Journal: Journal of the American Heart Association

Article Title: Super‐Enhancer–Driven Syndecan‐4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension

doi: 10.1161/jaha.124.036757

Figure Lengend Snippet: Figure 3. Silencing of syndecan-4 inhibited hypoxia-induced PASMC proliferation. A, Silencing of syndecan-4 blocked the effects of hypoxia on EdU incorporation and Ki67 expression in hPASMCs, EdU (red), Ki67 (green), 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 μm. B, hPASMCs viability was determined by Cell-Counting Kit-8 assay (n=6). C, Cell cycle analysis by flow cytometry indicated that hypoxia stimulated cell progression into G2/M+S phase, and this effect was inhibited by syndecan-4 small interfering RNA. D, Western blot analysis of syndecan-4, HIF-1α, PCNA, cyclin A, and cyclin D in hPASMCs (n=6). E, mPASMC viability was determined by Cell-Counting Kit-8 assay (n=6). F, Silencing of syndecan-4 blocked the effects of hypoxia on EdU incorporation in mPASMCs. EdU (red), 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 μm (n=6). G, Western blot analysis of syndecan-4, HIF-1α, PCNA, cyclin A, and cyclin D in mPASMCs (n=6). All values are presented as the mean±SEM. Statistical analysis was performed with 1-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. EdU indicates ethynyl-2′- deoxyuridine; hPASMCs, human pulmonary artery smooth muscle cells; HYP, hypoxic; mPASMCs, mouse pulmonary artery smooth muscle cells; NC, negative control; NOR, normoxia; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; and SDC4, syndecan-4.

Article Snippet: Cells were incubated with anti- syndecan- 4 antibody (1:100, sc- 12766; Santa Cruz Biotechnology, Santa Cruz, CA), anti- PRKCA (1:100, 21991- 1- AP, Proteintech, Rosemont, IL), anti- CD63 antibody (1:100, ab315108, Abcam), and anti- Ki67 antibody (1:100, M00254- 3; Boster) at 4 °C overnight.

Techniques: Expressing, Cell Counting, Cell Cycle Assay, Flow Cytometry, Small Interfering RNA, Western Blot, Negative Control

Journal: Cells

Article Title: Cistanoside F Ameliorates Lipid Accumulation and Enhances Myogenic Differentiation via AMPK-Dependent Signaling in C2C12 Myotubes.

doi: 10.3390/cells14120874

Figure Lengend Snippet: Figure 4. Expression of PGC-1α, MMP, and ROS in two adipogenic models after Cis adminis- tration. (A,B) PGC-1α expression. (C,D) Immunofluorescence of TMRE, the statistic was eval- uated by fluorescence spectroscopy, at λexcitation = 550 nm and λemission = 575 nm. (E,G) Im- munofluorescence of JC-1, statistic was evaluated by fluorescence spectroscopy, JC-1 monomer at λexcitation = 490 nm and λemission = 530 nm and JC-1 aggregate at λexcitation = 525 nm and λemission = 590 nm. (F,H) Immunofluorescence of ROS, the statistic was evaluated by fluorescence spectroscopy, at λexcitation = 488 nm and λemission = 525 nm. n = 6. ## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group.

Article Snippet: Antibodies against UCP1, CPT1b, ACC2, Desmin, and PGC-1α were purchased from Boster Biotech (Wuhan, China; #M00255; #A03558; #A03668; #PB9105; #BM4898; 1:1000).

Techniques: Expressing, Immunofluorescence, Fluorescence, Spectroscopy

Journal: Cells

Article Title: An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids

doi: 10.3390/cells8050481

Figure Lengend Snippet: Phase-contrast and confocal images of three lung F-PDOs. ( a ) Phase-contrast images of RLUN5, RLUN16, and RLUN21 were obtained using a ×4 objective. Scale bar: 200 µm. The upper panels show each F-PDO immediately after passage and the lower panels show each F-PDO before passage. ( b ) Confocal images of RLUN5, RLUN16, and RLUN21 were obtained using a ×10 objective. Scale bar: 100 µm. Ki67-expression images for RLUN5, RLUN16, and RLUN21, prepared using an anti-Ki67 antibody (green). DNA stained with DAPI (blue). ( c – e ) Confocal imaging data analyzed with NoviSight. ( c ) Box-and-whisker plot of the cell cluster volumes. ( d ) Box-and-whisker plot of the cell density expressed as the cell number/cell volume in a cell cluster. ( e ) Box-and-whisker plot of the Ki67-positive ratio expressed as 100× the number of positive cells/the number of total cells in a cluster.

Article Snippet: Immunofluorescence staining of F-PDOs was performed using an anti-Ki-67 antibody (1:250 dilution) and 4′,6-diamidino-2-phenylindole (DAPI) after fixation in phosphate buffer solution containing 4% paraformaldehyde (PFA; Fujifilm Wako Pure Chemical, Ltd.) and 1% Triton X-100 (Fujifilm Wako Pure Chemical, Ltd.).

Techniques: Expressing, Staining, Imaging, Whisker Assay

Journal: Cells

Article Title: An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids

doi: 10.3390/cells8050481

Figure Lengend Snippet: Dependency of F-PDO proliferation on epidermal growth factor (EGF). ( a ) Growth rate of RLUN5, RLU16, and RLUN21 in the presence or absence of EGF. Each F-PDO was seeded in a 96-well, round-bottomed, ultra-low-attachment microplate (Corning, Inc.). Twenty-four hours after seeding, EGF was added into the wells at a final concentration of 100 ng/mL. The wells without EGF were also set up to confirm the dependency of cell growth on EGF. One hour (culture start time) or 144 h later, the amount of ATP was measured as described in . Growth rates were calculated by dividing the amount of ATP in each well at 144 h by that at the culture start time. ( b ) Phase-contrast images of RLUN21 cultured with EGF (upper panels) or without EGF (lower panels) for six days. The images were obtained using a ×5 objective. Scale bar: 100 µm. ( c ) Images of Ki67 expression in RLUN21 cultured with EGF were obtained using an anti-Ki67 antibody (green). DNA stained with DAPI (blue). Magnification: ×10. Scale bar: 100 µm. Box-and-whisker plot of the Ki67-positive ratio ( d ) and cell density ( e ) of RLUN21 cells cultured with EGF.

Article Snippet: Immunofluorescence staining of F-PDOs was performed using an anti-Ki-67 antibody (1:250 dilution) and 4′,6-diamidino-2-phenylindole (DAPI) after fixation in phosphate buffer solution containing 4% paraformaldehyde (PFA; Fujifilm Wako Pure Chemical, Ltd.) and 1% Triton X-100 (Fujifilm Wako Pure Chemical, Ltd.).

Techniques: Concentration Assay, Cell Culture, Expressing, Staining, Whisker Assay