Journal: Neural Regeneration Research
Article Title: Hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect neurons from cardiac arrest–induced pyroptosis
doi: 10.4103/NRR.NRR-D-23-01922
Figure Lengend Snippet: HP-BMSCs suppress OGD-induced neuronal pyroptosis to some extent by inhibiting the MAPK and NF-κB pathways. (A) From left to right: BMSCs at passage 3 (P3), osteogenic differentiation BMSCs, and adipogenic differentiation BMSCs. The cells are typically spindle-shaped. Scale bars: 100 μm. (B) Flow cytometric analysis showing the expression of BMSC surface markers. The cells expressed CD29 and CD90 but did not express CD11b and CD45. (C) Immunofluorescence staining of primary cultured neurons for MAP2 (red, Alexa Fluor 594), whereas nuclei were labeled with DAPI (blue). Scale bars: 75 μm. (D) Western blotting analysis was performed to evaluate the expression of NLRP3, GSDMD-N, ASC, cleaved-caspase-1, NF-κB, JNK, and p38 MAPK in primary neurons from the indicated group ( n = 3). The results of parallel test were shown in Additional Figure 2 . (E–K) Quantitative analysis of NLRP3 (E), GSDMD-N (F), ASC (G), cleaved-caspase-1 (H), NF-κB (I), JNK (J), and p38 MAPK (K) protein expression levels as shown in D. Results are expressed as mean ± SD. * P < 0.05, vs . control group; # P < 0.05, vs . OGD group; & P < 0.05, vs . OGD + N-BMSCs group; $ P < 0.05, vs . OGD + HP-BMSCs group (one-way analysis of variance followed by Tukey’s multiple comparisons test). BMSCs: Bone marrow mesenchymal stem cells; HP-BMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; N-BMSCs: normal-cultured BMSCs; OGD: oxygen-glucose deprivation.
Article Snippet: Then, the samples were incubated at 4°C overnight with the appropriate primary antibodies: translocase of outer mitochondrial membrane 20 (TOM20, rabbit, 1:5000, Proteintech, Cat# 11802-1-AP, RRID: AB_2207530), fission 1 protein (FIS1, rabbit, 1:1000, Proteintech, Cat# 10956-1-AP, RRID: AB_2102532), cytochrome c oxidase subunit 4 (COX IV, rabbit, 1:5000, Proteintech, Cat# 11242-1-AP, RRID: AB_2085278), brain-derived neurotrophic factor (BDNF, rabbit, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab108319, RRID: AB_10862052), NLRP3 (rabbit, 1:1000, Immunoway, Cat# YT5382, RRID: AB_3095508), apoptosis-associated speck-like protein containing a CARD (ASC, rabbit, 1:1000, Immunoway, Cat# YT0365, RRID: AB_2750947), cleaved-caspase-1 (rabbit, 1:1000, Immunoway, Cat# YC0022, RRID: AB_3095510), gasdermin D N-terminal domain (GSDMD-N, rabbit, Abcam, 1:1000, Cat# ab219800, RRID: AB_2888940), p38 mitogen-activated protein kinase (p38 MAPK, rabbit, 1:1000, Immunoway, Cat# YT3513, RRID: AB_3095512), JNK (rabbit, 1:1000, Immunoway, Cat# YT2440, RRID: AB_2895034), NF-κB p65 (mouse, 1:1000, Immunoway, Cat# YM3111, RRID: AB_3073611), Bcl-2 (mouse, 1:1000, Immunoway, Cat# YM3041, RRID: AB_2814758), bak (mouse, 1:1000, Proteintech, Cat# 29552-1-AP, RRID: AB_2923596), activated-caspase-3 (mouse, 1:1000, Immunoway, Cat# YM3431, RRID: AB_3095515), PFKL (rabbit, 1:1000, Immunoway, Cat# YT3685, RRID: AB_3095516), and β-actin (rabbit, 1:5000, CST, Boston, MA, USA, Cat# 4970, RRID: AB_2223172).
Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Labeling, Western Blot, Control
