Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Androgens Accentuate TGF‐β Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice

doi: 10.1161/JAHA.119.015773

Figure Lengend Snippet: A , Phosphorylated‐Erk1/2 (p‐Erk1/2) (n=5, each group) and ( B ) phosphorylated‐Smad2 (p‐Smad2) (n=6, each group) in aortic root/ascending (ASC) aorta derived SMC from Fbn1 C1039G/+ (Fbn1) male mice treated with dihydrotestosterone 10 nmol/L (DHT), DHT+flutamide 1 μmol/L (Flu), TGF‐β 5 ng/mL, TGF‐β+DHT, TGF‐β+DHT+Flu or vehicle control. Relative protein level quantified using vinculin or Gapdh as loading control. Values expressed as fold difference compared with control for p‐Erk1/2. Values expressed as fold difference compared with TGF‐β for p‐Smad2 since no signal detected for control‐treated SMC. C , MMP‐2 activity in cell culture supernatants of ASC derived SMC from Fbn1 male mice treated with listed hormones or vehicle control (n=6, each group). Values expressed as fold difference compared with vehicle control or TGF‐β with representative pictures. Results presented as median±interquartile. Kruskal–Wallis test with Dunn's posttest was used for multiple comparison among the groups: control vs DHT vs DHT+Flu, TGF‐β vs TGF‐β+DHT vs TGF‐β+DHT+Flu. * P ≤0.05, ** P <0.01. Erk indicates extracellular‐signal‐regulated kinase; MMP, matrix metalloproteinase; and TGF‐β, transforming growth factor beta.

Article Snippet: SMC were treated with either dihydrotestosterone (DHT) (10 nmol/L) (Sigma‐Aldrich), flutamide (1 μmol/L) (Selleckchem, Houston, TX), mouse recombinant TGF‐β (5 ng/mL) (R&D Systems, Minneapolis, MN), or vehicle control for 24 hours for p‐ERK1/2 protein assay, 48 hours for MMP activity assay and 24 and 48 hours for androgen receptor protein assay.

Techniques: Derivative Assay, Control, Activity Assay, Cell Culture, Comparison

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Androgens Accentuate TGF‐β Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice

doi: 10.1161/JAHA.119.015773

Figure Lengend Snippet: Diameter (mm) and aortic growth (mm) of aortic root ( A ) and ascending aorta ( B ) in Fbn1 C1039G/+ (Fbn1) and littermate wild type control mice (WT) males treated with flutamide or vehicle control measured by transthoracic echocardiography at ages 6, 8, 12, and 16 weeks (Fbn1 vehicle n=9, Fbn1 flutamide n=9, WT vehicle, n=8, WT flutamide n=7). Results presented as mean±SD. Mixed effects model used for aortic growth comparison between groups. C , Average number of elastin lamina breaks per lamellae with representative elastin histological staining (EVG) in aortic root from Fbn1 treated with flutamide or vehicle control at 16 weeks (n=5, each group). Scale bars represent 50 μm. Results presented as median±interquartile. Mann–Whitney U test used for comparison between groups. * P ≤0.05, ** P <0.01, *** P <0.001.

Article Snippet: SMC were treated with either dihydrotestosterone (DHT) (10 nmol/L) (Sigma‐Aldrich), flutamide (1 μmol/L) (Selleckchem, Houston, TX), mouse recombinant TGF‐β (5 ng/mL) (R&D Systems, Minneapolis, MN), or vehicle control for 24 hours for p‐ERK1/2 protein assay, 48 hours for MMP activity assay and 24 and 48 hours for androgen receptor protein assay.

Techniques: Control, Comparison, Staining, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Androgens Accentuate TGF‐β Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice

doi: 10.1161/JAHA.119.015773

Figure Lengend Snippet: A , Automated immunoblotting of phosphorylated‐Erk1/2 (p‐Erk1/2) (flutamide‐treated Fbn1 males=8; vehicle control‐treated Fbn1 males=8) and ( B ) western blotting of phosphorylated‐Smad2 (p‐Smad2) (n=9, each group) in aortic root/ascending (ASC) and descending (DES) aortic specimens from Fbn1 C1039G/+ male mice treated with vehicle control or flutamide euthanized at age 16 weeks. Vinculin or Gapdh served as loading control. Values expressed as fold difference compared with littermate wild type control (WT) males. C , Gelatin zymography in ASC and DES aortic specimens from Fbn1 C1039G/+ male mice treated with vehicle control and flutamide (n=7, each group) at age 16 weeks. Values expressed as fold difference compared with WT males. Results presented as median±interquartile. Mann–Whitney U test was used for the comparison between the groups * P ≤0.05, ** P <0.01. Erk indicates extracellular‐signal‐regulated kinase; and MMP, matrix metalloproteinase.

Article Snippet: SMC were treated with either dihydrotestosterone (DHT) (10 nmol/L) (Sigma‐Aldrich), flutamide (1 μmol/L) (Selleckchem, Houston, TX), mouse recombinant TGF‐β (5 ng/mL) (R&D Systems, Minneapolis, MN), or vehicle control for 24 hours for p‐ERK1/2 protein assay, 48 hours for MMP activity assay and 24 and 48 hours for androgen receptor protein assay.

Techniques: Western Blot, Control, Zymography, MANN-WHITNEY, Comparison

Journal: Angiogenesis

Article Title: The influences of ApoE isoforms on endothelial adherens junctions and actin cytoskeleton responding to mCRP

doi: 10.1007/s10456-024-09946-4

Figure Lengend Snippet: The effects of recombinant and circulating APOE proteins for the mCRP toxic effects on actin cytoskeleton, adherens junctions and cerebrovascular integrity. We conducted i.p. injection of mCRP or i.p. injection of different recombinant APOE proteins followed by i.p. injection of mCRP into different ApoE mouse models. Double immunostaining of different proteins was conducted in the cortex. n = 9–15 for each condition. A The expression of LIMA1 under different treatments was quantified for each ApoE genotype mouse line. While recombinant ApoE 4 plus mCRP increased the expression of LIMA1 in ApoE 3 mice (p < 0.05), recombinant ApoE 2 protein suppressed the expression of LIMA1 induced by mCRP to the baseline level in ApoE 4 mice. B The expression of CD31 under different treatments was quantified for each ApoE genotype mouse line. mCRP decreased the expression of CD31 only in ApoE 4 mice (p < 0.05), and the co-injection of recombinant ApoE 2 protein tended to rescue the effect mediated by mCRP. C mCRP increased the intensity of colocalization of CD31 and LIMA1 only in ApoE 4 mice (p < 0.01), and the co-injection of recombinant ApoE 2 protein suppressed the effect of mCRP (p < 0.01). D The expression levels of pCD31 under different treatments were quantified. mCRP increased the expression of pCD31 only in ApoE 4 mice (p < 0.01). Recombinant ApoE 2 protein suppressed the expression of pCD31 induced by mCRP to the baseline level in ApoE 4 mice (p < 0.05). E The co-localization of pCD31 and LIMA1 under different treatments was quantified. mCRP increased the intensity of colocalization of pCD31 and LIMA1 only in ApoE 4 mice (p < 0.01), and the co-injection of recombinant ApoE 2 protein suppressed the effect of mCRP (p < 0.01). F PLA was performed on the cortex to further examine the binding of pCD31 and LIMA1 in mice. Treatment with mCRP alone significantly induced pCD31-LIMA1 binding in the cortex in ApoE 4 mice (p < 0.05) but not in ApoE -/-, ApoE 2 or ApoE 3 mice. However, the first injection of recombinant ApoE 4 protein followed by mCRP significantly induced pCD31-LIMA1 binding in the cortex in ApoE -/- mice (p < 0.001), ApoE 2 mice (p < 0.05) and ApoE 3 mice (p < 0.0001). On the other hand, the first i.p. injection of recombinant ApoE 2 protein followed by mCRP into ApoE 4 mice suppressed the binding of pCD31-LIMA1 induced by mCRP to baseline (p < 0.05). G Western blots showed that mCRP increased the brain level of pCD31 and the combined treatment of APOE 4 and mCRP further increased the brain levels of pCD31 and LIMA1 in ApoE2 mice; H The co-localization of CD31 and VE-cadherin under different treatments from 4B was quantified. mCRP decreased the intensity of colocalization of CD31 and VE-cadherin only in ApoE 4 mice (p < 0.001) and pre-injection of ApoE 2 protein rescued some. The first i.p. injection of recombinant ApoE 4 protein followed by mCRP into ApoE 2 or ApoE 3 mice further suppressed the binding of CD31-VE-cadherin induced by mCRP (p < 0.0001). I The microvascular length was qualitied and showed injection of recombinant ApoE4 protein followed by mCRP significantly shortened microvascular lengths in ApoE2 (p < 0.05) and ApoE3 (p < 0.01) mice at the cortex. Conversely, injecting ApoE2 protein followed by mCRP in ApoE4 mice rescued mCRP-induced microvascular damage (p < 0.05). Data are expressed as the mean ± SEM. Two-way ANOVA with Tukey’s post hoc test was applied. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for the comparisons among PBS and mCRP alone and ApoE protein plus mCRP treatments. The scale bar is 50 μm

Article Snippet: Human ApoE genetic knock-in mice and ApoE knockout mice were purchased from Taconic Biosciences, Inc. (APOE2: #1547-F, APOE3: #1548-F, APOE4: #1549-F, ApoE −/− : Rensselaer, NY, USA).

Techniques: Recombinant, Injection, Double Immunostaining, Expressing, Binding Assay, Western Blot

Journal: Angiogenesis

Article Title: The influences of ApoE isoforms on endothelial adherens junctions and actin cytoskeleton responding to mCRP

doi: 10.1007/s10456-024-09946-4

Figure Lengend Snippet: Characterization of the opposite roles of APOE isoforms in the endothelial pathology and the molecular pathway induced by mCRP. Primary brain endothelial cells (BECs) were isolated from WT mice and treated with different concentrations of mCRP for 24 h. In addition, recombinant APOE 2 or APOE 4 protein at a concentration of 1.0 μM was sequentially incubated with mCRP (10 μg/ml) for a 24-h incubation. The targeted proteins were detected using specific antibodies, and the cell nuclei were stained with DAPI for visualization. Scale bar: 10 μm. A The presented images demonstrate LIMA1 (red) and F-actin (green) expressions in BECs. The merged images (yellow) show the combined staining. mCRP induced a stretched cell shape in endothelial cells. However, when treated with APOE 2 protein plus mCRP, the endothelial cells regained a shape similar to those treated with PBS treatment. Conversely, the presence of APOE 4 protein did not appear to affect the cell shape induced by mCRP. B Quantification of Lima1 levels and F-actin area analyses from Fig. 6A are presented. The statistical results show that mCRP (10 μg/ml) alone, or in combination with APOE2 or APOE4, had varying effects on Liam1 and F-actin area. C Representative images displaying double immunostaining of F-actin (green) and Arpc4 (red), along with the merged images (yellow), are presented. The quantification analysis revealed that the expression of Arpc4 remained unaffected when comparing the addition of mCRP alone or mCRP plus APOE 2 to the PBS treatment. D Representative images displaying double immunostaining of F-actin (green) and Itgb1 (red), along with the merged images (yellow), are presented. Quantitative analysis revealed that APOE2 protein suppressed mCRP-induced Itgb1 production, while APOE4 protein significantly enhanced mCRP induce Itgb1 expression. E The displayed images show representative double immunostaining of Rox (green) and Oxct1 (red), along with the merged images (yellow). Quantitative analysis revealed significant findings regarding the production of Rox and Oxct1. APOE 2 protein suppressed mCRP-induced Rox production and Oxct1 level, while APOE 4 protein significantly enhanced mCRP induce Rox production and increase Oxct1 expression. F To investigate the relationship between F-actin formation and mitochondrial function in endothelial cells, images are shown with double immunostaining for F-actin (green) and Rox (red). Notably, the combination of mCRP and APOE 4 protein synergistically enhanced F-actin formation and ROS production in endothelial cells. G When compared to PBS treatment, the addition of mCRP resulted in an increased production of Rox (p < 0.05); coincubation with APOE 2 protein suppressed mCRP-induced Rox production, while APOE 4 protein significantly enhanced Rox production compared to PBS (p < 0.001), mCRP alone (p < 0.05), and mCRP plus APOE 2 (p < 0.001) (left panel). In the middle panel, it was observed that mCRP alone exhibited a dose-dependent tendency to increase Rox production, although statistical significance was not reached. In the right panel regarding the expression of Oxct1, adding mCRP alone or mCRP plus APOE 2 did not impact its expression with statistical significance compared to the PBS treatment. However, coincubation of mCRP and APOE 4 significantly increased Oxct1 expression compared to PBS (p < 0.001), mCRP alone (p < 0.01), and mCRP plus APOE 2 (p < 0.001)

Article Snippet: Human ApoE genetic knock-in mice and ApoE knockout mice were purchased from Taconic Biosciences, Inc. (APOE2: #1547-F, APOE3: #1548-F, APOE4: #1549-F, ApoE −/− : Rensselaer, NY, USA).

Techniques: Isolation, Recombinant, Concentration Assay, Incubation, Staining, Double Immunostaining, Expressing

Journal: Cell Stem Cell

Article Title: SARS-CoV-2 Infects the Brain Choroid Plexus and Disrupts the Blood-CSF Barrier in Human Brain Organoids

doi: 10.1016/j.stem.2020.10.001

Figure Lengend Snippet:

Article Snippet: rabbit anti-clusterin (D7N2K) , Cell Signaling Technology , Cat# 34642; RRID: AB_2799057.

Techniques: Virus, Isolation, Recombinant, Membrane, Plasmid Preparation, Expressing, Software

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: Brain Microenvironment-Induced Down-Regulation of DNMT1 Reactivates GAD1 Expression. A, qRT-PCR of DNMT1 mRNA expression after 48 hours co-culture with either CAF or glia cells. (left) MDA-MB-231; (right) A375SM. B, qRT-PCR of DNMT1 mRNA expression of MDA-MB-231 cultured either with 100% conditioned media from either CAF or glia cells or 50% mix of conditioned media and fresh media. C, Cytokine screen of glia and CAF conditioned media. (left) MA plot of Log [mean expression of Glia/CAF] of 73 cytokines analyzed. *: differentially expressed cytokines (adjusted p < 0.1) (right) Heatmap of differentially expressed cytokines. D, Network analysis of differentially expressed cytokines. E, Impact of extracellular clusterin on DNMT1 and GAD1 expression. (left) Cytokine expression profile of clusterin in conditioned media from CAFs or glia cells. (right) qPCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells treated with control or 200 ng of clusterin. F, qRT-PCR of GAD1 and DNMT1 mRNA expression in tumor cells genetic knockdown of glia derived-clusterin. (left) qRT-PCR of clusterin mRNA expression in glia cells. (right) qRT-PCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells co-cultured with control glia or siClusterin glia cells. G, qRT-PCR of mRNA levels in tumor cells after 48 hours co-culture with primary glia cells. Prior to co-culture, tumor cells were transfected with either vector control or DNMT1 over expression plasmid for 24 hours. (left) DNMT1 mRNA expression; (right) GAD1 mRNA expression under glia co-culture. H, Proliferation of MDA-MB-231 cells after DNMT1 overexpression and co-culture with glia cells for 48 hours.

Article Snippet: Human clusterin protein (2937-HS-050) was purchased from R&D systems.

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Cell Culture, Control, Knockdown, Derivative Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: GAD1-Mediated Glutamine Metabolism Enables Brain Metastatic Outgrowth. A, (top) Representative images of A375SM brain metastatic tumors arising from 1:1 mix of tumor cells expressing either control (labeled with GFP) or GAD1 targeted shRNA (labeled with RFP), (bottom) Quantification of metastatic incidence. B, (top) Representative images of MDA-MB-231 brain metastatic tumors with/without GAD1 knockdown, (bottom) Quantification of metastatic incidence. C, Representative images of brain metastatic tumors arising from arising from A375SM cell after treated with vehicle control or vigabatrin (4 mg/kg) for seven days beginning at seven days post injection. D, Reduction of metastatic tumor lesions by vigabatrin. (left) representative images of haemoxyolin and eosin staining of metastatic tumors treated with either control or vigabatrin (4 mg/kg); (right) Quantification of stained tumors per section. E, Ki-67 immunohistochemistry staining of tumors arising in mice treated with either vehicle control or vigabatrin (4 mg/kg). (left) Representative images of Ki-67 staining; (right) Quantification of ki-67 positive tumor cells per region of interest. F, Proposed model of brain microenvironment-induced tumor cell metabolic shifting via clusterin-mediated epigenetic upregulation of GAD1, which contributes to brain metastatic outgrowth.

Article Snippet: Human clusterin protein (2937-HS-050) was purchased from R&D systems.

Techniques: Expressing, Control, Labeling, shRNA, Knockdown, Injection, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Elevated extracellular and reduced cell associated Clusterin in Idiopathic Pulmonary Fibrosis. ( A ) Clusterin gene expression was quantitated using RT-PCR in lung tissue from healthy control lung tissue (n = 10), COPD patients (n = 19) and IPF patients (n = 54). ( B,C ) Circulating Clusterin protein levels were quantitated and compared between IPF (n = 60) and a cohort of age matched controls (n = 30) ( B ), and from COPD (n = 15) and a separate cohort of age matched controls (n = 25) ( C ). Levels were measured by Somascan analysis, each dot representing a different individual. ( D–J ) Clusterin expression was visualized (brown staining) by IHC analysis of three IPF lungs ( D – I ) and a representative normal lung ( J ) tissue, size bars are indicated on image. ( K ) The staining intensity of cell-associated Clusterin was quantified in airway epithelial cells using Aperio Scanscope software. Shown is the average Clusterin staining intensity in airway epithelial cells in normal, IPF and COPD lung tissue. Data are expressed as Mean ± SEM *P ≤ 0.05, ****P ≤ 0.001 significance to relevant control levels.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Software

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Induction of Clusterin in response to bleomycin-induced lung injury. ( A–H ) C57Bl/6 mice were given bleomycin intratracheally and CLU expression was visualized by IHC after 7 ( C , D ), 14 ( E , F ) and 21 ( G , H ) days of bleomycin instillation and compared to staining in the lungs of Day 21 saline treated mice ( A , B ). ( I ) Clusterin transcript expression was quantitated after 14, 21 and 28 days of bleomycin instillation by RT-PCR in whole lung samples. Data are expressed as Mean ± SEM*P ≤ 0.05, **P ≤ 0.01 significance to relevant control levels. ( J–Q ) Saline or bleomycin challenged C57Bl/6 mice were treated with 20 µg of recombinant Clusterin every three days starting at Day 2. Shown are representative histological images of the lungs stained with Masson’s trichrome from saline + PBS vehicle ( J , K ), saline + Clusterin ( L , M ), bleomycin + PBS ( N , O ) and bleomycin + Clusterin ( P , Q ) treated mice. The top images are of whole scans of the lungs and the bottom images are taken at 200x magnification. ( R ) Collagen content in the lungs of the recombinant Clusterin and vehicle treated mice was biochemically quantified using a hydroxyproline assay. Shown is the average hydroxyproline (µg) from right lung lobes. ( S , T ) Shown is the average total BAL cell number ( S ) and the total TGFß levels ( T ) in the lungs of vehicle or Clusterin treated mice 15 days after bleomycin challenge. ( U ) FN1, VEGFA, PDGFRA and MMP12 transcript expression was quantified in whole lung samples 15 days after bleomycin, vehicle and/or Clusterin treatment. Shown is the average expression from 3 Saline/PBS, 3 Saline/rClusterin, 7 Bleo/PBS and 7 Bleo/rClusterin treated mice.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Expressing, Staining, Saline, Reverse Transcription Polymerase Chain Reaction, Recombinant, Hydroxyproline Assay

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Clusterin deficient mice show persistent fibrosis in response to bleomycin. WT or CLU −/− received bleomycin intratracheally on Day 0. ( A–H ) Depicted is Masson’s Trichrome histological staining of WT saline ( A , B ), WT bleomycin ( C , D ), CLU−/− saline ( E,F ) and CLU−/− bleomycin ( G , H ) treated lungs fourteen days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( I ) Collagen content in the lungs of the of bleomycin treated wildtype and CLU−/− mice was biochemically quantified using a hydroxyproline assay, 14 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( J ) Shown is the average total TGFβ levels in the BAL from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( K ) Shown is the average IL-1β levels in whole lung lysates from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( L , M ) The proliferative status ( L ) and Caspase3 expression ( M ) in live CD45 − EpCAM + epithelial cells was assessed via Ki67 and activated Caspase-3 staining, respectively, 5 days after bleomycin challenge. Depicted is the fold change in cell number in response to bleomycin, relative to its relevant saline control. ( N , O ) IHC analysis for cleaved Caspase3 was performed on fibrotic murine lungs and quantified using Aperio Scanscope software. Depicted is the fold change in clusters, as defined by three or more adjacent positive cells, staining for cleaved Caspase3, 14 ( N ) and 28 ( O ) days after bleomycin challenge, compared to relevant saline control. ( P – W ) Depicted is Masson’s Trichrome histological staining of WT saline ( P , Q ), WT bleomycin ( R , S ), CLU−/− saline ( T,U ) and CLU−/− bleomycin ( V , W ) treated lungs 28 days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( X ) Collagen content in the lungs of the of wildtype and CLU−/− treated mice was biochemically quantified using a hydroxyproline assay 28 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( Y , Z ) Quantitative PCR analysis was performed on RNA purified from lungs of 28-day saline and bleomycin treated murine lungs. Shown is the average col3a1 ( Y ) and fn1 ( Z ) transcript expression. Data are Mean ± SEM., n = 8–13 mice/ group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001 significance, or as stated. ns = not significant.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Staining, Saline, Hydroxyproline Assay, Expressing, Software, Real-time Polymerase Chain Reaction, Purification

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Loss of transcripts for Clusterin, MMR, BER & DSB DNA repair pathway components in IPF SSEA4 + basal-like epithelial cells. ( A ) SSEA4 + and SSEA4 − cells were sorted from normal and IPF stromal cultures, RNA was extracted and subjected to RNAseq analysis. Fold changes in IPF relative to normal were calculated and the results were uploaded onto ingenuity IPA for pathway analysis ( A ). ( B ) Shown is an representative sort plot depicting the forward and side scatter localization of SSEA4 + cells. ( C ) Normalized RNA sequencing FPKM values were mined for transcripts that were enriched in SSEA4 + relative to SSEA4 − cells. Shown are enriched basal cell lineage markers in normal lung and IPF SSEA4 + cells. ( D ) Shown is the average CLU transcript FPKM values normal and IPF SSEA4 + cells. ( E–G ) Transcripts encoding for components of MMR ( E ), BER ( F ) and DSB ( G ) pathways were mined from SSEA4 + RNAseq data, clustered using Euclidean distance with complete linkage and heat maps were generated using Morpheus (Broad Institute). ( H–M ) Normal and IPF lung biopsies were stained with anti-SSEA4 and BRCA1 antibodies followed by fluorescent microscopic analysis. Representative images were taken at 200x magnification from two normal and four IPF lung biopsies stained with BRCA1 ( H , K respectively), SSEA4 ( I , L respectively) and the merged composites ( J , M respectively). ( N ) The number of SSEA4 + cells showing both SSEA4 and BRCA1 staining was quantified, averaged, and the percentage of cells was calculated from 28 normal and 84 IPF SSEA4 + cells from 3 normal and 4 IPF lungs, respectively.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: RNA Sequencing Assay, Generated, Staining

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Pictorial summary for the potential role of Clusterin variants in the modulation of epithelial cell survival and lung fibrosis.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: