Journal: Advanced Science

Article Title: Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes Functionally Recapitulated Damaged Skin

doi: 10.1002/advs.202103079

Figure Lengend Snippet: Activation of β 2 ‐AR signaling induced stemness acquisition in HEKs. a) Western blot analysis of β 2 ‐AR expression in human skin samples. Human liver tissues were used as positive controls ( n = 3); b) Immunohistochemistry of β 2 ‐AR in human developing glands at embryo day 26 (E26). Scale bar represents 100 µm; c) Representative immunofluorescence showing the co‐labeling of β 2 ‐AR with duct marker CK14, luminal markers CK18, CK19, and myoepithelial marker α ‐SMA in human mature SwGs. Boxed area is magnified to see β 2 ‐AR typically restricted in the CK18 + or CK19 + luminal cells (white arrows). Star showing SwG ducts. Scale bar represents 25 µm; d) Heatmap illustrating the genes involved in stemness regulation, skin development, gland organogenesis, and neuron development in HEKs with or without ISO treatment (FDR ≤ 0.05 and |FC| ≥ 2); e) Scatter plot of RNA‐seq transcriptome analyses between HEKs treated with or without ISO. Stemness‐associated genes were enriched in ISO‐treated HEKs versus controls (colored dots); f) Expression of stemness‐associated genes in ISO‐treated and untreated HEKs; g) For β 2 ‐AR blockage, starved HEKs were pre‐treated with 10 µ m ICI‐118551 for 3 h. The cells were subsequently incubated in the presence or absence of 5 µ m ISO for an additional 24 h. Then, relative mRNA expression levels of LGR5, LGR6, OCT4, SOX9 was assessed by quantitative RT‐PCR ( n = 3). The results were normalized to GAPDH expression; h) qRT‐PCR analysis showing LGR5, LGR6, OCT4, SOX9 expression in HEKs and si‐ β 2 ‐AR‐treated HEKs with or without ISO ( n = 3); i) Western blot analysis was performed to investigate the protein levels of LGR5, OCT4, SOX9 in HEKs after switched to the medium containing 0, 5 µ m ISO with or without ICI‐118551 ( n = 3); j) Quantitative western blot analysis of protein expression for LGR5, OCT4, and SOX9. The densitometric values were qualified with Image J software and the relative density (compared with untreated HEKs) of the indicated protein was calculated; k) Western blot analysis of LGR5, LGR6, OCT4, SOX9 expression levels in HEKs and si‐ β 2 ‐AR‐treated HEKs with or without ISO treatment ( n = 3); l) Qualification of LGR5, LGR6, OCT4, SOX9 immunoblots was performed using Image J software. GAPDH was used as internal loading control. Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blots were probed with the following primary antibodies overnight at 4 °C: rabbit anti‐SOX9 (ab185966, Abcam, 1:1000), rabbit anti‐LGR5 (ab75850, Abcam, 1:1000), rabbit anti‐OCT4 (ab19857, Abcam, 1:1000), mouse anti‐CK18 (4548s, CST, 1:2000), rabbit anti‐EDA (ab125233, Abcam, 1:500), mouse anti‐CEA (2383s, CST, 1:1000), rabbit anti‐BMP5 (ab38565, Abcam, 1:1000), rabbit anti‐CK19 (ab52625, Abcam, 1:50 000), rabbit anti‐CK5 (ab52635, 1:10 000), rabbit anti‐c‐MYC (ab32072, Abcam, 1:1000), rabbit anti‐ α ‐SMA (19245s, CST, 1:1000), rabbit anti‐AQP5 (ab92320, Abcam, 1:1000), rabbit anti‐ β 2 ‐AR (ab182136, Abcam, 1:1000), rabbit anti‐HA (ab9110, Abcam, 1:4000), and rabbit anti‐GAPDH (2118s, CST, 1:1000).

Techniques: Activation Assay, Western Blot, Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Marker, RNA Sequencing, Incubation, Quantitative RT-PCR, Software, Control

Journal: Advanced Science

Article Title: Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes Functionally Recapitulated Damaged Skin

doi: 10.1002/advs.202103079

Figure Lengend Snippet: Reprogramming of HEKs into SwG cells by combining the stimulation of β 2 ‐AR, forced transgenic expression of EDA and SGM culture. a) Scheme of ISO‐based reprogramming procedure. ISO‐treated HEKs were transduced with EDA and plated in Epilife. Then cells were transferred into SGM supplemented with 5 µ m ISO (Day 0) and cultured for indicated days; b) Phase contrast images showing the morphological changes of iSwGCs in optimized SGM containing ISO. Scale bar = 100 µm. Insets, higher magnification of the boxed areas; c) qPCR analysis of transcriptional expression of CK5, CK18, AQP5, α ‐SMA, and hair follicle‐specific genes LHX2, CDH3 in HEKs, and iSwGCs after 8 days of induction. Primarily isolated SwG cells from human skin samples (hSwGCs) were used as positive controls. The genes showing significant change in PCR array assay are presented; d) Representative immunofluorescence of CK5, CK10, AQP5, CK18, and α ‐SMA in HEKs, and iSwGCs at day 20 after SGM treatment with ISO. Scale bar = 50 µm; e) Percentages of CK5 + , CK10 + , AQP5 + , CK18 + , and α ‐SMA + cells in HEKs and iSwGCs calculated according to the immunostaining. Quantification was done with 5 randomly selected microscopy fields from each of the 3 independent experiments; f) FACS analysis showing the cell fractions labeled with antibodies against CK5, CK10, AQP5, CK18, and α ‐SMA in HEKs, iSwGCs and native hSwGCs. g) Proportions and absolute numbers of the CK5 + /CK10 + , AQP5 + /CK18 + , and CK5 + / α ‐SMA + cell population in HEKs, iSwGCs, and hSwGCs. n = 3. Data are mean ± SD and analyzed by two‐tailed t ‐tests, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Blots were probed with the following primary antibodies overnight at 4 °C: rabbit anti‐SOX9 (ab185966, Abcam, 1:1000), rabbit anti‐LGR5 (ab75850, Abcam, 1:1000), rabbit anti‐OCT4 (ab19857, Abcam, 1:1000), mouse anti‐CK18 (4548s, CST, 1:2000), rabbit anti‐EDA (ab125233, Abcam, 1:500), mouse anti‐CEA (2383s, CST, 1:1000), rabbit anti‐BMP5 (ab38565, Abcam, 1:1000), rabbit anti‐CK19 (ab52625, Abcam, 1:50 000), rabbit anti‐CK5 (ab52635, 1:10 000), rabbit anti‐c‐MYC (ab32072, Abcam, 1:1000), rabbit anti‐ α ‐SMA (19245s, CST, 1:1000), rabbit anti‐AQP5 (ab92320, Abcam, 1:1000), rabbit anti‐ β 2 ‐AR (ab182136, Abcam, 1:1000), rabbit anti‐HA (ab9110, Abcam, 1:4000), and rabbit anti‐GAPDH (2118s, CST, 1:1000).

Techniques: Transgenic Assay, Expressing, Transduction, Cell Culture, Isolation, Immunofluorescence, Immunostaining, Microscopy, Labeling, Two Tailed Test

Journal: Advanced Science

Article Title: Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes Functionally Recapitulated Damaged Skin

doi: 10.1002/advs.202103079

Figure Lengend Snippet: Establishment of human SwG organoids from iSwGCs. a) Representative phase contrast images showing iSwGO cultures. Scale bar = 100 µm; b) Representative images of lumen‐containing organoids derived from iSwGCs. d and l represent the diameter (lumen width) and long axis of the lumen, respectively; c) Signal distribution acquired by confocal microscopy showing the quantification of luminal‐containing organoids derived from reprogrammed HEKs ( n = 23). Organoids with similar size were analyzed independently from 3 biological replicates; d,e) Scatter plots representing the features of iSwGOs. HEKs cultured under the same 3D condition were controls; f) Immunofluorescence assay of ductal markers CK5 and CK10, luminal markers AQP5 and CK18, and myoepithelial markers CK5 and α ‐SMA in iSwGOs. The iSwGOs were obtained at passages 2–4 after the initiation of 3D culture. Scale bar = 50 µm; g–i) Percentages of CK5 + /CK10 + ‐, AQP5 + /CK18 + ‐, and CK5 + / α ‐SMA + ‐expressing organoids in each GFP‐positive population were shown. Quantifications involved > 100 organoids from 3 independent experiments. Data are mean ± SD; j) Immunofluorescence co‐staining of α ‐SMA with CK18 or CK19 in iSwGOs generated from CD49f hi CD29 hi cells. Scale bar = 75 µm; k) Fluorescence live cell imaging of intracellular Ca 2+ activity in iSwGOs after ACh addition. Scale bar = 25 µm, n = 3; l) Immunofluorescence analysis of expression of ductal markers CK5 and CK10, luminal markers AQP5 and CK18 and myoepithelial markers CK5 and α ‐SMA in tubular structures generated from iSwGOs in response to bFGF gradients. Scale bar = 25 µm.

Article Snippet: Blots were probed with the following primary antibodies overnight at 4 °C: rabbit anti‐SOX9 (ab185966, Abcam, 1:1000), rabbit anti‐LGR5 (ab75850, Abcam, 1:1000), rabbit anti‐OCT4 (ab19857, Abcam, 1:1000), mouse anti‐CK18 (4548s, CST, 1:2000), rabbit anti‐EDA (ab125233, Abcam, 1:500), mouse anti‐CEA (2383s, CST, 1:1000), rabbit anti‐BMP5 (ab38565, Abcam, 1:1000), rabbit anti‐CK19 (ab52625, Abcam, 1:50 000), rabbit anti‐CK5 (ab52635, 1:10 000), rabbit anti‐c‐MYC (ab32072, Abcam, 1:1000), rabbit anti‐ α ‐SMA (19245s, CST, 1:1000), rabbit anti‐AQP5 (ab92320, Abcam, 1:1000), rabbit anti‐ β 2 ‐AR (ab182136, Abcam, 1:1000), rabbit anti‐HA (ab9110, Abcam, 1:4000), and rabbit anti‐GAPDH (2118s, CST, 1:1000).

Techniques: Derivative Assay, Confocal Microscopy, Cell Culture, Immunofluorescence, Expressing, Staining, Generated, Fluorescence, Live Cell Imaging, Activity Assay

Journal: Advanced Science

Article Title: Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes Functionally Recapitulated Damaged Skin

doi: 10.1002/advs.202103079

Figure Lengend Snippet: Engraftment of iSwGOs functionally repopulated damaged skin. a) Schematic diagram representing the experimental procedure; b) Starch–iodine sweat tests on paw skin of thermal‐injured mice showed that only paws of iSwGO‐treated mice responded by displaying indigo‐black dots at day 21 after transplantation; c) Starch–iodine assessment of newly formed SwGs in the defect regions of thermally injured mice at day 30 after SGM, iSwGC and iSwGO treatment. The number of mice positive for starch‐iodine reaction increased in a time‐dependent manner. After 30 days treatment with iSwGOs, 34.4 ± 13.0% of the recipient mice ( n = 60 per group) exhibited sweat production as compared with those treated with iSwGCs (13.3 ± 10.6%, n = 60), SGM (0, n = 60) and vehicle control (0, n = 60). Data are mean ± SD of 6 independent experiments; d) H&E staining was conducted to visualize SGM‐, iSwGC‐, iSwGO‐treated wounds at day 21 post‐injury. Emerging glandular structures were seen in the dermis of iSwGO‐treated mice. Dotted line represents the ridges of epidermis where the sweat pores open. Note that the rete ridges in iSwGO‐engrafted paw skin were elongated and intertwined with underlying dermal tissues comparable to those derived from normal skin. Scale bar = 100 µm; e) Immunofluorescence and quantification of SwG marker expression in nascent glands at day 21 after iSwGO transplantation (left). EDA‐GFP + cells were seen in the newly generated glandular structures. Newly emerged glands showed typical SwG morphological features and expressed SwG specific markers, for example, ductal markers CK5 and CK10, luminal markers AQP5, CK18, and myoepithelial markers CK5 and α ‐SMA. Scale bar = 25 µm. Quantification (right) involved > 150 cells from 3 independent experiments, and GFP + cells within the duct, luminal, and myoepithelial domain were measured, respectively; f) Immunofluorescence of human‐specific histone protein expression in nascent SwGs at day 21 after iSwGO transplantation. Scale bar = 25 µm; g) Wound healing curves for quantification of the wound coverage at different times in SGM‐, iSwGC‐, iSwGO‐treated mice ( n = 15, 3 independent experiments). Mice treated with DMEM/F‐12 were vehicle controls. Data are mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Blots were probed with the following primary antibodies overnight at 4 °C: rabbit anti‐SOX9 (ab185966, Abcam, 1:1000), rabbit anti‐LGR5 (ab75850, Abcam, 1:1000), rabbit anti‐OCT4 (ab19857, Abcam, 1:1000), mouse anti‐CK18 (4548s, CST, 1:2000), rabbit anti‐EDA (ab125233, Abcam, 1:500), mouse anti‐CEA (2383s, CST, 1:1000), rabbit anti‐BMP5 (ab38565, Abcam, 1:1000), rabbit anti‐CK19 (ab52625, Abcam, 1:50 000), rabbit anti‐CK5 (ab52635, 1:10 000), rabbit anti‐c‐MYC (ab32072, Abcam, 1:1000), rabbit anti‐ α ‐SMA (19245s, CST, 1:1000), rabbit anti‐AQP5 (ab92320, Abcam, 1:1000), rabbit anti‐ β 2 ‐AR (ab182136, Abcam, 1:1000), rabbit anti‐HA (ab9110, Abcam, 1:4000), and rabbit anti‐GAPDH (2118s, CST, 1:1000).

Techniques: Starch, Transplantation Assay, Control, Staining, Derivative Assay, Immunofluorescence, Marker, Expressing, Generated

Journal: Kidney Research and Clinical Practice

Article Title: Human CD36 overexpression in renal tubules accelerates the progression of renal diseases in a mouse model of folic acid-induced acute kidney injury

doi: 10.23876/j.krcp.2018.37.1.30

Figure Lengend Snippet: (A) A representative image of PAS staining (×100) showing normal histology of the kidneys of vehicle-treated WT and Tg mice and severely dilated tubules with casts in the kidneys of folic acid-treated WT and Tg mice. (B) A representative image of PAS staining (×400) showing marked renal damage, including renal tubular epithelial cell flattening and casts in the tubular lumens, in all folic acid-treated mice. Severely dilated tubules with casts were observed in the kidneys of folic acid-treated Tg mice. (C) A representative image of immunohistochemical analysis with anti-α-SMA antibody (×400). No significant difference was observed between the kidneys of folic acid-treated WT and Tg mice. (D) The first representative image of semiquantitative scoring of tubular tissue injury showed more damage in folic acid-treated Tg mice compared with folic acid-treated WT mice and vehicle-treated Tg mice; however, the area fraction of positive α-SMA staining of α-SMA between folic acid-treated WT and Tg mice did not show a significant difference in the second representative image. (E) A representative image of quantitative data for albuminuria, as determined using Albuwell M ELISA kit (Exocell Inc., Philadelphia, PA, USA) and creatinine ELISA kit (Exocell Inc.). Albuminuria was higher in folic acid-treated Tg mice (n = 7) than in vehicle-treated WT (n = 7) and Tg mice (n = 7) and folic acid-treated WT mice (n = 7). α-SMA, mouse α-smooth muscle actin; PAS, periodic acid Schiff; Tg, transgenic; UACR, the urinary albumin to creatinine ratio; WT, wild type. * P < 0.05 compared with vehicle-treated WT mice, † P < 0.05 compared with vehicle-treated Tg mice, ‡ P < 0.01 compared with folic acid-treated WT mice, § P < 0.05 compared with folic acid-treated WT mice, and || P < 0.01 compared with vehicle-treated Tg mice; n, number of mice. Significance was analyzed using the Mann–Whitney U test.

Article Snippet: The following antibodies were used in this study: anti-hCD36 antibody (Santa Cruz Bio, Paso Robles, CA, USA) and mouse anti-α-smooth muscle actin (α-SMA) antibody (Santa Cruz Bio).

Techniques: Staining, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Mouse Assay, MANN-WHITNEY

Journal: Kidney Research and Clinical Practice

Article Title: Human CD36 overexpression in renal tubules accelerates the progression of renal diseases in a mouse model of folic acid-induced acute kidney injury

doi: 10.23876/j.krcp.2018.37.1.30

Figure Lengend Snippet: (A) A representative image of relative Mcp1 mRNA level. Mcp1 mRNA level was significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice. However, no significant difference was observed in Mcp1 mRNA level between folic acid-treated Tg and WT mice. (B) A representative image of relative Cd68 mRNA level. Cd68 mRNA level was significantly higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (C) A representative image of relative Acta2 and Fn1 mRNA levels. Acta2 and Fn1 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (D) A representative image of relative Col1a1 and Col3a1 mRNA levels. Col1a1 and Col3a1 mRNA levels were higher in folic acid-treated Tg mice than in vehicle-treated and folic acid-treated WT mice. In particular, the Col3a1 mRNA level was significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice. (E) A representative image of relative Apaf1 and Bax/Bcl2 mRNA levels. Apaf1 and Bax/Bcl2 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. Moreover, the Bax/Bcl2 mRNA level increased in folic acid-treated Tg mice. However, there was no significant difference in Bax/Bcl2 mRNA levels between folic acid-treated Tg and WT mice. Acta2 , α-smooth muscle actin; Apaf1 , apoptotic protease activating factor 1; Bax/Bcl2 , B-cell lymphoma 2-associated X/B-cell lymphoma 2; Col1a1 , collagen 1a1; Col3a1 , collagen 3a1; Fn1 , fibronectin; Mcp1 , monocyte chemoattractant protein-1; Tg, transgenic; WT, wild type. * P < 0.05 compared with vehicle-treated WT mice, † P < 0.05 compared with vehicle-treated Tg mice, and ‡ P < 0.05 compared with folic acid-treated WT mice; a P < 0.01. Significance was analyzed using the Mann–Whitney U test.

Article Snippet: The following antibodies were used in this study: anti-hCD36 antibody (Santa Cruz Bio, Paso Robles, CA, USA) and mouse anti-α-smooth muscle actin (α-SMA) antibody (Santa Cruz Bio).

Techniques: Transgenic Assay, Mouse Assay, MANN-WHITNEY

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Serotonin Activated Hepatic Stellate Cells Contribute to Sex Disparity in Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2017.01.002

Figure Lengend Snippet: Sex disparate increase of HSCs and activated HSCs after kras V12 induction. ( A ) Overlap of hand2+ and Gfap expression. Gfap expression was examined in liver sections of hand2+ transgenic fish with 4′,6-diamidino-2-phenylindole (DAPI) staining for nuclei (blue) 8 days after fertilization. ( B ) Percentage of Gfap+ HSCs as marked by hand2:gfp expression in liver sections (n = 20). ( C ) Percentage of hand2+ HSCs as marked by Gfap+ expression in liver sections (n = 20). ( D ) IF co-staining of GFAP (red, general HSCs) and α-Sma (green, activated HSCs) in liver sections of adult zebrafish. Three-month-old kras+ and WT, male and female zebrafish were treated with 30 μg/mL dox for 7 days. Total HSCs, activated HSCs, and serotonin were examined. White boxes indicate enlarged regions shown on the right of each photograph. Arrows indicate Gfap+ cells. ( E ) Quantification of HSC density (Gfap+, top ) and percentages of activated HSCs (Gfap+/α-Sma+, bottom ) in liver sections (n > 8 in each group). * P < .05. Scale bar : 20 μm.

Article Snippet: For IHC and IF stainings, the primary antibodies were derived mostly from rabbits, including anti-proliferating cell nuclear antigen (PCNA) (FL-261; Santa Cruz Biotechnology, Dallas, TX), anti–caspase 3 (C92-065; BD Biosciences, Singapore), antiserotonin (C5545; Sigma), anti-TgfB1 (04-953; EMD Millipore, Billerica, MA), anti-PTph (SC135716; Santa Cruz), anti–α-smooth muscle actin (α-Sma) (ab15734; Abcam, Singapore), anti-PSmad2 (3101; Cell Signaling Technology, Danvers, MA) and anti–collagen I (ab23730; Abcam), except for anti-glial fibrillary acidic protein (Gfap) (154474; Abcam) from the mouse.

Techniques: Expressing, Transgenic Assay, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Serotonin Activated Hepatic Stellate Cells Contribute to Sex Disparity in Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2017.01.002

Figure Lengend Snippet: Manipulation of serotonin level and HSC density affects HSC activation and apoptosis. ( A ) IF staining and quantification of HSC density (Gfap+) in liver sections (n > 8 in each group). Three-month-old adult zebrafish were treated with dox with or without serotonin, BW23C86, PCPA, and SB204741 for 7 days. Percentages of activated HSCs (Gfap+/α-Sma+) in the liver sections (n > 8 in each group). ( B ) Expression of tgfb1 in hepatocytes, HSCs, neutrophils, macrophages, and cholangiocytes. These cells were isolated by fluorescence-activated cell sorting based on DsRed, GFP, DsRed, mCherry, and GFP expression, respectively, from fabp10+ , hand2+ , lyz+ , mpeg+ , and tp1+ transgenic zebrafish. Total RNAs were extracted and htr2b expression was determined by reverse-transcription quantitative PCR. Relative expression levels are with the values with female hepatocytes set as 1. ( C and D ) IF staining and quantification of percentages of ( C ) TgfB1+ HSCs (Gfap+/Tgfb1+) and ( D ) apoptotic HSCs in the liver sections as described in panel A . n > 8 in each group. * P < .05. Scale bar : 20 μm.

Article Snippet: For IHC and IF stainings, the primary antibodies were derived mostly from rabbits, including anti-proliferating cell nuclear antigen (PCNA) (FL-261; Santa Cruz Biotechnology, Dallas, TX), anti–caspase 3 (C92-065; BD Biosciences, Singapore), antiserotonin (C5545; Sigma), anti-TgfB1 (04-953; EMD Millipore, Billerica, MA), anti-PTph (SC135716; Santa Cruz), anti–α-smooth muscle actin (α-Sma) (ab15734; Abcam, Singapore), anti-PSmad2 (3101; Cell Signaling Technology, Danvers, MA) and anti–collagen I (ab23730; Abcam), except for anti-glial fibrillary acidic protein (Gfap) (154474; Abcam) from the mouse.

Techniques: Activation Assay, Staining, Expressing, Isolation, Fluorescence, FACS, Transgenic Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells

doi: 10.1084/jem.20180752

Figure Lengend Snippet: Identification of EPCs in VEGF-induced kidney. VEGF-induced kidney stained with Epo antibody. EPCs were visualized using HRP or immunofluorescent markers, highlighting perivascular Epo expression. Note Epo expression in previously identified EPCs (proximal tubules and peritubular interstitial fibroblasts; marked by asterisks), as well as by SMA + perivascular cells (marked by arrows). (A and B) Epo-HRP immunostaining with eosin counterstaining. (C–H) Immunofluorescent Epo staining with the indicated cell type–specific markers: isolectin B4 (staining endothelial cells); CD73 (staining peritubular interstitial fibroblasts); SMA (staining pericytes and VSMC-like cells); LTL (staining distal tubules); and DBA (staining proximal tubules). Bar, 50 µm. Some images were cropped to more closely highlight Epo staining. Data are representative of at least two independent experiments ( n = 3–5 animals per experiment).

Article Snippet: Primary antibodies included rabbit Epo antibody (1:50, H-162, sc-7956; Santa Cruz), mouse Epo antibody (1:50, 7D10, sc-80995; Santa Cruz), rabbit SMA (1:200, ab32575; Abcam), mouse SMA (1:200, ab7817; Abcam), rabbit phospho-PDGFRβ (1:50, phospho Y1021, ab62437; Abcam), rabbit GFP (1:200, G10362; Invitrogen), mouse CD68 (1:200, KP1, ab955; Abcam), rat CD73 (1:50, 14-0731; eBioscience), rat Ter119 (1:100, ab91113; Abcam), rabbit Ki67 (1:200, SP6, RM9106S; Thermo Scientific), rat PDGFRβ (1:00, 14-1402, APB5; eBioscience), and rabbit VCAM1 (1:50, EPR5047, ab134047; Abcam).

Techniques: Staining, Expressing, Immunostaining

Journal: The Journal of Experimental Medicine

Article Title: VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells

doi: 10.1084/jem.20180752

Figure Lengend Snippet: Epo expression by perivascular SMA + cells in both kidney and liver is VEGF dependent. (A and B) Kidney (A) and liver (B) sections derived from VEGF-induced mice (TetVEGF164-LAPtTa) and control littermates (LAPtTa) were costained for Epo, SMA, IB4, and DAPI. Representative confocal images are shown at two magnifications each. Bar, 50 µm. The percentage of EPC-containing SMA + vessels was determined per each genotype ( n = 5 mice per group). Each data point represents the mean over five fields for each animal. Midline represents mean per group; error bars represent ±SEM. Data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001 by unpaired two-tailed Student’s t test.

Article Snippet: Primary antibodies included rabbit Epo antibody (1:50, H-162, sc-7956; Santa Cruz), mouse Epo antibody (1:50, 7D10, sc-80995; Santa Cruz), rabbit SMA (1:200, ab32575; Abcam), mouse SMA (1:200, ab7817; Abcam), rabbit phospho-PDGFRβ (1:50, phospho Y1021, ab62437; Abcam), rabbit GFP (1:200, G10362; Invitrogen), mouse CD68 (1:200, KP1, ab955; Abcam), rat CD73 (1:50, 14-0731; eBioscience), rat Ter119 (1:100, ab91113; Abcam), rabbit Ki67 (1:200, SP6, RM9106S; Thermo Scientific), rat PDGFRβ (1:00, 14-1402, APB5; eBioscience), and rabbit VCAM1 (1:50, EPR5047, ab134047; Abcam).

Techniques: Expressing, Derivative Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells

doi: 10.1084/jem.20180752

Figure Lengend Snippet: VEGF recruits alternative EPCs from Gli1 + perivascular MSC-like cells. (A) Experimental scheme depicting Gli1 lineage tracing in Gli1-CreER::Ai9TdTomato::Tet-VEGF164::LAP-tTA mice. VEGF was induced, and circulating VEGF and Epo levels were measured weekly by ELISA. A tamoxifen pulse was initiated after 3 wk of VEGF induction to stably label Gli1-expressing cells and their descendants with TdTomato. Triple transgenic mice missing the VEGF responder transgene served as littermate controls. (B) Representative confocal images of kidney sections derived from mice of the indicated genotype after Gli1 lineage tracing immunostained for TdTomato to highlight Gli1-expressing and descendant cells together with SMA and CD31 staining to examine a perivascular localization. Note a fraction of TdTomato + cells also expressing SMA. (C) Quantification of Gli1 + cells based on immunostaining. Each data point represents 10 fields averaged over one animal ( n = 7–8 mice per group). (D) Percentage of Gli1 + cells in contact with endothelial cells as determined by TdTomato/CD31 immunostaining. Each data point represents 10 fields averaged over one animal ( n = 7–8 mice). (E) Representative confocal images obtained as described above and costained for TdTomato and Epo to highlight Epo-expressing Gli1-descendant cells, with Gli1-CreERT2 TdTomato-Ai9 LAP-tTa as the littermate control. Bar, 50 µm. (F) Percentage of Gli1 + cells that express Epo in kidney ( n = 5–7 mice). Midline represents mean of each group; error bars represent ±SEM. *, P < 0.05; ***, P < 0.001, by unpaired two-tailed Student’s t test. In B–F, results are representative of two independent experiments.

Article Snippet: Primary antibodies included rabbit Epo antibody (1:50, H-162, sc-7956; Santa Cruz), mouse Epo antibody (1:50, 7D10, sc-80995; Santa Cruz), rabbit SMA (1:200, ab32575; Abcam), mouse SMA (1:200, ab7817; Abcam), rabbit phospho-PDGFRβ (1:50, phospho Y1021, ab62437; Abcam), rabbit GFP (1:200, G10362; Invitrogen), mouse CD68 (1:200, KP1, ab955; Abcam), rat CD73 (1:50, 14-0731; eBioscience), rat Ter119 (1:100, ab91113; Abcam), rabbit Ki67 (1:200, SP6, RM9106S; Thermo Scientific), rat PDGFRβ (1:00, 14-1402, APB5; eBioscience), and rabbit VCAM1 (1:50, EPR5047, ab134047; Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Transgenic Assay, Derivative Assay, Staining, Immunostaining, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells

doi: 10.1084/jem.20180752

Figure Lengend Snippet: Epo induction by VEGF is not mediated by canonical VEGF receptors. (A–C) Immunostaining of spleen, kidney, and liver sections from VEGFR2-GFP knock-in mice. Note no VEGFR2 activity in SMA perivascular cells (i.e., in the cells that produce Epo under VEGF in the three organs). Results are representative of two independent experiments ( n = 3 animals per experiment). Bar, 50 μm. (D) Scheme for VEGFR1 and VEGFR2 blockade using an i.p.-injected VEGFR1-neutralizing antibody (MF1), a VEGFR2-neutralizing antibody (DC101), or a vehicle PBS control. Reagents were injected at the indicated dose and schedule, starting 3 wk from VEGF induction and in age-matched controls ( n = 4–5 animals per group). (E and F) Circulating Epo levels were monitored weekly from the onset of VEGF induction (E; n = 4–5 animals per group; midlines represent mean per group and error bars represent ±SEM) and at sacrifice (F; each data point represents the mean of 4–5 animals per group; error bars represent ±SEM). Results are representative of two independent experiments. n.s., P > 0.05, by unpaired two-tailed Student’s t test in comparing treatment to control. n.s., not significant.

Article Snippet: Primary antibodies included rabbit Epo antibody (1:50, H-162, sc-7956; Santa Cruz), mouse Epo antibody (1:50, 7D10, sc-80995; Santa Cruz), rabbit SMA (1:200, ab32575; Abcam), mouse SMA (1:200, ab7817; Abcam), rabbit phospho-PDGFRβ (1:50, phospho Y1021, ab62437; Abcam), rabbit GFP (1:200, G10362; Invitrogen), mouse CD68 (1:200, KP1, ab955; Abcam), rat CD73 (1:50, 14-0731; eBioscience), rat Ter119 (1:100, ab91113; Abcam), rabbit Ki67 (1:200, SP6, RM9106S; Thermo Scientific), rat PDGFRβ (1:00, 14-1402, APB5; eBioscience), and rabbit VCAM1 (1:50, EPR5047, ab134047; Abcam).

Techniques: Immunostaining, Knock-In, Activity Assay, Injection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: VEGF expands erythropoiesis via hypoxia-independent induction of erythropoietin in noncanonical perivascular stromal cells

doi: 10.1084/jem.20180752

Figure Lengend Snippet: VEGF-induced perivascular Epo production is PDGFRβ dependent. (A) Representative confocal images of immunostaining of VEGF-induced (upper panels) and control (lower panels) kidney, liver, and spleen with the indicated markers from two independent experiments. To better visualize phosphorylated PDGFRβ in perivascular SMA + cells, single staining is shown below the merged image. (B) Scheme of PDFGRβ blockade experiments using an i.p.-injected PDFGRβ-neutralizing antibody (1B3) or a vehicle PBS control. Reagents were injected at the indicated dose and schedule, starting 3 wk from VEGF induction and in age-matched controls ( n = 5–9). (C–E) Relative gene expression for Epo normalized to GAPDH in the indicated organ at the end of the treatment period. Each data point represents a single animal, with the midline representing mean for the group and error bars representing ±SEM. **, P < 0.01; ***, P < 0.001, by one-way ANOVA with Tukey post-test. (F) Fold change in plasma Epo levels, with midlines representing the median for each group and error bars extending from the first quartile to the minimum and from the third quartile to the maximum values. **, P < 0.01 by one-way ANOVA with Tukey post-test. (G) Confocal images of kidney sections treated with PDGFRβ-neutralizing antibody 1B3 (or vehicle control) and immunostained with the indicated markers. Higher magnification shown in insets; bars, 50 µm. (H) Percentage of SMA-surrounded kidney vessels containing Epo-expressing cells calculated on the basis of images such as the one shown in G. Each data point represents the average for multiple vessels of a single animal. Error bars represent ±SEM. *, P < 0.05 by one-way ANOVA with Tukey post-test. Results are representative of two independent experiments.

Article Snippet: Primary antibodies included rabbit Epo antibody (1:50, H-162, sc-7956; Santa Cruz), mouse Epo antibody (1:50, 7D10, sc-80995; Santa Cruz), rabbit SMA (1:200, ab32575; Abcam), mouse SMA (1:200, ab7817; Abcam), rabbit phospho-PDGFRβ (1:50, phospho Y1021, ab62437; Abcam), rabbit GFP (1:200, G10362; Invitrogen), mouse CD68 (1:200, KP1, ab955; Abcam), rat CD73 (1:50, 14-0731; eBioscience), rat Ter119 (1:100, ab91113; Abcam), rabbit Ki67 (1:200, SP6, RM9106S; Thermo Scientific), rat PDGFRβ (1:00, 14-1402, APB5; eBioscience), and rabbit VCAM1 (1:50, EPR5047, ab134047; Abcam).

Techniques: Immunostaining, Staining, Injection, Expressing

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Significant expression of α-SMA in cTnI-ND mouse heart. Ventricular muscle homogenates of wild type, cTnI-ND, and cTnI-K118C (a double transgenic line expressing a cTnI mutant on the endogenous cTnI gene knockout background ) mice (∼80 μg total protein/lane), or mouse bladder (∼5 μg total protein/lane) were analyzed by SDS–PAGE ( upper panel, Coomassie blue protein stain) and immunoblotting ( lower three panels ). The results detected a significant level of α-SMA in cTnI-ND hearts but not in the wild type and cTnI-118C control hearts. Smooth muscle-specific protein markers h1 and h2 calponins (RAH2 blot) and SM22 were examined as controls.

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Expressing, Transgenic Assay, Mutagenesis, Gene Knockout, SDS Page, Staining, Western Blot

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Analysis of vasculature in wild type and cTnI-ND mouse ventricle . (A) Immunohistochemical analysis of transverse heart sections from wild type and cTnI-ND hearts stained for α-SMA revealed vascular structures. A representative slice from a wild type heart is shown with magnified portions from wild type and cTnI-ND hearts to demonstrate the level of resolution used for quantification as described in . (B) Vessel density ( upper plot ) was increased in cTnI-ND hearts in comparison with that in wild type hearts. However, total vessel size ( middle ) and vessel wall area ( bottom ) was not significantly different between the two groups. Data are presented as mean ± SE. * P < 0.05. Wild type, 1149 vessels examined from 14 sections; cTnI-ND, 885 vessels examined from 11 sections. Wild type, N = 5; cTnI-ND, N = 4.

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Immunohistochemical staining, Staining

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Significant expression of α-SMA in cTnI-ND mouse cardiomyocytes . Cardiomyocytes were isolated from 2 to 3 months old wild type and cTnI-ND mouse hearts and examined by SDS–PAGE ( upper panel , Coomassie blue protein stain, ∼50 μg/lane) and immunoblot analyses using anti-α-SMA ( lower panel ). The results detected a significant level of α-SMA expressed in cTnI-ND but not wild type cardiomyocytes. Mobilities corresponding to molecular weight (MW) standards (in kDa) are indicated on the left.

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Expressing, Isolation, SDS Page, Staining, Western Blot, Molecular Weight

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Immunofluorescence microscopy analysis of α-SMA expression in cTnI-ND cardiomyocytes. (A) Freshly isolated cardiomyocytes from wild type and cTnI-ND hearts were fixed on glass cover slips, stained with anti-α-SMA antibody and Alexa Fluor 488-conjugated second antibody. No-primary-antibody controls were included. Immunostained myocytes, also shown in phase contrast ( left panels ), were examined using identical imaging and off-line processing, which revealed significant levels of intracellular α-SMA in cTnI-ND but not wild type cardiomyocytes. (B) Total fluorescence intensity was determined for cells from wild type and cTnI-ND hearts by normalizing to the average level measured in wild type cells, to correct for the variable but low levels of autofluorescence within cardiomyocytes. Relative fluorescence intensities per cell area showed the significantly increased expression of α-SMA in cTnI-ND cardiomyocytes, while cell areas were similar to that of wild type cardiomyocytes (C), wild type, n = 35; cTnI-ND, n = 47. Data are presented as mean ± SE, ** P < 0.01. (D) To determine whether anti-α-SMA immunofluorescence correlated with cell surface area, a measure of hypertrophic growth, cardiomyocyte area-normalized intensity was plotted against the area of each cell. Linear regression correlation confirmed that immunofluorescence intensity is independent of the cell size. Low levels of background fluorescence intensity measured for wild type cells was also independent of normalized cell area (least-squares fit of data, dashed line ). Inset panels show representative cells with relative fluorescent intensity indicated on the bottom left. Wild type, N = 3; cTnI-ND, N = 3.

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Immunofluorescence, Microscopy, Expressing, Isolation, Staining, Imaging, Fluorescence

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Limited myofibril-integration of α-SMA. (A) Ventricular muscle homogenates (H, 40 μg/lane) and isolated ventricular myofibrils (MF, protein levels adjusted to achieve similar actin bands on gel) were prepared from wild type and cTnI-ND mouse hearts and analyzed by SDS–PAGE ( upper panel) and immunoblotting ( lower two panels) using anti-α-SMA or anti-TnI antibody. (B) Anti-α-SMA immunoblot densitometry of total ventricular homogenates and isolated cardiac myofibrils from cTnI-ND hearts normalized to total actin band in the SDS–PAGE gel showed significantly lower proportion of α-SMA in the myofibrils than that in the total muscle homogenates. * P < 0.01. Wild type, N = 3; cTnI-ND, N = 3. Data are presented as mean ± SE.

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Isolation, SDS Page, Western Blot

Journal: FEBS Open Bio

Article Title: Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

doi: 10.1016/j.fob.2013.11.002

Figure Lengend Snippet: Intracellular localization of α-SMA shows non-complete overlap with the sarcomeric protein TnT . Cardiomyocytes from cTnI-ND hearts were immunostained for α-SMA (green) and TnT (red), followed by confocal imaging. Image of the cell shows apparent overlap of the two antigens across the cell plane. However, enlarged inset shows a non-complete overlap of the two antigens within individual sarcomeres, consistent with a non-identical association with the thin filaments. (For the interpretation of color mentioned in the caption of figure, the reader is requested to view the online version of this paper.)

Article Snippet: The sections were incubated with 3% H 2 O 2 at room temperature for 15 min, washed three times with PBS-T, and incubated with an anti-α-SMA rabbit polyclonal antibody (AbCam, ab5694) diluted 1:100 in PBS-T at room temperature for 2 h. Following three washes with PBS-T, sections were incubated with HRP-conjugated goat anti-rabbit antibody diluted 1:2000 in PBS-T at room temperature for 2 h, washed three times again in PBS-T, and developed at room temperature in DAB substrate solution (Sigma FAST 3,3′-Diaminobenzidine Tablet set).

Techniques: Imaging

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 1. Adenoviral-mediated overexpression of CTGF in the chamber angle of the mouse eye. A: Light microscopy: eyes treated with control virus (Ad5-GFP) or Ad5-CTGF show no structural abnormalities in the ciliary body (CB), iris (Ir), TM (arrows), and Schlemm’s canal (SC, asterisk). B: GFP labeling in TM and corneal endothelium (arrows) after Ad5-GFP injection. C: Real-time RT-PCR for CTGF mRNA in limbal tissues of Ad5-GFP– (Co) and Ad5-CTGF–treated eyes 1 to 24 days after treatment. Mean value of Ad5-GFP–treated eyes was set at 1 (*P 0.05, **P 0.01; reference gene: GNB2L). D: Western blot analysis for fibronectin of CTGF in limbal tissues of eyes 2 months after injection of Ad5-GFP or Ad5-CTGF. Coomassie blue staining to confirm equal loading of proteins. E: Immunohistochemistry (IHC) for CTGF (red) in the anterior eye of animals 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling is seen in virus-transduced cells (green). In both experimental and control eyes, immunostaining for CTGF is seen in the corneal endothelium and in the stroma of the ciliary processes. In control eyes, no staining for CTGF is seen in the TM (arrows), whereas the TM of experimental eyes shows distinct immunoreactivity for CTGF (arrows) F: IHC for fibronectin (red) in the chamber angle of eyes 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling in virus-transduced cells (green). In control eyes, fibronectin is seen adjacent to the SC and in the stroma of the ciliary processes. After Ad5-CTGF injection, immunoreactivity for fibronectin around SC is more intense and detectable in the Ir root throughout the CB and the sclera close to the outer wall of SC. G: IHC for -SMA (red) in eyes 2 months after treatment with Ad5-GFP or Ad5-CTGF. In Ad5-GFP–injected eyes, -SMA is detectable in several cells in the TM and around ciliary process vessels. In contrast, in the experimental eye -SMA staining is seen in cells throughout the entire TM and in the stroma of the CB and Ir. Scale bar 50 m.

Article Snippet: After blocking with 3% bovine serum albumin in PBS for 1 hour at room temperature, frozen sections were incubated with anti-fibronectin rabbit IgG (1:500; Dako, Glostrup, Denmark) or anti– -SMA mouse IgG (1:100; Serotec, Kidlington, England) at 4°C overnight.

Techniques: Over Expression, Light Microscopy, Control, Virus, Labeling, Injection, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, Immunostaining

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 5. Depletion of CTGF in HTM cells by RNA interference attenuates the actin cytoskeleton. A: Real-time RT-PCR analysis for CTGF mRNA in HTM-N cells or HTM-pSiCTGF cells with stable depletion of CTGF. Mean value of HTM-N cells was set at 1 (**P 0.02, reference gene: GNB2L). B: Western blot analysis for CTGF or fibronectin in HTM-N or pSiCTGF cells; Coomassie blue staining was used to confirm equal loading of proteins. IHC for vinculin (green) and phalloidin labeling (red) of HTM-N or pSiCTGF cells 24 hours after seeding (C) or in confluent areas (D). In confluent areas, HTM-N cells show numerous thick and longitudinally arranged actin stress fibers that are associated with vinculin-labeled focal contacts. In marked contrast, in pSiCTGF cells longitudinally arranged actin stress fibers are barely detectable. After treatment of pSiCTGF cells with 50 ng/mL of CTGF for 12 hours, number and thickness of longitudinally arranged actin stress fibers significantly increases. E: Western blot analysis for -SMA, -actinin, or profilin-1 in HTM-N or pSiCTGF cells after serum starvation for 24 hours. F: Real-time RT-PCR analysis for mRNA of -SMA or profilin-1 in HTM-N or pSiCTGF cells after 24 hours. Mean value of HTM-N cells was set at 1 (**P 0.02; reference gene: GNB2L). G: Western blot analysis for RhoA/GTP and total RhoA, pMLCs and total MLCs, or pFAK and total FAK in HTM-N or pSiCTGF cells after serum free incubation for 3 hours. Coomassie blue staining was used to confirm equal loading of proteins.

Article Snippet: After blocking with 3% bovine serum albumin in PBS for 1 hour at room temperature, frozen sections were incubated with anti-fibronectin rabbit IgG (1:500; Dako, Glostrup, Denmark) or anti– -SMA mouse IgG (1:100; Serotec, Kidlington, England) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Labeling, Incubation

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 6. Analysis of CTGF overexpressing transgenic mice. A: Northern blot analysis for CTGF mRNA in transgenic and WT lenses and whole eyes without lenses. Methylene blue staining used to confirm equal loading. B: Western blot analysis for CTGF in aqueous humor of 1-month-old WT and transgenic B1-CTGF1 mice. C: Light microscopy. In WT and transgenic B1-CTGF1 mice, the iridocorneal angle is wide open, and no structural abnormalities are present in ciliary body (CB), iris (Ir), TM (arrow), and Schlemm’s canal (SC, asterisks). D: IHC for CTGF in the anterior eye of 2-month-old WT and transgenic B1-CTGF1 mice. In the WT animal, immunoreactivity for CTGF in the TM is weak or absent (arrows). In contrast, in the transgenic B1-CTGF1 mouse immunoreactivity for CTGF is seen in the TM (arrows), in the corneal endothelium, and along the inner surface of the cornea. E: IHC for fibronectin in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In WT animals, fibronectin is seen adjacent to the SC and close to the capillaries in the Ir and CB. In contrast, in transgenic B1-CTGF1 mice, immunoreactivity for fibronectin around the SC and in the stroma of the CB is more intense. F: IHC for -SMA in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In transgenic B1-CTGF1 mice, intense immunoreactivity for -SMA is present in the TM and the stroma of the CB in contrast to the WT littermate. G: IOP measurement of 1- to 3-month-old WT and transgenic B1-CTGF1 mice. Medians (red) with maximum and minimum (lines) and upper and lower quartile (n 15, *P 0.05). H: Optic nerve sections of 3-month-old WT and transgenic B1-CTGF1 mice showing increased areas devoid of axons in transgenic B1-CTGF1 animals (left; arrows). Number of optic nerve axons in 1- to 3-month-old WT and transgenic B1-CTGF1 mice (right; n 5; **P 0.01). Scale bars: 50 m (C–F); 20 m (H).

Article Snippet: After blocking with 3% bovine serum albumin in PBS for 1 hour at room temperature, frozen sections were incubated with anti-fibronectin rabbit IgG (1:500; Dako, Glostrup, Denmark) or anti– -SMA mouse IgG (1:100; Serotec, Kidlington, England) at 4°C overnight.

Techniques: Transgenic Assay, Northern Blot, Staining, Western Blot, Light Microscopy

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 1. Adenoviral-mediated overexpression of CTGF in the chamber angle of the mouse eye. A: Light microscopy: eyes treated with control virus (Ad5-GFP) or Ad5-CTGF show no structural abnormalities in the ciliary body (CB), iris (Ir), TM (arrows), and Schlemm’s canal (SC, asterisk). B: GFP labeling in TM and corneal endothelium (arrows) after Ad5-GFP injection. C: Real-time RT-PCR for CTGF mRNA in limbal tissues of Ad5-GFP– (Co) and Ad5-CTGF–treated eyes 1 to 24 days after treatment. Mean value of Ad5-GFP–treated eyes was set at 1 (*P 0.05, **P 0.01; reference gene: GNB2L). D: Western blot analysis for fibronectin of CTGF in limbal tissues of eyes 2 months after injection of Ad5-GFP or Ad5-CTGF. Coomassie blue staining to confirm equal loading of proteins. E: Immunohistochemistry (IHC) for CTGF (red) in the anterior eye of animals 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling is seen in virus-transduced cells (green). In both experimental and control eyes, immunostaining for CTGF is seen in the corneal endothelium and in the stroma of the ciliary processes. In control eyes, no staining for CTGF is seen in the TM (arrows), whereas the TM of experimental eyes shows distinct immunoreactivity for CTGF (arrows) F: IHC for fibronectin (red) in the chamber angle of eyes 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling in virus-transduced cells (green). In control eyes, fibronectin is seen adjacent to the SC and in the stroma of the ciliary processes. After Ad5-CTGF injection, immunoreactivity for fibronectin around SC is more intense and detectable in the Ir root throughout the CB and the sclera close to the outer wall of SC. G: IHC for -SMA (red) in eyes 2 months after treatment with Ad5-GFP or Ad5-CTGF. In Ad5-GFP–injected eyes, -SMA is detectable in several cells in the TM and around ciliary process vessels. In contrast, in the experimental eye -SMA staining is seen in cells throughout the entire TM and in the stroma of the CB and Ir. Scale bar 50 m.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Over Expression, Light Microscopy, Control, Virus, Labeling, Injection, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, Immunostaining

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 3. CTGF induces contraction and formation of actin stress fibers in HTM cells. A: Collagen contraction assay with HTM cells seeded in a matrix of collagen type I and treated with CTGF or TGF-2. After 24 hours, culture wells were photographed (bottom) and area of matrices measured (top). The area obtained in controls was set at 1 (n 4; *P 0.05). B: Phalloidin labeling (red) of actin stress fibers in control HTM cells or 24 hours after treatment with CTGF. Nuclear DNA was stained with DAPI (blue). Scale bar 50 m. C: Western blot analysis for -SMA or -actinin in proteins of cultured HTM cells after treatment with 5 and 50 ng/mL of CTGF for 2 hours. Coomassie blue staining to confirm equal loading of proteins. D: Length of phalloidin-labeled stress fibers 24 hours after treatment with CTGF (n 4; *P 0.05). E: Phalloidin labeling (red) of actin stress fibers in HTM cells or 24 hours after treatment with control virus (Ad5-GFP) or Ad5-CTGF. GFP labeling (green) shows virus-infected cells; nuclear DNA is labeled with DAPI (blue). Scale bar 50 m.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Contraction Assay, Labeling, Control, Staining, Western Blot, Cell Culture, Virus, Infection

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 5. Depletion of CTGF in HTM cells by RNA interference attenuates the actin cytoskeleton. A: Real-time RT-PCR analysis for CTGF mRNA in HTM-N cells or HTM-pSiCTGF cells with stable depletion of CTGF. Mean value of HTM-N cells was set at 1 (**P 0.02, reference gene: GNB2L). B: Western blot analysis for CTGF or fibronectin in HTM-N or pSiCTGF cells; Coomassie blue staining was used to confirm equal loading of proteins. IHC for vinculin (green) and phalloidin labeling (red) of HTM-N or pSiCTGF cells 24 hours after seeding (C) or in confluent areas (D). In confluent areas, HTM-N cells show numerous thick and longitudinally arranged actin stress fibers that are associated with vinculin-labeled focal contacts. In marked contrast, in pSiCTGF cells longitudinally arranged actin stress fibers are barely detectable. After treatment of pSiCTGF cells with 50 ng/mL of CTGF for 12 hours, number and thickness of longitudinally arranged actin stress fibers significantly increases. E: Western blot analysis for -SMA, -actinin, or profilin-1 in HTM-N or pSiCTGF cells after serum starvation for 24 hours. F: Real-time RT-PCR analysis for mRNA of -SMA or profilin-1 in HTM-N or pSiCTGF cells after 24 hours. Mean value of HTM-N cells was set at 1 (**P 0.02; reference gene: GNB2L). G: Western blot analysis for RhoA/GTP and total RhoA, pMLCs and total MLCs, or pFAK and total FAK in HTM-N or pSiCTGF cells after serum free incubation for 3 hours. Coomassie blue staining was used to confirm equal loading of proteins.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Labeling, Incubation

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 6. Analysis of CTGF overexpressing transgenic mice. A: Northern blot analysis for CTGF mRNA in transgenic and WT lenses and whole eyes without lenses. Methylene blue staining used to confirm equal loading. B: Western blot analysis for CTGF in aqueous humor of 1-month-old WT and transgenic B1-CTGF1 mice. C: Light microscopy. In WT and transgenic B1-CTGF1 mice, the iridocorneal angle is wide open, and no structural abnormalities are present in ciliary body (CB), iris (Ir), TM (arrow), and Schlemm’s canal (SC, asterisks). D: IHC for CTGF in the anterior eye of 2-month-old WT and transgenic B1-CTGF1 mice. In the WT animal, immunoreactivity for CTGF in the TM is weak or absent (arrows). In contrast, in the transgenic B1-CTGF1 mouse immunoreactivity for CTGF is seen in the TM (arrows), in the corneal endothelium, and along the inner surface of the cornea. E: IHC for fibronectin in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In WT animals, fibronectin is seen adjacent to the SC and close to the capillaries in the Ir and CB. In contrast, in transgenic B1-CTGF1 mice, immunoreactivity for fibronectin around the SC and in the stroma of the CB is more intense. F: IHC for -SMA in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In transgenic B1-CTGF1 mice, intense immunoreactivity for -SMA is present in the TM and the stroma of the CB in contrast to the WT littermate. G: IOP measurement of 1- to 3-month-old WT and transgenic B1-CTGF1 mice. Medians (red) with maximum and minimum (lines) and upper and lower quartile (n 15, *P 0.05). H: Optic nerve sections of 3-month-old WT and transgenic B1-CTGF1 mice showing increased areas devoid of axons in transgenic B1-CTGF1 animals (left; arrows). Number of optic nerve axons in 1- to 3-month-old WT and transgenic B1-CTGF1 mice (right; n 5; **P 0.01). Scale bars: 50 m (C–F); 20 m (H).

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Transgenic Assay, Northern Blot, Staining, Western Blot, Light Microscopy