Journal: PLoS ONE

Article Title: In Vitro Hepatic Trans-Differentiation of Human Mesenchymal Stem Cells Using Sera from Congestive/Ischemic Liver during Cardiac Failure

doi: 10.1371/journal.pone.0092397

Figure Lengend Snippet: (A) In vitro cytotoxicity assay showing MTT activity across 9 days of induction in different culture conditions. Error bars represents mean ± S.D. The differences in cell proliferation between the groups were considered statistically significant at p <0.05 and p -values are indicated on the graph. (*) indicates significant increase in cell proliferation at day 9 compared to day 3 and day 6 in 5% patient sera group. (#) indicates no significant difference in hMSC proliferation at different time points across 9 days of culture. (B) Cell proliferation assay by direct cell counting reveals higher proliferation rate in 10% FBS, which was comparable to that of 10% normal sera (NS) induction group. 10% patient sera (PS) induction group did not show any increase in cell proliferation. However, 5% PS group showed increase in proliferation, which was significantly more compared to 10% PS group (* p <0.05). (C) Cell death assay by annexinV-FITC/PI flow cytometric quantification revealed that 10% patient sera caused mostly necrotic cell death with increase in cell death across 9 days, whereas in 5% PS group cell death was comparably minimal. However, there was negligible cell death in control groups.

Article Snippet: Cell death assay was performed by annexin V-FITC/PI apoptosis detection kit (Cell Signaling Technology) as per manufacturer’s protocol.

Techniques: In Vitro, Cytotoxicity Assay, Activity Assay, Proliferation Assay, Cell Counting

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Isoimperatorin Induces Apoptosis of Nasopharyngeal Carcinoma Cells via the MAPK/ERK1/2 Signaling Pathway

doi: 10.1155/2020/2138186

Figure Lengend Snippet: (a) Dual-fluorescence flow cytometry cellometer image cytometer (K2) was used to detect the change of apoptotic rate of CNE2 cells after isoimperatorin intervention. The apoptotic rate in the 10 μ M group was 23.06 ± 2.00%, the 20 μ M group was 22.90 ± 3.47%, and the 40 μ M group was 29.06 ± 1.25% vs control group: ∗∗ P < 0.01. Cytation™ 5 cell imaging multifunctional detection system detects changes in the nucleus (b) and cell membrane potentials (c) of the cells after the intervention of isoimperatorin. (A) Control; (B)ISOIMP 10 μ M; (C) ISOIMP20 μ M; (D)ISOIMP 40 μ M; (E) CIS 4.0 μ g·mL –1 (bar=100 μ m, 200x). The nucleus appears bright blue with nuclear condensation. The increase in green fluorescence indicates a decrease in CNE2 cell membrane potentials and cell apoptosis. Isoimperatorin inhibits the expression of proliferation-related and apoptosis-related proteins in CNE2 cells. Western blot analysis of the effects of isoimperatorin at different concentrations on the relative expression of PCNA, XIAP, and survivin (d) and Bax and Bcl-2 (e) in CNE2 cells vs control group: ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: RPMI-1640 medium (Hyclone), fetal bovine serum (Gibco), DMSO (Amresco), MTT (Biosharp), Annexin V-FITC/PI apoptosis kit (MULTI SCIENCES), β -actin Mouse mAb, Survivin Rabbit mAb, XIAP Rabbit mAb, Phospho-c-Raf Rabbit mAb, Phospho-MEK Rabbit mAb, Phospho-p44/42 MAPK (ERK1/2) Rabbit mAb (Cell Signaling TECHNOLOGY), Rabbit Anti-PCNA Polyclonal Antibody, Rabbit Anti-Bax polyclonal antibody, and Rabbit Anti-Bcl-2 Polyclonal Antibody (Bioss) were used.

Techniques: Fluorescence, Flow Cytometry, Cytometry, Imaging, Expressing, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Isoimperatorin Induces Apoptosis of Nasopharyngeal Carcinoma Cells via the MAPK/ERK1/2 Signaling Pathway

doi: 10.1155/2020/2138186

Figure Lengend Snippet: Effect of isoimperatorin on CNE2 cell apoptosis is attenuated by ISO. Western blot analysis shows the expression of p-c-RAF, p-MEK, and p-ERK1/2 (a), PCNA, XIAP, and survivin (b), and Bax and Bcl-2 (c) in CNE2 cells. vs control group: ∗∗ P < 0.01; vs ISOIMP group, # P < 0.05; ## P < 0.01; dual-fluorescence flow cytometry cellometer image cytometer (K2) was used to detect the change of apoptotic rate of CNE2 cells after ISO and isoimperatorin intervention (d) vs control group: ∗∗ P < 0.01; vs ISOIMP group, # P < 0.05; ## P < 0.01.

Article Snippet: RPMI-1640 medium (Hyclone), fetal bovine serum (Gibco), DMSO (Amresco), MTT (Biosharp), Annexin V-FITC/PI apoptosis kit (MULTI SCIENCES), β -actin Mouse mAb, Survivin Rabbit mAb, XIAP Rabbit mAb, Phospho-c-Raf Rabbit mAb, Phospho-MEK Rabbit mAb, Phospho-p44/42 MAPK (ERK1/2) Rabbit mAb (Cell Signaling TECHNOLOGY), Rabbit Anti-PCNA Polyclonal Antibody, Rabbit Anti-Bax polyclonal antibody, and Rabbit Anti-Bcl-2 Polyclonal Antibody (Bioss) were used.

Techniques: Western Blot, Expressing, Fluorescence, Flow Cytometry, Cytometry

Journal: Molecules

Article Title: Cell Cycle Arrest and Apoptosis-Inducing Ability of Benzimidazole Derivatives: Design, Synthesis, Docking, and Biological Evaluation

doi: 10.3390/molecules27206899

Figure Lengend Snippet: The apoptotic effect in A549, MDA−MB 231, and SKOV3 cancerous cells after treatment with vehicle control and compounds 10 and 13 by annexin V−FITC/PI staining using flow cytometry. One-way ANOVA was used to test for statistical differences (* p < 0.05, ** p < 0.01).

Article Snippet: The assessment of apoptosis was performed using the Annexin V-FITC/PI analysis Kit, Cell Signaling Technology (CST), as instructed by the manufacturer [ ].

Techniques: Staining, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis

doi: 10.1002/ctm2.711

Figure Lengend Snippet: Ldlr knockout exacerbated BLM‐induced PF. (A–C) Pulmonary tissue sections were stained with H&E and Masson's trichrome, and the Ashcroft score was calculated. Scale bar: 500 μm. (C) Soluble collagen synthesis in lung homogenate. (D) qRT‐PCR analysis of fibrosis‐related genes in mouse lungs. (E) Plasma lipid levels. (F) Measurement of TGF‐β1 and ET‐1 levels at day 7 and 21 after BLM treatment. (G) Heatmap of all of the differentially expressed genes in Ldlr− /− mice and WT mice in response to BLM administration. (H) The expression profiles of inflammation‐, ECM‐, apoptosis‐, migration‐, junction‐ and surfactant homeostasis‐related genes. mRNA levels, cell counts and lipid levels were normalized to the saline‐treated group. N = 6–10 per group in data (A) to (F) and N = 4–6 per group in data (G) and (H). * p < .05, ** p < .01, *** p < .001 versus control. Data are presented as the mean ± SEM

Article Snippet: The Sircol Assay Kit (S5000, Biocolor), Annexin V‐Alexa Fluor 488/PI Apoptosis Detection Kit (6592, CST, Massachusetts, USA), Endothelin 1 ELISA Kit (DET100, R&D), TUNEL Kit (11684795910, Roche, Indianapolis, IN), Mouse PCSK9 SimpleStep ELISA Kit (ab215538, Abcam, Cambridge, UK), Human PCSK9 SimpleStep ELISA Kit (ab209884, Abcam), Mouse TGF beta 1 ELISA Kit (ARG80211, Arigo) and Human TGF beta 1 ELISA Kit (ARG80123, Arigo) were obtained from indicated manufacturers.

Techniques: Knock-Out, Staining, Quantitative RT-PCR, Expressing, Migration

Journal: Clinical and Translational Medicine

Article Title: LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis

doi: 10.1002/ctm2.711

Figure Lengend Snippet: Increased apoptosis in lungs of BLM‐treated Ldlr− /− mice. (A) Representative TUNEL staining images of lung sections at day 7. Scale bar: 250 μm. (B) Co‐localization of positive TUNEL labelling with anti‐CD31 or anti‐SP‐C staining from BLM‐treated Ldlr− /− mice at day 7. Scale bar: 25 μm. (C) Numbers of apoptotic cells at day 7. (D) Western blot analysis of cleaved caspase‐3 and total caspase‐3 levels in the lungs at day 7. D1: caspase 3 activation in WT mouse lungs; D2: caspase 3 activation in Ldlr− /− mouse lungs; D3: The endogenous levels of cleaved and total caspase 3 in WT and Ldlr− /− mice without BLM treatment. D4: The levels of cleaved and total caspase 3 in WT and Ldlr− /− mice with BLM treatment. (E−F) Densitometric values of cleaved caspase‐3 (E) and total caspase‐3 (F) at both days 7 and 21 in the lungs. N ≥ 6 per group in (A to E). N = 24 per group in (F). * p < .05, ** p < .01, *** p < .001 versus control. Data are presented as the mean ± SEM

Article Snippet: The Sircol Assay Kit (S5000, Biocolor), Annexin V‐Alexa Fluor 488/PI Apoptosis Detection Kit (6592, CST, Massachusetts, USA), Endothelin 1 ELISA Kit (DET100, R&D), TUNEL Kit (11684795910, Roche, Indianapolis, IN), Mouse PCSK9 SimpleStep ELISA Kit (ab215538, Abcam, Cambridge, UK), Human PCSK9 SimpleStep ELISA Kit (ab209884, Abcam), Mouse TGF beta 1 ELISA Kit (ARG80211, Arigo) and Human TGF beta 1 ELISA Kit (ARG80123, Arigo) were obtained from indicated manufacturers.

Techniques: TUNEL Assay, Staining, Western Blot, Activation Assay

Journal: Clinical and Translational Medicine

Article Title: LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis

doi: 10.1002/ctm2.711

Figure Lengend Snippet: LDL and LDLR knockdown induced apoptosis, fibroblast‐like endothelial and ATII cells and fibrosis. Examination of apoptosis in pHLEC cells (A–B) and pHLATII cells (C–D) after LDL stimulation by flow cytometry and western blot. (E–F) Effects of si‐LDLR on the induction of fibroblast‐like endothelial cells based on western blot and immunofluorescent assay. (G–H) Effects of si‐LDLR on the induction of fibroblast‐like epithelial cells based on western blot and immunofluorescent assay. (I–J) Immunofluorescence of α‐SMA in PHLF after incubation with conditioned medium from LDL‐ or si ‐ LDLR ‐treated endothelial cells. (K–L) Collagen, α‐SMA and p‐SMAD2/3 levels, as analyzed by western blot. (M–N) Immunofluorescence and western blot analyses were performed on cells treated with LDL or si‐LDLR for 12 and 48 h, respectively. (O–P) Collagen, α‐SMA and p‐SMAD2/3 levels, as analyzed by western blot. (Q–R) ELISA analysis of TGF‐β1 in culture medium from LDL‐treated pHLEC and PHFL cells, respectively. (S–T) ELISA analysis of ET‐1 levels in the culture medium from LDLR‐deficient pHLEC and PHFL cells. Lipoprotein deficient serum (LPDS) and control siRNA were used as controls. Scale bar: 200 μm. * p < .05, ** p < .01, *** p < .001 versus control. Data are presented as the mean ± SEM of three independent experiments

Article Snippet: The Sircol Assay Kit (S5000, Biocolor), Annexin V‐Alexa Fluor 488/PI Apoptosis Detection Kit (6592, CST, Massachusetts, USA), Endothelin 1 ELISA Kit (DET100, R&D), TUNEL Kit (11684795910, Roche, Indianapolis, IN), Mouse PCSK9 SimpleStep ELISA Kit (ab215538, Abcam, Cambridge, UK), Human PCSK9 SimpleStep ELISA Kit (ab209884, Abcam), Mouse TGF beta 1 ELISA Kit (ARG80211, Arigo) and Human TGF beta 1 ELISA Kit (ARG80123, Arigo) were obtained from indicated manufacturers.

Techniques: Flow Cytometry, Western Blot, Immunofluorescence, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis

doi: 10.1038/s41467-021-25079-4

Figure Lengend Snippet: A Representative microscopy images of amino acid-starved U2OS cells incubated in absence or presence of LQ, AICAR, metformin, or A769662 for 72 h, as indicated. Scale bar represents 100 µm. B Cell viability as estimated by a trypan blue exclusion assay of amino acid-starved U2OS cells incubated in the presence or absence of LQ and AICAR during 72 h. C Representative images of clonogenic assay of U2OS cells treated as in ( B ). D Colony quantification of images obtained in ( C ). E Quantification of late apoptosis population of three biologically independent experiments (double positive, annexin V and PI) as obtained in ( F ) for the indicated condition. F Flow cytometry analysis of annexin V/PI staining of amino acid-starved U2OS cells incubated with or without LQ in combination with AICAR, metformin, or A769662 during 72 h. G Immunoblot of the pro-apoptotic markers (BAX, cleaved caspase 3, and cleaved PARP), mTORC1 activity markers (Raptor, S6K, and S6 phosphorylation), and AMPK phosphorylation of amino acid-starved U2OS cells incubated in the presence or absence of LQ, with or without AICAR, during 72 h. H Cell viability as estimated by a trypan blue exclusion assay of ATG5 +/+ and ATG5 −/− MEFs incubated in the presence or absence of amino acids (AA) and AICAR during 72 h. I Immunoblot analysis of pro-apoptotic markers of ATG5 +/+ and ATG5 −/− MEFs incubated as in ( H ). J – L Representative immunohistochemistry microscopy pictures (×40 magnification) of xenograft tumors of mice treated as indicated (TEM: Temsirolimus; MET: Metformin). Samples were stained against S6-pS235/236 ( J ) and cleaved caspase 3 ( L ). Scale bars represent 100 µm. K – M IHC visual score of S6-pS235/236 ( K ) and caspase 3 ( M ) of images from ( J ) and ( L ), respectively. The upper and lower limits of the boxes represent quartiles, with the line within the boxes indicating the median and the whiskers showing the extremes ( n ≥ 10 images per treatment). Graphs show mean values ± SEM ( n = 3 biologically independent experiments). * p < 0.05 (ANOVA analysis followed by a post hoc Bonferroni test). Source data are provided as a .

Article Snippet: Cells were seeded the day before treatment, treated for the indicated time and stained following with annexin V and propidium iodide (PI) (Annexin V early apoptosis detection kit, #6592 Cell Signaling Technology) according to the manufacturer’s instructions.

Techniques: Microscopy, Incubation, Trypan Blue Exclusion Assay, Clonogenic Assay, Flow Cytometry, Staining, Western Blot, Activity Assay, Immunohistochemistry