EMBO/soda-nel huggingface dataset:
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 5

Figure 5. OTUD1 sustains SMAD3 stability and facilitates SMAD3/SMAD4 complex formation and nuclear translocation. (A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates). (C-D) 293T cells transfected with GFP-SMAD3 and Flag-OTUD1 were treated with cycloheximide (CHX, 50ug/ml) at the indicated time points. Western blot analysis (C) and densitometric quantification (D) of GFP are shown. GAPDH was used as the loading control (n=3 biological replicates). (E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates). (G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. (H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G. (I) HUVECs transfected with OTUD1 siRNA or NC siRNA were challenged with 1 μM Ang II for 24 h and with 10 μM MG132 6 h before harvest. Protein levels of SMAD3 in cytoplasm and nucleus were measured by Western blot assay. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions. (J-K) HUVECs transfected with Flag-OTUD1 (or EV) and si-SMAD3 were challenged with 1 μM Ang II for 24 h. mRNA levels of Cdh5, Vim, Acta2, Twist1, Snai1 (J), Tgfb1 and Col1a1 (K) were determined by RT-qPCR assay (n=3 biological replicates). The values were normalized to those of β-actin. Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 6

Figure 6. OTUD1 overexpression aggravates vascular remodeling and collagen deposition through regulating SMAD3 in vivo. The methodology of this animal experiment was described in Methods. (A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates. (E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates). (G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues were examined by real-time qPCR. The values were normalized to Rn18s (n=5 biological replicates). Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle (2022)
Figure 1

Figure 1. Interferon signalling regulates stem cells in the young and old brain and decreases in neural progenitors

A. Experimental layout for scRNA-Seq in young and old mice lacking interferon receptors. All mice represent TiCY (Tlx reporter, see methods) that are either IFNAGR WT or KO. TAM (Tamoxifen). See also Fig EV1.

B. UMAP embedding showing the 15,548 single cells in this analysis with their cell types. See also Fig EV1. n = 2 biological replicates per age and genotype.

C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. See also Fig EV1. n = 3 biological replicates.

D. Schematic representation of Ribo-Seq pipeline in NSCs.

E. MA plot showing differential expression between IFN-β 16h treated bulk cells and Control cells. Light red dots are significantly upregulated (one-sided Wald test), dark red dots are the top 300 genes selected as the "NSC Type-I Interferon Response". n = 4 biological replicates

F. Scores computed for the NSC Type-I Interferon Response signature displayed in the UMAP embedding for young cells (with colours clipped to the range seen in the lineage cells) and averaged for the cell types in our analysis at varying ages in IFNAGRWT and IFNAGRKO cells. n = 2 biological replicates per age and genotype.

G. Scores computed for the Wu et al. (2018) intrinsic interferon response gene set displayed in the UMAP embedding for young cells (with colours clipped to the range seen in the lineage cells) and averaged for the cell types in our analysis at varying ages in IFNAGRWT and IFNAGRKO cells. n = 2 biological replicates per age and genotype.
Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle (2022)
Figure 2

Figure 2. IFN-β controls mTORC1 shaping a biphasic regulation of mRNA translation in NSCs

A,B. Representative polysome profiles of NSCs treated with IFN-β for 2h and 14h treatment. Arrows indicate the 40S, 60S and 80S subunits of the ribosome.

C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. For associated GO terms see also Fig EV3. n = 4 biological replicates.

E,F. Ribo-Seq results of 2h and 14h IFN-β treatments. 5' terminal oligopyrimidine motif-containing mRNAs (TOP-mRNAs) highlighted in blue. n = 4 biological replicates.

G,H. Coverage profile of ribosome-protected reads (footprints) of TOP-mRNAs in 2h and 14h IFN-β treatment. Nucleotide 0 depicts the start codon (AUG) marking the interface between 5' untranslated region (5' UTR) and coding sequence (CDS).

I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test (See Methods for details). p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.
Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle (2022)
Figure 3

Figure 3. IFN-β's biphasic control of mTORC1 uncouples growth and cell cycle and represses Sox2 translation via its 5' PRM motif

A. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test (See Methods for details). p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates.

B. Schematic representation of the type-I interferon signalling pathway and its crosstalk with the receptor tyrosine kinase (RTK) pathway. Activatory (blue) and inhibitory (red) phosphosites are depicted. Red inhibitory lines depict late mTORC1-derived inhibitory feedback loops.

C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates.

D. Coverage profile of ribosome-protected reads (footprints) in genes with upregulated canonical (upper) or alternative upstream (lower) ORFs upon extended IFN-β treatment. Nucleotide 0 depicts the start codon (AUG) marking the interface between 5' untranslated region (5' UTR) and coding sequence (CDS).

E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates.

F. Schematic representation of the prevailing signalling pathways driving the early inhibition and late activation of mTORC1.

G. Polysome profiling (RT-qPCR) of Sox2 mRNA upon 2h and 14h IFN-β treatment. Hyphens represent mean of biological replicates. Arrows represent the 40S, 60S and 80S subunits of the ribosome. Two-way ANOVA with Šídák's multiple comparison test was computed. 2h fraction 11 * (p=0.0206), 14h fraction 3 **(p=0.0042), fraction 9 **(p=0.0028), fraction 10 ***(p=0.0003). n = 4 (2h) or 3 (14h) biological replicates.

H. Experimental layout of IFN and Torin1 treatments (upper panel). Torin1 was administered 12 and 9 hours before collection. Schematic representation (lower panel) of the 5'UTR constructs priming the renilla luciferase. TOP = 5'Terminal Oligopyrimidine motif; PRM = 5' Pyrimidine Rich Motif.

I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified).

Data information: p ≡ p-value.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 1

miR-203 restores drug sensitivity

Immunoblots and qRT–PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test.

Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).

Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.

MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).

Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells.

Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann–Whitney U-test.

Source data are available online for this figure.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 2

ZEB1-dependent epigenetic modifications

A Schemes for the genomic loci of the miRNA and E-cadherin genes, showing regions of the CpG islands (yellow), of the qRT–PCR amplicon for chromatin immunoprecipitation (ChIP) analysis (blue) and of the bisulfite sequencing (red).

B, C Histone marks were analyzed using ChIP coupled to qRT–PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 3

Screening of epigenetic drugs for upregulation of miRNAs and downregulation of ZEB1

Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT–PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.

Relative expression of indicated genes in Panc1 measured by qRT–PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.

Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.

Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.

Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann–Whitney U-test.

Source data are available online for this figure.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 4

Mocetinostat sensitizes to gemcitabine in vitro

Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.

MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).

Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).

MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01–0.05; for exact P-values, see Supplementary Table S4).

Source data are available online for this figure.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 5

Mocetinostat sensitizes to gemcitabine in vivo

Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann–Whitney U-test.

Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.

Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.

Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann–Whitney U-test.
ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat. (2015)
Figure 6

Clinical relevance of miR-203 expression

A, B Relative expression levels of miR-203 and miR-200c in pancreatic adenocarcinomas. (A) In normal versus tumor tissue of the same case (n = 6 cases), (B) in the non-recurrence (n = 10 cases) versus the recurrence group (n = 11 cases). The mean value of the lower group in each figure was set to 1. Nonparametric Mann–Whitney U-test.
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 1

Figure 1. TFEB and TFE3 translocate from the cytosol to the nucleus in response to ER stress.

(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments.

(B) Immunoblot of protein lysates of ARPE-19 cells treated as shown in (A). Data are representative of three independent experiments.

(C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments.

(D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition).

(E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.

(F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.

(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student’s t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition).

(H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 2

Figure 2. Tunicamycin-induced TFE3 activation requires PERK.

(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.

(B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.

(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).

(G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001).

(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).