Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 1. Effects of IgE-FcεR1 signaling on CFs. (A) FcεR1a expression levels in CFs of WT and FcεR1-KO mice measured by immunoblot. (B-D) FcεR1a expression levels after IgE treatment (5 μg/mL) for 0, 3, and 24 h measured by qPCR (B) and immunoblot (C-D). (E-F) mRNA expression of key fibrotic genes (a-SMA, Col1a1 and Col3a1) in IgE-stimulated FcεR1-WT (E) and FcεR1-KO (F) CFs at 0, 3, and 24 h. (G) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-WT CFs after IgE treatment for 0, 3, and 24 h. (H) Statistical analysis of α-SMA, COL1A1, and COL3A1 protein expression in FcεR1-WT CFs, normalized to GAPDH (fold change versus 0 h). (I) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs after IgE treatment. (J) Statistical analysis of the relative protein expression of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 2. Effects of CF FcεR1 deletion on Ang II-induced cardiac fibrosis. (A) Representative images of Masson staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 200X magnification. Scale bars, 50 μm. (B) Quantification of myocardial interstitial fibrosis by Masson staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (C) Representative images of Sirius Red staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 400X magnification. Scale bars, 50 μm. (D) Quantification of myocardial interstitial fibrosis by Sirius Red staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (E) qPCR analysis of periostin (Postn) and a-SMA expression in the heart tissues of the four indicated groups. (F) Representative immunoblot analysis showing the expression of COL1A1 and COL3A1 in the hearts of Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. (G) Statistical analysis of COL1A1 and COL3A1 expression normalized to GAPDH. Total n = 5 (Saline/FcεR1-Flox), n = 5 (Saline/cKO), n = 10 (Ang II/FcεR1-Flox), or n = 9 (Ang II/cKO) per group. Results are shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 by Two-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Staining, Saline, Expressing, Western Blot

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 3. IgE alters miRNAs profile in CFs and down-regulates miR-486a-5p. (A) Heatmap illustrates differentially expressed miRNAs. The expression is normalized as row z-scores, up-regulation is indicated in red and down-regulation is indicated in blue. Each row represents a miRNA. (B) Three-way Venn diagram indicating the total number of miRNAs predicted to directly target Col1a1 and Col3a1. The numbers of shared miRNAs are indicated in the intersections of the Venn diagram. Two miRNAs in the intersections are shown as black stars in (A). (C) Two-way Venn diagram indicating the miRNAs that may be involved in fibrosis. Seven miRNAs in the intersection are shown as red stars in (A). (D) qPCR analysis of eight candidate miRNAs from the intersections shown in (B-C). The expression levels of miRNAs in FcεR1-WT (left panel) and FcεR1-KO (right panel) CFs after IgE stimulation were normalized to U6. Three independent experiments were performed. (E) Expression of miR-486a-5p in IgE-stimulated FcεR1-WT and FcεR1-KO CFs at 0, 3, and 24 h measured by qPCR (fold change versus 0 h) in three independent experiments. Results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s indicates no significance in One-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 5. IgE promotes collagen expression via miR-486a-5p/Smad1 in CFs. (A-B) Representative immunoblot (upper panel) and quantification (lower panel) of SMAD1 protein expression in IgE-stimulated WT (A) and FcεR1-KO (B) CFs at indicated lengths of time (0, 3, 24 h). (C-D) Representative immunoblot (C) and quantification (D) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with small interference RNA (siRNA) against Smad1 or scrambled control. (E-F) Representative immunoblot (E) and quantification (F) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with Smad1 ORF clone or scrambled control. (G-H) Representative immunoblot (G) and quantification (H) of SMAD1, COL1A1, and COL3A1 protein expression in CFs transfected with miR-486a-5p mimic and/or IgE. (I-J) Immunoblot (I) and quantification (J) showing protein levels of SMAD1, COL1A1, and COL3A1 in CFs transfected with siRNA against Smad1 and/or miR-486a-5p inhibitor. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni’s post hoc test (A-B), Student’s t-test (D, F) or Two-way ANOVA with Bonferroni’s post hoc test (H, J).

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Cell reports

Article Title: Opening of cohesin's SMC ring is essential for timely DNA replication and DNA loop formation.

doi: 10.1016/j.celrep.2021.108999

Figure Lengend Snippet: Figure 5. Head opening is essential for cohesin translocation and stable DNA loop formation. (A) SYPRO-Ruby staining of purified head-tetherable human cohesin tetramer harboring Smc1-FKBP-3HA, Smc3-FRB*-3FLAG, Scc1-Halo, and 10His-SA1. Alexa 488 dye was conjugated to a Halo tag. (B) Head-tetherable cohesinA488 was loaded on DNA by NIPBL-Mau2 and ATP in the presence or absence of AP21967. After high-salt washing, the intensities of the salt-stable cohesin on the DNA were quantified and shown as boxplots (R50 DNAs per experiment; results from three experiments were combined. Mann- Whitney U test). (C) MSD versus time for DNA-bound head-tetherable cohesinA488 in the presence or absence of AP21967. Head-tetherable cohesinA488 was loaded onto DNA in the presence of NIPBL-Mau2 and ATP. After high-salt washing, the cohesin particles on the DNA were observed in the presence or absence of AP21967. The D indicates the diffusion coefficient (n R 33, means ± SEM). (D) DNA loop formation frequencies in the presence or absence of AP21967 or ADP/AlFx (n = 3, R71 DNAs per condition, means ± SEM). (E) Examples of stable and unstable DNA loops seen in the experiments shown in (D). DNA was visualized by SYTOX orange staining. Inserted numbers indicate time (in seconds). Bar: 2 mm. (F) Quantification of the loop-maintaining duration. In the DNA loops formed in (D), durations of maintained loops were quantified in the presence or absence of AP21967 and shown as boxplots (n = 3, R77 DNAs per condition, Mann-Whitney U test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Smc3 Bethyl Cat#A300-060A; RRID: AB_67579 Rabbit polyclonal anti-Smc1 Bethyl Cat#A300-055A; RRID: AB_2192467 Rabbit polyclonal anti-Rad21 Bethyl Cat#A300-080A; RRID: AB_2176615 Rabbit polyclonal anti-Wapl Kueng et al., 2006 N/A Rabbit polyclonal anti-Sororin Schmitz et al., 2007 N/A Rabbit polyclonal anti-Pds5A Bethyl Cat#A300-089A; RRID: AB_2162213 Goat polyclonal anti-Histone H3 (C-16) Santa Cruz Cat#Sc8654; RRID: AB_2118303 Mouse monoclonal anti-PCNA (PC10) Santa Cruz Cat#SC56; RRID: AB_628110 Rat monoclonal anti-HA (3F10) Roche Cat#11867423001; RRID: AB_390918 Mouse monoclonal anti-FLAG M2 Sigma-Aldrich Cat#F18041MG; RRID: AB_262044 Chicken polyclonal anti GFP Abcam Cat#ab13970; RRID: AB_300798 Rabbit monoclonal anti-p53 (EP42Y) Abcam Cat#ab239880; RRID: AB_1524137 Rabbit monoclonal anti-Phospho-SMC1 (D7S8Y) CST Cat#58052S; RRID: AB_2799538 Mouse monoclonal anti-Phospho-ATM (Ser1981) CST Cat#4526S; RRID: AB_2062663 Rabbit polyckonal anti-CHK1 (phospho S345) Genetex Cat#GTX100065; RRID: AB_1949975 Mouse monoclonal anti-H2A.X Phospho (Ser139) Biolegend Cat#613401; RRID: AB_315794 Anti-rabbit IgG (HRP-conjugated) GE Healthcare Cat#NA934V; RRID: AB_2722659 Anti-mouse IgG (HRP-conjugated) GE Healthcare Cat#NA931V; RRID: AB_772210 Anti-rat IgG (HRP-conjugated) GE Healthcare Cat#NA935; RRID: AB_772207 Anti-Chicken IgY, Alexa Fluor 488 Invitrogen Cat#A11039; RRID: AB_142924 Anti-Mouse IgG, Alexa Fluor 488 Invitrogen Cat#A21202; RRID: AB_141607 Anti-Rabbit IgG, Alexa Fluor 488 Invitrogen Cat#A11008; RRID: AB_143165 Chemicals, peptides, and recombinant proteins Thymidine Sigma-Aldrich Cat#T1895-5G Nocodazole Sigma-Aldrich Cat#M1404-10MG Aphidicolin Sigma-Aldrich Cat#M1404-10MG Camptothecin Sigma-Aldrich Cat#C9911-100MG Mirin Sigma-Aldrich Cat#M9948 3-Indoleacetic acid (IAA) Sigma-Aldrich Cat#15-0330-1 RO-3306 Millipore Cat#217699-5MG Doxycycline Clontech Cat#631311 A/C Heterodimerizer Clontech Cat#635057 Lipofectamine RNAiMAX Invitrogen Cat#13778-030 Opti-MEM GIBCO Cat#31985-062 BSA (Albumin, from Bovine Serum, Fraction V Ph7.0) Wako Cat#013-27054 Triton X-100 Sigma-Aldrich Cat#30-5140-5-500ML-J Goat-serum GIBCO Cat#16210-064 Paraformaldehyde Millipore Cat#104005 Prolong Gold Antifade Reagent Invitrogen Cat#P36930 Cellstain - DAPI solution DOJINDO Cat#D523 cOmplate EDTA-Free Protease Inhibitor Cocktail Roche Cat#5056489001 Benzamidene Sigma-Aldrich Cat#12072-10G ATP Sigma-Aldrich Cat#A2383-1G (Continued on next page) e1 Cell Reports 35, 108999, April 27, 2021

Techniques: Translocation Assay, Staining, MANN-WHITNEY, Diffusion-based Assay