EMBO/soda-nel huggingface dataset:
Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 1

Figure 1. Activin A is overexpressed in benign skin tumor lesions and promotes HPV8-induced skin tumor development in mice

A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test).

B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies.

C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E).

F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test.

G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor.

H. Representative macroscopic pictures of ear tumors (left panel) and H&E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).
Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 2

Figure 2. Depletion of epidermal γδ T cells and accumulation of αβ T cells in the ear skin of activin-overexpressing mice

A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice.

C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice.

D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.

Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons test (B-F).

G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm.

H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.
Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 3

Figure 3. Activin promotes migration of CCR2+ macrophage precursors to the skin

A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.

C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.

D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).

E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.

F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.

Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test (B-E) or one sample t-test after log-transformation (F).
Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 5

Figure 5. Activin induces expression of pro-tumorigenic genes in macrophages, associated with a proteolytic microenvironment and vascular remodeling

A,B,F. Expression of Cx3cr1 (A), Spp1 (B), and Arg1 (F) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.

C,D. Western blot analysis of total ear protein lysates of 12-14 week-old mice for osteopontin (OPN) and GAPDH (C). Each lane represents an individual mouse (n=3). (D) Band intensities were quantified and normalized to GAPDH. The expression level in one of the wt mice was arbitrarily set to 1.

E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.

G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm.

H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.

J. Representative pictures of ear skin from 10 week-old mice stained for MECA32 (red), LYVE1 (green); counterstained with Hoechst (blue). Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.

K,L. Quantification of MECA32+ area (K) and average distance between MECA32+ vessels and basement membrane (BM) (L).

Statistical significance was determined using one-sample t test of log-transformed data (A,B,F), 1-way ANOVA and Bonferroni`s Multiple Comparisons test of log-transformed data (D) or 1-way ANOVA and Bonferroni`s Multiple Comparisons test (I,K,L).
Activin promotes skin carcinogenesis by attraction and re-programming of macrophages (2016)
Figure 6

Figure 6. Depletion of macrophages delays spontaneous tumorigenesis in HPV8/Act mice by reducing keratinocyte proliferation and angiogenesis

A. Kinetics of tumor incidence in HPV8/Act mice treated for 5 weeks with 50mg/kg anti-CSF1R antibody (AFS98) or with control rat IgG. N=10 mice per treatment group.

B. Representative pictures of anti-CD68- or CD206-stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar = 100μm.

C. Quantification of the CD68+ or CD206+ area relative to total dermis area. N=10 mice per treatment group.

D. Representative pictures of anti-BrdU (green) and anti-K14 (red) stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar: 50μm.

E. Quantification of BrdU+ keratinocytes per mm of basement membrane. N=8 anti-CSF1R-injected mice; N=4 rat IgG-injected mice.

F. Expression of Mmp12 relative to Rps29 was analyzed by qRT-PCR in total ear skin from mice treated with anti-CSF1R antibody or with rat IgG. Expression in one of the IgG-injected mice was set to 1. N=10 per group.

G. Representative pictures of the ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R or control rat IgG, stained for CD31 (red), LYVE1 (green), and counterstained with Hoechst (blue). Boxed areas are shown at higher magnification in the top-right corner. Scale bar: 100μm.

H,I. Quantification of CD31+LYVE1- area (H) and number of CD31+LYVE1-vessels (I).

White dotted lines in (B), (D) and (G) indicate the basement membrane.

Statistical significance was determined using Log-rank (Mantel-Cox) test (A),

Mann-Whitney test (C,E,H,I) or unpaired t test after log-transformation (F).
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 1

Figure 1. ISX expression induces TWIST1 and Snail1 expression, and promotes EMT.

a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.

b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.

c and d, ISX transcriptionally activates luciferase activity driven by TWIST1 (c) and Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.

e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.

f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.

g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.

h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi (described in Material and Methods). Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.

i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 2

Figure 2. ISX interacts with both BRD4 and PCAF in vitro and in vivo.

a, BRD4 peptide (291-314 a.a) obtained from anti-ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography‒tandem mass spectrometry (LC-tandem -MS).

b, ISX association proteins (PCAF, BRD4, CBP, CREB, RNA Pol II, and Histone H3) determined by Western blot in immunoprecipitation of A549 cells.

c, ISX association proteins (PCAF, BRD4, CBP, CREB, and RNA Pol II) determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with lung cancer.

d, Proximity ligation assay (PLA) of ISX and BRD4(PCAF) interaction in A549 cells. Red foci indicate close proximity of the two proteins. NC: negative control. Blank arrows indicate interactions of ISX with BRD4 (PCAF) in the cells.

e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction.

f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control.

g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction.

h and i, Cell migration (wound healing, h) and invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.

Data information: Each experiment was repeated at least three times.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 3

Figure 3. Acetylation of ISX at lysine 69 is critical for ISX-BRD4 association.

a and b, Schematic representation of the potential acetylation domain organization of ISX and its lysine mutants (AC1‒AC3).

c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody.

d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody.

f, The protein levels of total and acetylated histone H3 were determined in anti-histone H3 immunoprecipites of A549 cells by Western blotting.

g and h, The cell migration (wound healing, g) and invasion (transwell, h) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.

i, Schematic representation of tumor xenograft metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA. Modified A549 cells were directly injected into lung to form xenograft tumor mass.

j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks .

k, Kaplan-Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP, ISX-GFP and ISX Ac-GFP (AC3) (n = 10). p = 0.0003. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.

Data information: Each experiment was repeated at least three times.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 4

Figure 4. Acetylation of BRD4 at lysine 332 is critical for ISX-BRD4 association.

a and b, Schematic representation of the potential acetylation domain organization of BRD4 and its lysine mutants (AC1‒AC4).

c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody.

d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells.

e and f, The mRNA levels of TWIST1(e) and Snail1(f) were verified in A549 cells co-expressing mCherry-tagged WT or mutant BRD4 and GFP-tagged ISX by RT-PCR. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate.

g and h, The DNA binding activity of TWIST1 and Snail1 were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190/+40 bp, negative control) (10% input of each groups were pulled down and used for qPCR analysis). Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate.

i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate.

Data information: Each experiment was repeated at least three times.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 5

Figure 5. Homeobox of ISX is a critical domain required for ISX-BRD4 interaction through co-immunoprecipitation (Co IP) analysis.

a, Schematic depiction of ISX and deletion constructs used.

b GFP-tagged truncated ISX mutant proteins or HA-tagged BRD4 were detected in anti-HA or GFP immunoprecipitates by Western blotting analysis.

c - f, A putative interaction surface of the ISX/BRD4 complex was presented through structural modeling. The electrostatic surfaces are drawn either blue for positive or red for negative charge (e and f).

Data information: Each experiment was repeated at least three times.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 6

Figure 6. BD2 domain of BRD4 is a critical domain that facilitates ISX-BRD4 complex formation.

a mCherry-tagged truncated BRD4 mutant proteins and GFP-tagged ISX were detected in anti-GFP immunoprecipitates by Western blot.

b and c, The DNA binding activity of Snail (b) and TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate.

d and e, The cell migration (wound healing, d) and invasion (transwell, e) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate.

Data information: Each experiment was repeated at least three times.
PCAF-mediated acetylation of ISX recruits BRD4 to promote epithelial-mesenchymal transition (2019)
Figure 7

Figure 7. ISX, BRD4 and PCAF expression are correlated with lung cancer metastasis. a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X).

b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.

c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.

d and e, Correlation analysis of mRNA expression for, (d) ISX and BRD4, (e) ISX and PCAF in 157 lung cancer samples.

f, Protein expression of ISX, ISX(69Kac) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting. N, normal tissue; T, tumor mass; Arrow, target protein; *, unspecific band.

g and h, The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and ISX (g) and PCAF (h) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.

Expanded View Figure legends
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 1

Figure 1. The knockout of hepsin with CRISPR/Cas9 inhibits TGFβ signaling in the mouse mammary gland. (A) Hepsin knockout mice harbor a 50bp frameshift deletion in the 4th exon of the Hpn gene (TM= Transmembrane domain, SRCR= Scavenger Receptor Cysteine Rich domain, SPD= Serine Protease Domain; Red bar indicates gRNA binding site). Immunoblot from whole mammary lysates against hepsin protein (Hpn+/+ =Wt, Hpn+/- = heterozygous deletion, Hpn-/- = homozygous deletion) (representative of 3 mice). (B) Weight of indicated organs isolated from Wt (N=3) and Hpn-/- (N=4) mice. (C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice. (D) Cytoscape enrichment map of pathways affected in Hpn-/- whole mammary glands compared to Wt controls (N=3 each). Node size correlates with the number of genes in the signature; node color correlates with either gene set enrichment (red) or reduction (blue) in Hpn-/- mammary glands. A full list of gene signatures affected in Hpn-/- mammary glands is shown in Table EV1. (E) Gene set enrichment analysis (GSEA) graphs showing enrichment of indicated TGFβ1 signaling gene sets in Hpn-/- mammary glands compared to Wt mammary glands. (F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt, normalized to total Smad2/3. (G) Representative Carmine alum stained mammary gland whole mounts from Wt and Hpn-/- mice. The histogram depicts quantification of duct length normalized to duct length in Wt mammary glands. The scale bar represents 1mm. Data in (B, C, F, G) are represented as mean±SD, a student's t-test was used for statistical analyses.