d. from at least three biological replicates. See also Figure EV2.
Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells (2022)
Figure 5

Figure 5. Optimization of minimal circuit.

A) Circuit schematics (left). Chart shows normalized Output production in response to changing amounts of plasmids encoding Act2, tTA (left) and PIT2 (right). Two transactivator constructs have been tested, onecontains miR302a/b target sites (Off), the other by mock miRFF3/FF6 target sites (ON).

B) Comparison of the maximal dynamic range across the two generic circuit versions and the two miRlow only circuit versions. For miRlow only circuits and PIT2-tTA circuit, the configuration with highest dynamic range in Output has been selected. For rtTA-PIT2 circuit, because of the low On state in the circuit with highest dynamic range, the circuit configuration with highest Off/On ratio in mCitrine / FF4 and highest On level in mCherry has been selected.

Data information: Charts and color plots show mean ± s.d. of at least three biological replicates.
Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells (2022)
Figure 6

Figure 6. Circuit-mediated BMP4 secretion enables control over cell composition and pattern formation in micropatterned hPSC colonies

A,B) Discrete circuit-mediated control of BMP4 by endogenous miRNAs A) Circuit schematic. Minimal circuit controlling BMP4 in response to the endogenous miRNAs miR-302a/b target sites (Off) or mock target sites TFF3/TFF6 (On).B) Micropatterned RUES2 have been transfected with On and Offcircuits. Bar chart shows BMP4 concentration as determined by ELISA and average iRFP expression as measured by Flow Cytometry (left). Corresponding bar chart showing the average fraction of cells expressing a given germ-layer marker (right).

C,D,E, F) Circuit-mediated tuning of BMP4 =using miSFITs library. C) Circuit schematic. Minimal circuit in mock On state controlling untargeted iRFP and miSFITs-targeted BMP4. Five miSFITs variants have been transfected on micropatterned RUES2 cells. D). Bar chart on the left shows BMP4 concentration for each circuit variant determined from culture media by ELISA. Bar chart on the right shows the same secreted BMP4 concentration along cell internal iRFP expression signal, measured by Flow Cytometry in comparison to untransfected controls in the absence (-BMP4) and presence (+ BMP4) of BMP4 (right).E) Bar chart showing the relative fraction of cells expressing a given germ-layer marker as analyzed from confocal microscopy images (right). Dose response chart of the marker fraction in response to measured BMP4 concentration (left). See also representative Flow Cytometry scatter plots in Figure EV5C.F) Radial marker profiles of each germ-layer marker analyzed from confocal microscopy images. Charts on the left show normalized intensity of marker expression at a given colony position, where x=0 is the center of the colony and x=1 is the edge of the colony.Representative microscopy image of a single colony (top) showing composite of Sox2/mCitrine (yellow), Sox17/mCherry (red) and TBXT/AF647 (blue).

Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm. See also Figure EV5.

Expanded View Figure Legends
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 1

Figure 1 - The RNase activity of IRE1α is required for activation of myofibroblasts.

A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.

B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.

C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.

D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.

E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.

F Immunohistochemical staining of αSMA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.

G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.

H αSMA mRNA expression in TGFβ-treated HFL1 cells overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. *P K599A = 0.013, *P K907A = 0.0008 compared to WT.

I αSMA mRNA expression in TGFβ-treated IRE1α -/- or WT MEF cells. *P = 0.025.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values are given. Error bars indicate s.e.m. n = 3 in A-C and G-I.
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 2

Figure 2 - miR-150 exhibits anti-fibrotic effects and is directly cleaved by IRE1α.

A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.

B miR-150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. * P = 0.0043 (K599A), P = 0.043 (K907A), compared to control.

C miR-150 levels in TGFβ-treated WT (IRE1α +/+) and IRE1α -/- MEF cells. *P = 0.018. Results are expressed as fold change compared to the Ctrl.

D miR-150 levels in IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR-150-5p miRNA inhibitor. * P = 0.034 compared to UT.

E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.

F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.

G Western blot of c-Myb in TGFβ-treated HFL1 fibroblasts that were either untransfected (UT), transfected with a control plasmid (pCtrl) or a miR150 expression plasmid (miR150). *P = 0.0206 in UT Ctrl vs. UT TGFβ and P = 0.0126 in pCtrl Ctrl vs. pCtrl TGFβ .

H Western blot of αSMA in the same experimental setup as in G. * P = 0.0405 in UT Ctrl vs. UT TGFβ, and P = 0.0284 in pCtrl Ctrl vs. pCtrl TGFβ.

I c-Myb mRNA levels in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or an siRNA targeting c-Myb (sicMyb). * P = 0.00001 compared to UT.

J αSMA mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or with a siRNA targeting c-Myb (sicMyb). * P = 0.0042 in UT Ctrl vs. UT TGFβ.

K Putative IRE1α cleavage sites in miR-150 based on sequence analysis are indicated with arrows.

L In vitro cleavage assay with recombinant IRE1α and miR-150 (left) or XBP-1 (right).

M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values given. Error bars indicate s.e.m. n = 3 in A-E, G-J, L-M and n=4 in F.
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 3

Figure 3 - ER expansion in myofibroblasts is coupled to XBP-1 splicing.

A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.

B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.

C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001.

D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values given. Error bars indicate s.e.m. n = 3 in B-C and n=4 in D.
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 4

Figure 4 - Pharmacological inhibition of IRE1α attenuates liver fibrosis.

A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6 mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.

B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.

C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.

D Liver to body weight ratio from the experimental setup in A. * P = 0,0031.

E BiP mRNA expression in liver tissue from mice presented in A. * P = 0,020.

F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.

G miR150 expression in liver tissue from mice in A. * P = 0,032 compared to untreated controls.

H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.

I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.

J Microscopic images of primary mouse hepatic stellate cells stained for BiP (top) or αSMA (bottom). Scale bars = 20 μm.

K Western blot showing BiP protein expression in primary mouse hepatic stellate cells.

L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values given. Error bars indicate s.e.m.
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 5

Figure 5 - Inhibition of IRE1α reduces skin fibrosis

A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.

B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.

C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.

D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).

E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.

F miR-150 expression levels in the same samples as shown in E.

G Immunohistochemical staining of αSMA in primary skin fibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.

H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.

I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.

J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.

K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.

L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values given. Error bars indicate s.e.m.
Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing. (2016)
Figure 6

Figure 6 - Inhibition of IRE1α reverts the fibrotic phenotype of myofibroblasts isolated from scleroderma patients.

A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).

B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc.

C Quantification of collagen protein levels in the same samples as in A. * P = 0,010.

D Immunohistochemical staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.

E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.

F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.

Data information: Statistical significance was evaluated using Student’s t-test. Significant differences are indicated with * and exact P values given. Error bars indicate s.e.m. n = 3 in E and F.
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 1

Figure 1. HAT1 is able to modulate succinylation on multiple proteins

A. Total cell lysates from wild type (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis (left). The expression levels of HAT1, Histone H3 (H3), and beta-actin (β-actin) were measured by Western blot analysis in the cells (right).

B. Quantitative proteomics of lysine (K) succinylation was performed in HepG2 and HAT1 knockout HepG2 cells. Graphs show the overlap of the total number of succinylated sites (left) and proteins (right) identified with those targeted by HAT1 (>1.2-fold decrease and P < 0.05, Student's t-test).

C. Volcano plot depicting the relative fold change of Ksucc levels in the succinylation quantitative proteomics of WT versus HAT1 KO cells. The vertical blue, dashed lines represent the threshold of 1.2-fold and the horizontal blue, dashed lines represents the threshold of P = 0.05. Student's t-test.

D. The diagram showing the cellular distribution of succinylated proteins targeted by HAT1 identified in the quantitative succinyl-proteome.

E. GSEA analysis showing the GO processes for the succinylated proteins targeted by HAT1 in the succinylation quantitative proteomic. BP, biological process; CC, cellular component; MF, molecular function.
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 2

Figure 2. HAT1 is a novel histone succinyltransferase

A. Histones were extracted from HepG2 (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells. The total histone succinylation and acetylation levels were analyzed by Western blot analysis. Total histone levels were measured by GelCode Blue staining. The efficiency of HAT1 knockout was measured by Western blot analysis.

B. Histone H3 was immunoprecipitated from HepG2 cells with or without endogenous HAT1 depletion (by knockout). The levels of succinylation, acetylation, histone H3, HAT1, and β-actin were analyzed by Western blot analysis in the cells.

C. HAT1-catalyzed histone H3 succinylation was determined by mixing purified HAT1, histone H3, and succinyl-CoA in the in vitro succinylation assays. Heat-inactivated HAT1 was used as a negative control. Western blot analysis was performed with the indicated antibodies.

D. HAT1-catalyzed histone H3 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.

E. HAT1-mediated histone H3 succinylation was assessed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without acetyl-CoA (2 μM). Western blot analysis was performed with the indicated antibodies.

F. Diagram illustrating the catalytic pocket of HAT1 bound to succinyl-CoA. Structures of HAT1 (PDB ID 2P0W) and succinyl-CoA (PDB ID 5TRL) were used for the modeling by Discovery Studio.

G. Histone H3 was immunoprecipitated from HAT1 KO HepG2 cells that were either reconstituted with wildtype HAT1 or mutant HAT1 (T188A). The levels of succinylation, acetylation, histone H3, HAT1, and β-actin were tested by Western blot analysis in these cells.
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 3

Figure 3. HAT1 catalyzes histone H3K122 succinylation for epigenetic regulation

A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A).

B. HAT1-catalyzed histone H3K122 succinylation was analyzed by mixing purified HAT1, histone H3 or histone H3 mutant (K122R), and succinyl-CoA in the in vitro succinylation assays. Heat-inactivated HAT1 was used as a negative control. Western blot analysis was performed with indicated antibodies.

C. HAT1-catalyzed histone H3K122 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.

D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR in WT HepG2 cells, HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance.

E. The mRNA levels of CREBBP, BPTF, and RPTOR were measured by RT-qPCR in HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

F. A model illustrating how HAT1-catalyzed histone H3K122 succinylation modulates epigenetic regulation and gene expression.
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 4

Figure 4. HAT1-mediated non-histone succinylation specifically targets glycolysis

A. Diagram showing 7 of 10 key glycolytic enzymes that were identified as HAT1 succinylation targets (red boxes) in the succinylation quantitative proteomics.

B. Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in (A). N = 3 biological replicates. Data were presented as mean ± SD.

C. The diagram showed the MS/MS spectra of PGAM1 K99succ peptide for its identification and quantification. The b and y represented the fragment ions of the N and C termini in peptide backbone, respectively. m/z, mass-to-charge ratio.

D. The decrease of glucose and increase of lactate in culture medium were measured by ELISA assays in HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

E, F. The relative amounts of indicated metabolites were quantified by ELISA assays in HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01; ns, no significance.