488 pi apoptosis detection kit  (Cell Signaling Technology Inc)

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