Journal: The American Journal of Pathology

Article Title: MDM2

doi: 10.2353/ajpath.2006.051351

Figure Lengend Snippet: A: Representative RT-PCR analysis of total RNA from cultured human VSMCs (HASMC). RNA was amplified in the presence of oligonucleotide primers specific for MR (top panel) and 11β-HSD type 2 (bottom panel). Extracts from human kidney were used as a positive control (P). B: Representative immunoblotting studies demonstrating MR and 11β-HSD type 2 proteins in HASMC. Extracts from human kidney were used as a positive control (P). C and D: Immunocytochemical analysis demonstrated that immunopositive cells for MR appear brown as a result of diaminobenzidine colorimetric reaction in HASMC without ligand stimulation (C) or treated with 10 nmol/L aldosterone (D) (Original magnification, ×400).

Article Snippet: Cell Culture and Characterization A cultured human VSMC cell line, ie, HASMC (derived from human abdominal aorta) was commercially obtained from Kurabo Corp. (Osaka, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Positive Control, Western Blot

Journal: Scientific Reports

Article Title: Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency

doi: 10.1038/s41598-018-21602-8

Figure Lengend Snippet: Accumulation of autophagosomes in the media of muscular arteries in Lamp2 KO mice and cytoplasm of cultured human brain VSMC in LAMP-2 deficiency. Appearance of autophagosomes visualized by LC3 (red) (arrowheads) in the media of the femoral ( a ), coronary ( b ), and cerebral ( c ) arteries in Lamp2 KO mice. Nuclei are counterstained by DAPI (blue) . Staining of α-SMA showed the localization of VSMC in the media of femoral arteries (green in a ) ( n = 4 per group). Scale bars: 100 µm. ( d ) LC3 staining in cultured human brain VSMC of control or LAMP-2 siRNA-transfected cells showed accumulation of autophagosomes (red) (arrowheads) in the cytoplasm of cells under LAMP-2 deficiency. Successful silencing of LAMP-2 RNA via siRNA treatment was proven by the attenuation of cytoplasmic LAMP-2 signals (green) . Nuclei are counterstained by DAPI (blue) . Scale bars: 5 µm. ( e ) Western blot analysis of LAMP-2 and LC3 in cultured human brain VSMC. LAMP-2 siRNA-treated vs control groups are compared. Quantified data from 3 independent experiments are shown. *** P < 0.001. A two-tailed Student’s t -test was used. Ctrl, control; KO, knockout; WT, wild-type.

Article Snippet: The human brain VSMC were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA, #1100) and cultured using smooth muscle cell medium (#1101, ScienCell) consisting of 500 mL basal medium, 10 mL fetal bovine serum (#0010), 5 mL of smooth muscle cell growth supplement (#1152), and 5 mL of penicillin/streptomycin solution (#0503).

Techniques: Cell Culture, Staining, Control, Transfection, Western Blot, Two Tailed Test, Knock-Out

Journal: Scientific Reports

Article Title: Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency

doi: 10.1038/s41598-018-21602-8

Figure Lengend Snippet: Impaired autophagic flux in LAMP-2–deficient human brain VSMC. ( a ) Fluorescent visualization of autophagosomes (GFP-positive puncta , green or yellow) and autolysosomes ( GFP-non-merged RFP-positive puncta , green-non-merged red) by the use of the autophagy tandem sensor RFP-GFP-LC3B. ( b ) Compared with control (top panel) , chloroquine alone (second panel) , LAMP-2 siRNA alone (third panel) , and double treatment (bottom) showed an increase in autophagosomes with an inverse reduction in autolysosomes (arrowheads) . * P < 0.05, ** P < 0.01, and *** P < 0.001. The nuclei and LAMP-2 protein were stained by DAPI (blue) and LAMP-2 (turquoise) , respectively. Scale bars: 5 µm. A two-tailed one-way ANOVA was used followed by Tukey’s post-hoc test for all panels.

Article Snippet: The human brain VSMC were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA, #1100) and cultured using smooth muscle cell medium (#1101, ScienCell) consisting of 500 mL basal medium, 10 mL fetal bovine serum (#0010), 5 mL of smooth muscle cell growth supplement (#1152), and 5 mL of penicillin/streptomycin solution (#0503).

Techniques: Control, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency

doi: 10.1038/s41598-018-21602-8

Figure Lengend Snippet: Morphologic and functional characteristics of VSMC under LAMP-2 deficiency. ( a ) Immunostaining of VSMC in the femoral arteries of Lamp2 KO and WT mice. In Lamp2 KO mice, the synthetic marker vimentin was increased ( green ), whereas the contractile marker α-SMA was conversely reduced (green) . Endothelial cells were stained by CD31 (red) , and nuclei were visualized by DAPI (blue) . Scale bars: 50 µm. The graphs show quantitation of the vascular markers from the immunostaining. The media of Lamp2 KO mice expressed more vimentin but less α-SMA than the media of WT mice. *** P < 0.001, compared with Lamp2 WT mice ( n = 4 per group). ( b ) Phenotypic switch of LAMP-2–deficient human brain VSMC. The LAMP-2 mRNA suppression led to increased vimentin expression (white) with an inverse reduction in α-SMA (white) , compared with control cells. The nuclei and cytoplasmic LAMP-2 protein were stained by DAPI (blue) and LAMP-2 (green) , respectively. Scale bars: 10 µm. ( c ) Western blot analysis of vimentin and α-SMA protein in LAMP-2 siRNA-treated human brain VSMC vs control cells. The graphs show quantified data from the Western blots. * P < 0.05, *** P < 0.001, compared with control cells. ( d ) Evaluation of apoptosis (left upper and lower panels) . Nuclei of LAMP-2 siRNA treated and control cells were stained with Hoechst 33342 (turquoise) . Both subgroups showed a normal nuclear appearance (right panel) . The actin filaments and LAMP-2 protein were stained by Phalloidin (white) and LAMP-2 (green) , respectively. The morphological features of nuclei appeared normal in both siRNA-treated (arrow) and control cells (arrowhead) . Scale bars: 20 µm. There was no difference in the frequency of apoptotic cells between the two groups. ns , not significant. ( e ) Proliferation activity was assessed by detection of Ki67-positive cells using flow cytometery. Histograms show an increased Ki67-positive population in LAMP-2 siRNA-treated human brain VSMC, compared with control cells. The bar graph data were obtained from 3 independent experiments. ** P < 0.01. A two-tailed Student’s t -test was used for all panels. Ctrl, control; CTTF, corrected total tissue fluorescence; KO, knockout; MFI, mean fluorescence intensity; WT, wild-type.

Article Snippet: The human brain VSMC were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA, #1100) and cultured using smooth muscle cell medium (#1101, ScienCell) consisting of 500 mL basal medium, 10 mL fetal bovine serum (#0010), 5 mL of smooth muscle cell growth supplement (#1152), and 5 mL of penicillin/streptomycin solution (#0503).

Techniques: Functional Assay, Immunostaining, Marker, Staining, Quantitation Assay, Expressing, Control, Western Blot, Activity Assay, Two Tailed Test, Fluorescence, Knock-Out

Journal: Scientific Reports

Article Title: Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency

doi: 10.1038/s41598-018-21602-8

Figure Lengend Snippet: Alterations in mitochondrial dynamics and function of VSMC under LAMP-2 deficiency. ( a ) Morphology of mitochondria in live VSMC. Mitotracker staining (red) revealed more marked fragmentation of mitochondria in LAMP-2 siRNA human brain VSMC, compared with control cells. The nuclei and cytoplasmic LAMP-2 protein were stained by DAPI (blue) and LAMP-2 (green) , respectively. Scale bars: 10 µm. The graphs show that LAMP-2 deficiency induced significantly decreased individual mitochondrial area and significantly increased number of mitochondria in ROI per cell. *** P < 0.001, compared with control cells. ( b ) Double immunostaining of DRP-1 ( turquoise ) and Mitotracker ( red ). The nuclei were visualized by DAPI (blue) . The colocalization of DRP-1 protein and mitochondria is indicated by white . Scale bars: 10 µm. Colocalization of DRP-1 with MitoTracker is more obvious in VSMC under LAMP-2 deficiency, compared with control cells. *** P < 0.001. ( c ) 3D time-lapse reconstructed images of mitochondria in live VSMC. Time-lapse tracking during 3 minutes revealed that mitochondria in LAMP-2–deficient VSMC changed their shape to become smaller, swollen, and more spherical, thereby resulting in a fragmented pattern ( arrowheads ). In contrast, control cells maintained normal mitochondrial dynamics, with fine tubular networks. Scale bars: 5 µm. ( d ) Immunocytochemical detection of H 2 O 2 in LAMP-2 siRNA human brain VSMC. Green fluorescence indicates DCF production. Nuclei were visualized by DAPI (blue) . Bars: 50 µm. ( e ) H 2 O 2 content was evaluated in flow cytometry analysis by quantification of DCF. Histograms showed enhanced DCF expression in LAMP-2 siRNA-treated human brain VSMC, compared with control cells. Quantified data from 3 independent experiments are shown. * P < 0.05. ( f ) Mitochondrial respiration assay. Oligomycin, FCCP, and a mix of rotenone/antimycin A were sequentially added to measure basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. ( g ) Representative line graphs of mitochondrial respiration assays are shown. ( h ) LAMP-2 siRNA human brain VSMC showed a significant increase in mitochondrial respiration. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns , not significant, compared with control cells. A two-tailed Student’s t -test was used for all panels. Ctrl, control; MFI, mean fluorescence intensity.

Article Snippet: The human brain VSMC were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA, #1100) and cultured using smooth muscle cell medium (#1101, ScienCell) consisting of 500 mL basal medium, 10 mL fetal bovine serum (#0010), 5 mL of smooth muscle cell growth supplement (#1152), and 5 mL of penicillin/streptomycin solution (#0503).

Techniques: Staining, Control, Double Immunostaining, Fluorescence, Flow Cytometry, Expressing, Respiration Assay, Two Tailed Test

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Flow cytometry gates for cell viability were set based on living and EtOH-killed controls (A). Incubation for 2 h (grey bar) with pDNA had no impact on vascular smooth muscle cell (vSMC) viability. Fifty-percent pCsM (C) and Cs (D) stabilize vSMC viability by 12 h post-incubation, whereas Vehicle (E) exposure drives protracted declines. Cell viability was unchanged 24 h after 20% treatments (F). (*p≤0.01, §p≤0.05)

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry, Incubation

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Flow cytometry gates to determine Live CD59-positive vSMC were set based on living and EtOH-killed controls (A). Chitosan (pCsM, CsM, Cs) initially decreased the number of CD59-positive cells (B, p≤0.001), an effect that was rapidly, potently and durably reversed, especially in the case of Cs (C, *p≤0.01, §p≤0.05).

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Mean fluorescent intensity from flow cytometry was used to approximate average CD59 expression vSMC (A). CD59 is transiently depressed by pCsM, CsM and Cs (B, p≤0.001) but recovers to baseline by 24 h despite the presence of Vehicle (C). In the absence of chitosan, Vehicle decreases CD59 expression.

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry, Expressing