Journal: Journal of Neuroinflammation

Article Title: Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke

doi: 10.1186/s12974-023-02888-6

Figure Lengend Snippet: Ischemia-induced upregulation of the expression of certain immunomodulatory membrane receptors as assessed by flow cytometry. A Gating strategy for flow cytometry analysis. B Quantification of the % of CD31 + endothelial cells expressing the receptors in the contralateral (contra) and ipsilateral (ipsi, ischemic) hemispheres 24h post-ischemia ( n = 6 per group) by flow cytometry. Ischemia increased the % of CD31 + cells expressing CD262 ( Tnfrsf10b) (* p = 0.0156), CD155 ( Pvr ) (* p = 0.0156), and CD146 ( Mcam ) (* p = 0.0156). Changes in the % of CD31 + cells expressing CD354 ( Trem1 ) ( p = 0.1562) were not significant. Analysis was performed with the Wilcoxon matched-pairs signed rank test. C , D Immunofluorescence for CD146 (red in C ) and CD155 (green in D ) in the brain vessels (Glut1 + ) of sham-operated and ischemic mice at 1- and 4-day post-ischemia. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Images in the bottom row are magnifications of the squares shown in the merged images at day 4 post-ischemia. Quantification of mean fluorescence intensity (MFI) of CD146 and CD155 in brain vessels (number of vessels ranging from 145 to 250 obtained from 3 to 5 mice per group) **** p < 0.0001 (Kruskal–Wallis and Dunn’s tests)

Article Snippet: Cells were incubated during 30 min at 4 °C with the following primary antibodies: CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949; or clone 390, BV421, #102423, Biolegend, RRID:AB_2562186), CD11b (clone M1/70, PE, #553311, BD Pharmingen, RRID:AB_394775), CD45 (clone 30-F11, FITC, #553080, BD Horizon, RRID:AB_394610), CD155 (clone TX56, BV421, #131517, Biolegend, RRID:AB_2716159), CD354 (clone 174031, BV711, #747902, BD Pharmingen, RRID:AB_2872364), CD146 (clone ME-9F1, PE-Cyanine7, #134713, Biolegend, RRID:AB_2563108), CD262 (clone MD5-1-3C2, PerCP-Vio700, #130-105-697, Miltenyi Biotec, RRID:AB_2656753).

Techniques: Expressing, Membrane, Flow Cytometry, Immunofluorescence, Staining, Fluorescence

Journal: EMBO Reports

Article Title: Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

doi: 10.15252/embr.202154133

Figure Lengend Snippet:

Article Snippet: Mouse α‐human CD253 (TRAIL) APC (clone RIK‐2.1) , Miltenyi Biotec , Cat#130‐097‐314; RRID:AB_2656681.

Techniques: Generated, Recombinant, Control, Sequencing, Staining, Software, Selection, Enzyme-linked Immunosorbent Assay, Marker

Journal: Cancers

Article Title: The Impact of TRAIL on the Immunological Milieu during the Early Stage of Abdominal Sepsis

doi: 10.3390/cancers15061773

Figure Lengend Snippet: Effect of 6h CASP treatment on the expression of TRAIL receptors. TRAIL receptors DR5, DcR1, and DcR2 were stained and assessed by FACS. Representative FACS plots show ( a ) DR5 staining in neutrophils derived from BM and spleen, ( c ) DcR2 staining in neutrophils of the spleen, and ( e ) DcR1 staining in neutrophils of the BM. Quantification of ( b ) DR5, ( d ) DcR2, and ( f ) DcR1 expression in neutrophils of the BM and spleen of n = 5 animals in each group. Neutrophils of septic WT and TRAIL–/– mice were compared with naïve ones. Graphs show mean ± SEM (* p < 0.05, ** p < 0.01).

Article Snippet: Surface staining was performed using antibodies against DcR1 (REA759, Milteny Biotech), DcR2 (mDcR2-1, Biolegend, San Diego, CA, USA), DR5 (REA1233, Milteny Biotech), TRAIL (CD 253) (N2B2, Biolegend), CD45 (30-F11, BD Bioscience), CD172 (P84, eBioscience), CD11b (M1/70, Biolegend), F4/80 (BM8, Biolegend), Ly6G (1A8, Biolegend), and DUMP (live/dead, CD3 (17A2, Biolegend), CD19 + (6D5, Biolegend) and CD49b (HMalpha2, BD Bioscience)).

Techniques: Expressing, Staining, Derivative Assay

Journal: Cell Death & Disease

Article Title: Carboxypeptidase A4 negatively regulates HGS-ETR1/2-induced pyroptosis by forming a positive feedback loop with the AKT signalling pathway

doi: 10.1038/s41419-023-06327-5

Figure Lengend Snippet: a After screening HLCZ01 cells for drug resistance, 2 μg/mL HGS-ETR1/2 was added to treat HLCZ01 and drug-resistant HLCZ01 cells (HLCZ01-ETR1/2 R) for 8 h, and the pyroptosis of the cells was observed by bright field photography and PI/Hoechst fluorescence photography. Scale bar, 125 μm. b, c HLCZ01 and HLCZ01-ETR1/2 R cells were treated with 2 μg/mL HGS-ETR1/2 for 8 h. GSDME cleavage was detected by WB, and β-actin was used as the internal reference protein. d The mRNA expression levels of DR4 , DR5 and GSDME in HLCZ01 and HLCZ01-ETR1/2 R cells were detected by qPCR, with GAPDH as the internal reference gene. e The protein expression levels of DR4, DR5 and GSDME in HLCZ01 and HLCZ01-ETR1/2 R cells were detected by western blotting, and β-actin was used as the internal reference protein. Three independent biological replicates were performed for each of the above experiments. Two-sided Student’s t test was used to analyse significant differences ( b-e ) (* p <0.05, ** p <0.01).

Article Snippet: DR4 (1139) and DR5 (2019) were purchased from ProSci.

Techniques: Fluorescence, Expressing, Western Blot

Journal: Carcinogenesis

Article Title: Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

doi: 10.1093/carcin/bgp008

Figure Lengend Snippet: Kinetics of expression of functional receptors and ligands of death receptors pathways after a 10 Gy irradiation of T-47D cells. ( a ) The kinetics of protein expression of receptors (Fas, TRAIL-R1, TRAIL-R2, TNF-R1 and TNF-R2) and ligands (FasL and TRAIL) of death receptors pathways were studied by western blot analysis using cellular extracts from 10 Gy-irradiated T-47D cells. β-Actin was used as loading control. ( b ), ( c ) and ( d ) Analysis of soluble forms of death receptors ligands in culture medium of 10 Gy-irradiated T-47D cells. TNF-α and TRAIL were detected by ELISA in supernatant of irradiated and non-irradiated cells each day for 7 days (b and c, left panel). To analyze ligand secretion over 24 h, medium was changed on day 5 and ELISA was performed on supernatant on day 6 (b and c, right panel). Each value corresponds to the mean of at least two independent measurements from two independent experiments. Error bars correspond to standard deviation. (d) Soluble FasL was detected by western blot after direct loading cell culture supernatants (see Materials and Methods). The representative results from three independent experiments are shown. ( e ) Irradiation sensitizes T-47D cells to TNF-α, TRAIL and anti-Fas agonist antibody. Cells were treated with anti-Fas agonist antibody (2 μg/ml), TRAIL (1 ng/ml) and TNF-α (0.5 ng/ml) 24 h after 10 Gy irradiation and 24 h later, viable and dead cells were counted after trypan blue staining. Results correspond to the mean ± SD of three independent experiments.

Article Snippet: Anti-PARP1 antibody (AM30) and anti-TRAIL antibody (# 556468) was from BD Biosciences (Le Pont-De-Claix, France), anti-β-actin antibody (A-5316) was from Sigma, anti-TNF-α antibody (AF-210-NA) was from R&D Systems, anti-TRAIL-R1 antibody (# 06-744) was from Upstate (Millipore, France) and anti-TRAIL-R2 antibody (# 2019) was from ProSci (Coger S.A., France).

Techniques: Expressing, Functional Assay, Irradiation, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Cell Culture, Staining

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: AS-IV ameliorated the senescence of BLM-induced VSMCs. BLM-treated VSMCs were given different doses of AS-IV. A Western blot exhibited the expression changes of p16, p21, and DcR2, and the protein levels were quantified on the gray scale. B The population ofβ-galactosidase was tested using flow cytometer, and quantitative analysis was conducted. C After processing with AS-IV, the SA-β-galactosidase kit was used to evaluate the senescence of BLM-induced VSMCs. Magnification, 200 × , scale bar = 50 μm. L low dose, M medium dose, H high dose. * P < 0.05, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: Mitophagy mediators regulated the functions of AS-IV on BLM-induced VSMC senescence. BLM-treated VSMCs were given AS-IV, and then exposed to the autophagy inducer (Torin 1) or mitophagy inhibitor (Mdivi-1). A Western blot was used to assess the protein levels of P62, p21, and DCR2, and quantitative analysis of the protein was also determined. B Flow Cytometer was used to analyze the population ofβ-galactosidase in each group. C Cellular senescence was evaluated using a SA-β-galactosidase kit in each group. Magnification, 200 × , scale bar = 50 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Western Blot, Flow Cytometry

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: Parkin-dependent mitophagy attenuated the cellular senescence induced by BLM. A Expression changes in p16, p21, DcR2 were monitored by Western blot. B The change in TMRM + fluorescence was confirmed through flow cytometer. C Western blot was used to assess the changes of p62 expression. D Western blot was used to evaluate expression of Parkin in VSMCs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Expressing, Western Blot, Fluorescence, Flow Cytometry

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: AS-IV attenuated senescence and mitophagy in the aging mouse model The aging mouse model was induced by d -Gal, and then given three doses of AS-IV. A Pathological change of vascular tissue was evaluated through H&E staining. Magnification, 200 × , scale bar = 100 μm. B – E Expressions of p16 ( B ), p21 ( C ), and Parkin ( D ) were accessed by immunofluorescence and Western blot (E). Magnification, 200 × , scale bar = 100 μm. E Western blot analysis shows the changes in p16, p21, DcR2 expressions. G The concentration of β-galactosidase was determined through the ELISA kit. F Western blot was also used to confirm p62 and Drp1 expressions. * P < 0.05, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Staining, Immunofluorescence, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay