Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Antibodies used for western blotting

Article Snippet: rabbit anti-ATF4 , 1: 1000 , Proteintech , 10835-1-AP , AB_2058600.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Microglia treated with tryptanthrin suppresses neuronal apoptosis through ER stress–related signaling. (A) Schematic of BV2 and N2a cell treatments and the conditional co-culture system. (B) Double immunostaining of CC-3 (green) and NeuN (red) in N2a cells incubated with conditional medium (CM) of BV2 cells treated by Ctrl (Ctrl-CM), LPS + DMSO (LD-CM) and LPS + Tryp (LT-CM). CC-3 + NeuN + cells were marked by white arrows. Scale bar: 100 μm. (C) Percentages of CC-3 + NeuN + cells shown in B ( n = 6 individual samples of cells per group). (D) Western blot analysis of Bcl-2 and Bax expression in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (E, F) Quantitative analysis of Bcl-2 (E) and Bax (F) protein levels shown in D (normalized to β-actin, n = 6 individual samples of cells per group). (G) Western blot analysis of p-eIF2α, eIF2α, ATF4 and CHOP in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (H–K) Quantitative analysis of p-eIF2α (H, normalized to eIF2α), eIF2α (I, normalized to β-actin), ATF4 (J, normalized to β-actin) and CHOP (K, normalized to β-actin) protein levels shown in G ( n = 6 individual samples of cells per group). (L) Double immunostaining of CHOP (green) and NeuN (red) in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. The areas marked by boxes were shown in the ‘Enlarge’ panels. Scale bars: 50 μm and 20 μm (enlarge). (M) Percentages of CHOP + NeuN + cells according to L ( n = 6 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. ATF4: Activating transcription factor 4; CC-3: cleaved caspase-3; CHOP: CCAAT-enhancer-binding protein homologous protein; DMSO: dimethyl sulfoxide; eIF2α: eukaryotic initiation factor 2α; ER: endoplasmic reticulum; LPS: lipopolysaccharide; NeuN: neuron-specific nuclear protein; ns: not significant; p: phosphorylated; Tryp: tryptanthrin.

Article Snippet: rabbit anti-ATF4 , 1: 1000 , Proteintech , 10835-1-AP , AB_2058600.

Techniques: Co-Culture Assay, Double Immunostaining, Incubation, Western Blot, Expressing, Binding Assay