EMBO/soda-nel huggingface dataset:
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 9

Figure 9. Overexpression of Rbd2 bypasses requirement for Cdc48 binding to Rbd2

A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).

C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-Flag IgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).

D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.
Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7) (2021)
Figure 2

Figure 2. Progressively increased pro-inflammatory immunity during the course of RDEB. (A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice.

(B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6.

(C) Plot from flow cytometry analysis of CD38+ and Egr2+ macrophages in forepaw skin from 10-wk-old WT and RDEB mice.

(D) Bar graph of flow cytometry analyses as in C of inflammatory macrophages (CD38+Egr2-) or tissue-repair macrophages (CD38-Egr2+) macrophages in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age; n = 5-6 (CD38+Egr2-); n = 4-5 (CD38-Egr2+).

(E) Plots of MHC II staining intensity in neutrophils (top) and in macrophages (bottom) from flow cytometry analysis of forepaws from 10-wk-old WT and RDEB mice.

(F-G) The mean fluorescence intensity (MFI) of MHC II staining on (F) neutrophils (n = 3-4 biological replicates) and (G) macrophages (n = 6-7 biological replicates).

(H) Systemic inflammation in RDEB mice. The percentage abundance of neutrophils and monocytes in blood of 4-wk-old WT and RDEB mice, (n = 5-7 mice).

(I) Analyses of percentage CD3ε+ positive CD45+ cells (T-cells) in paws, and percentage CD4 or CD8 single positive T cells, (n = 3-4 biological replicates),

(J) Staining of back skin from 10-wk-old WT and RDEB mice for IgG (red). Keratinocytes are stained with syndecan-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to visualize nuclei, scale bar = 100 μm.

Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction (B, D, F, G, and I) or unpaired t-test (H). P values < 0.05 are considered significant.
Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7) (2021)
Figure 3

Figure 3. Translational validation of stage-dependent inflammatory and fibrosis-associated protein changes in human RDEB skin.

(A-D) Skin from healthy young (3-8 years old) and adult donors ( > 25 years old) and from donors with RDEB with different stages of fibrosis - early (0-3 years old), mild (6-10 years old) and advanced ( > 15 years old) - stained for CD68 (green), CD3ε (green), periostin (green) and tenascin-C (red). Nuclei were counterstained with DAPI (blue). A and B, scale bar = 50 μm; C and D, scale bar = 100 μm. The bar graphs at the right to the stained sections, represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D, the mean intensity of staining from 3-6 donors per group. Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction. P values < 0.05 are considered significant.
Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7) (2021)
Figure 4

Figure 4. Ang-(1-7) exerts sustained, inflammatory cell-mediated effects to deactivate fibroblasts.

(A) Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor‎), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control.

(B) Densitometric quantification of blots as in A. The abundance was normalized to β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates).

(C) Western blots of cell lysates from THP-1 monocytes treated daily with 10 μM LY2109761 or 100 nM Ang-(1-7) for two days. The blots were probed with antibodies against the indicated proteins. β-tubulin or β-actin was used as loading control.

(D) Densitometric quantification of western blots from THP-1 monocytes treated daily with 10 μM LY2109761 or 100 nM Ang-(1-7) for two days. The abundance was normalized to β-tubulin or β-actin and then expressed as % of untreated (-). P values are shown; data were analyzed with one-way ANOVA with Dunnett's correction (n = 3-8 technical replicates) .

(E) Schematic illustration of fibroblast- THP-1 monocyte co-culture assay to assess the sustained effect of Ang-(1-7) and their principal cellular mediators.

(F) Western blots of fibroblast lysates from co-cultures as described in E. The blots were probed with antibodies detecting fibronectin, thrombospondin-1 (THBS-1) and pSMAD-2/3. β-tubulin was used as loading control.

(G) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments, n = 4 -5 using cells from 4 donors with RDEB. The abundance was normalized to β-tubulin then and then expressed as % of both untreated. The data were analyzed by one-way ANOVA with Tukey's correction.

Data information: P values < 0.05 are considered significant and shown, ns = not significant. For B, D and G individual data points, mean ± S.E.M are shown.
Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7) (2021)
Figure 6

Figure 6. Modulation of dermal inflammation by Ang-(1-7).

(A) Haemotoxylin and Eosin (H&E) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm.

(B-D) Flow cytometry analysis of the number of CD45 positive cells (B), percentage of neutrophils (C) and of inflammatory macrophages (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) (D) in forepaw skin of RDEB mice treated as in A.

(E) The mean fluorescence intensity (MFI) of MHC II staining on neutrophils and macrophages, inflammatory macrophage (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) from mice as in B-D.

(F) Staining for IgG (red) in forepaws, nuclei counterstained with DAPI.

(G) Quantification of fluorescence staining intensity of samples as in F.

Data information: Panels A and F, scale bar = 100 μm. For B-E and G, individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.
Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7) (2021)
Figure 7

Figure 7. Dermal and systemic fibrosis in RDEB is effectively attenuated by low-dose Ang-(1-7).

(A) Picrosirius red staining visualized under polarized light and Elastica van Gieson (EvG) staining) of forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red in RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the EvG staining).

(B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown.

(C) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A. α-SMA, α-smooth muscle actin; Thbs-1, thrombospondin-1. Quantifications of multiple samples are shown in Figure EV4.

(D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained with DAPI (blue).

(E) Western blot for the indicated proteins on tissue lysates from eyes, esophagus or tongue from RDEB mice treated as in A. Quantifications of multiple samples are shown in Figure EV5.

Data information: β- actin or β-tubulin was used as loading control for C and E. A, scale bar = 100 μm; D, scale bar = 20 μm.

Expanded view
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 1

Figure 1. Multiple growth factors upregulate AURKB in severe fibrotic lung disease.

(A) Quantification of AURKB transcripts in non-IPF lung derived resident-fibroblasts treated with indicated mitogens for 16hrs. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1-way ANOVA, (n=4).

(B) Quantification of AURKB gene transcripts in isolated fibroblasts from non-IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t-test, (n=6).

(C) IPF and non-IPF lung sections were immunostained using AURKB antibody (n=4). Representative images were obtained at 40X magnification. Scale bar: 40µm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.

(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).

(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.

(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).

Data information: All data was presented as mean ± SEM. Exact P values are shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 2

Figure 2. WT1 regulates AURKB expression.

(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).

(B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).

(C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3).

(D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4).

(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).

(F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).

Data information: All data was presented as mean ± SEM. The above data was a representative of two independent experiments with similar results. P values were shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 3

Figure 3. AURKB functions as a positive regulator of fibroproliferation and survival.

(A) Venn diagram depicting the comparison and overlap of differentially expressed genes in IPF lungs and AURKB siRNA-treated fibrotic fibroblasts. The dashed box indicates genes that were up- (141 genes) or down-regulated (DN, 50 genes) in IPF lungs compared with AURKB siRNA knockdown gene expression signatures.

(B) AURKB-driven genes activated in IPF were analyzed using ToppFun and visualized using Cytoscape. Red- and blue-colored circles represent genes that are up or downregulated, respectively in IPF lungs. The turquoise-colored circle represents enriched biological processes for the inversely correlated genes between AURKB siRNA knockdown and IPF.

(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).

(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).

(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).

(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).

(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).

(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).

(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).

(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).

(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).

Data information: All data was presented as mean ± SEM. The above data was a representative of two independent experiments with similar results. Exact P values are shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 4

Figure 4. Blockade of AURKB-activity impacts fibroblast proliferation and survival.

(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).

(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).

(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).

(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).

(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).

Data information: All data were presented as mean ± SEM. The above data was a representative of two independent experiments with similar results. P values were shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 5

Figure 5. In vivo Barasertib treatment prevents from TGFα induced lung fibrosis.

(A) Schematic illustration of barasertib preventive treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40mg/kg; twice a day) for 4 wks while they were fed with Dox containing food.

(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.

(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).

(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).

(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 6

Figure 6. In vivo barasertib treatment attenuates mesenchymal proliferation.

(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.

(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).

(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 7

Figure 7. Therapeutic barasertib treatment reduces established and ongoing lung fibrosis.

(A) Schematic illustration of barasertib treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40mg/kg; twice a day) for last 3 wks while they were fed with Dox containing food for total of 6 wks.

(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).

(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).

(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.

(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.
Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis (2020)
Figure 8

Figure 8. Therapeutic barasertib treatment attenuates bleomycin-induced lung fibrosis

(A) Schematic illustration of barasertib treatment protocol. Mice were treated with either vehicle or barasertib (40mg/kg; twice a day) for last 2 wks while they were injected intradermally with either saline or bleomycin five days per wk for total of 4 wks.

(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.

(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).

(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).

(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.

Expanded view figure legends