Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 2. (A) CTGF-immunoreactivity (A, red) was detected in the corneal epithelium, mainly concentrated in basal layers (blue: DAPI). (B) Single superficial epithelial cells were detected with rather intense immunoreactivity for CTGF (red, arrowhead; blue: DAPI). (C) CTGF-immunoreactivity (red) was also detectable in corneal endothelial cells (arrows), as well as in keratinocytes of the stroma (arrowheads; blue: DAPI). (D) CTGF-immunoreactivity was absent in corresponding negative controls (here: corneal epithelium and stroma; blue: DAPI). (E, F) Conjunctival epithelial cells displayed immunoreactivity for CTGF (E), but CTGF-immunoreactivity was absent in the corresponding negative control (F). Arrowheads: goblet cells; blue: DAPI). (G, H) In the trabecular meshwork (asterisk) and Schlemm’s canal (arrowheads), CTGF immunoreactivity (red) revealed an identical staining pattern (G) as compared with corresponding negative controls (H).

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques: Negative Control, Staining

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 3. (A, B) In the iris, CTGF-immunoreactivity (A, red) was present in anterior layers of the iris (arrows) as well as muscle fibers of the iris sphincter (arrowheads), as detected with alpha-smooth-muscle actin (green), but was absent in the corresponding negative control (B). DAPI: white. (C, D) In C, iris vessels displayed immunoreactivity for CTGF (red) in the vascular endothelium (blue, CD31), as seen by an association of both signals (purple color), while an overlap of CTGF with vascular smooth muscle cells (green, ASMA) was not observed, as seen by absence of yellow-mixed color. Immunoreactivity was absent in corresponding negative controls (D). Asterisks indicates vessel lumen; DAPI: white. (E, F) CTGF-immunoreactivity (E, red) was present in the muscle fibers of the ciliary body (asterisk), as detected with alpha – smooth muscle actin (green), but was absent in the corresponding negative control (E). DAPI: white. (G, H) CTGF-immunoreactivity was present in the non-pigmented ciliary epithelium (G, arrowheads), but was absent in corresponding negative controls (F; red signal here, arrows, corresponds to autofluorescence). DAPI: white. (I, J) CTGF-immunoreactivity (red) in the lens was absent in the lens epithelium (I, arrowheads), but present in superficial layers of the lens (I, asterisk), while it was absent in the corresponding negative control (J, asterisk). DAPI: blue.

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques: Negative Control

Journal: Current eye research

Article Title: Immunohistochemical Detection of CTGF in the Human Eye.

doi: 10.3109/02713683.2016.1143014

Figure Lengend Snippet: Figure 4. (A, B) In the retina, CTGF-immunoreactivity (red) was present in the nerve fiber layer (NFL; A: cross-section close to the optic nerve head), and further a weak signal was also present in the IPL and OPL, while immunoreactivity was absent in corresponding negative controls (F). DAPI: blue. (C, D) In the choroid, CTGF- immunoreactivity (red) was present in the choriocapillaris (C, arrowheads) and in blood vessels of the choroidal stroma (C, arrows), but was absent in corresponding negative controls (D). DAPI: blue; RPE: retinal pigment epithelium; CC: choriocapillaris. (E, F) In the optic nerve head, CTGF-immunoreactivity (red) was present in endothelial cells of the central retinal artery (E, asterisks) and was detected in single cells within connective tissue strands of the optic nerve (F, arrowhead; inset represents magnified situation in F). DAPI: blue. All images in Figures 2, 3, and 4 represent confocal images in single optical section mode.

Article Snippet: After a 5 min rinse, slides were incubated overnight at RT with an anti-human connective tissue growth factor (CTGF; raised in goat, 1:100, AF660, R&D Systems, Minneapolis, MN, USA) in TBS, containing 1% BSA and 0.5% Triton X-100.

Techniques: