Fig 2: miR-23a-3p attenuates caspase-dependent neuronal apoptosis after irradiation. Neurons were transfected with miR-23a-3p mimics and miR-ve mimics and collected at 6 h after 8 Gy irradiation. Cytosolic fractions were separated by SDS-polyacrylamide gel and immunoblotted with antibodies against cytochrome c and GAPDH. Image of a representative experiment (A). Protein levels were quantified by densitometry, normalized to an approximately 37 kDa band of GAPDH, and presented as a fold change compared to control levels, n = 4 for control, n = 5 for other groups in each experiment. The experiment was repeated three times. Data represent mean ± SD. Significance assigned by one-way ANOVA and Tukey post-hoc analysis, **** p < 0.0001 vs. control RCNs; ^^^^ p < 0.0001 vs. 8 Gy + miR-ve mimic. RCNs were transfected with miR-23a-3p mimics, and negative control mimics before irradiation. Neurons were collected at 30 min, 6 h, and 24 h after 8 Gy irradiation. Whole-cell lysates were separated by SDS-polyacrylamide gel and immunoblotted with antibodies against Puma, a-fodrin, and cleaved caspase-3. Image of a representative experiment. (B). Protein levels were quantified by densitometry, normalized to ß-actin, and presented as fold change compared with untreated control levels (C). The experiment was repeated 3 times with similar results, n = 3/group in each experiment. Data represent mean ± SD of one-way ANOVA and Tukey post hoc analysis, *** p < 0.001, **** p < 0.0001 vs. control RCN, ^^^^ p < 0.0001 vs. negative control mimic transfected cells (-ve mim) RCN.

Fig 3: Irradiation decreased levels of cellular miR-23a-3p, and miR-23a-3p, PUMA, and Noxa mRNAs within the RNA-induced silencing complex. Neurons collected at 30 min, 6 h, and 24 h after 2, 8, and 32 Gy irradiation. Total RNA was used for qPCR analysis. qPCR quantification of miR-23a-3p (A) and miR-27a-3p (B) levels in a representative experiment. The experiment was repeated three times. N = 3/group in each experiment for miR-23a-3p, n = 5/group in each experiment for miR-27a-3p, with 2 technical replicates. Data represent the mean ± SD of one-way ANOVA and Tukey post-hoc analysis * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control RCN. Neurons were collected 3 h after 8 Gy treatment, subjected to RIP with Ago2 antibodies, and samples used for qPCR analysis. qPCR quantification of miR-23a-3p (C), Puma (D) and Noxa (E) levels in precipitates after RIP in a representative experiment. The experiment was repeated two times, n = 3/group in each experiment with 2 technical replicates. Data represent the mean ± SD. Statistical significance assigned by one-tailed t-test, *** p < 0.001 versus control, n = 3/group.

Fig 4: The IRE1α-mediated activation of the mitochondrial pathway plays an important role in apoptosis induction by FB1 in liver cells. (A) The effect of FB1 on the expression of JNK, Mcl-1, Bak, Bax, and Puma in the protein level. The cells were exposed to FB1 with or without TUDCA for 48 h, and the phosphorylation of JNK, Mcl-1, Bak, Bax, and Puma were analyzed by Western blotting. n = 3. (B) FB1 significantly induced cell death in wild-type MEF cells but not in Bax/Bak knockout MEF cells. The bars denote standard errors from three experiments. (C) The effect of the IRE1α specific inhibitor 4μ8C on the expression of apoptosis-related proteins. The cells were exposed to FB1 with or without 4μ8C for 24 h, and the phosphorylation of JNK, Mcl-1, Bak, Bax, and Puma were analyzed by Western blotting. n = 3. ** p < 0.01 compared with the corresponding control.

Fig 5: miR-23a-3p mimic reverses the decrease of endogenous miR-23a-3p in irradiated primary cortical neurons and attenuates neuronal cell death. RCNs were transfected with miR-23a-3p mimics and negative control mimics before irradiation. At 3 h after exposure to 8 Gy, neurons were harvested for RNA isolation. qPCR quantification of miR-23a-3p in a representative experiment (A). The experiment was repeated two times. N = 3/group in each experiment with 2 technical replicates. Data represent mean ± SD. Statistical significance assigned by one-way ANOVA and Tukey post hoc analysis; * p < 0.05, *** p < 0.001 vs. control RCN; ^ p < 0.05 vs. irradiated negative control mimics. LDH release was measured at 24 h after irradiation as a percentage of control untreated neurons as well as completely permeabilized cells (100% cell death). Analysis of a representative experiment (B). The experiment was repeated four times with similar results, n = 6/group in each experiment. Data represent mean ± SD. Statistical significance assigned by one-way ANOVA and Tukey post hoc analysis, * p < 0.05, *** p < 0.001, vs. control RCN. ^ p < 0.05, ^^^^ p < 0.0001 vs. negative control mimic transfected cells (-ve mim) RCN. RCNs were treated as described above and harvested 3 h after 8 Gy irradiation. Lysates from each sample were subjected to RNA-binding protein immunoprecipitation (RIP) using Ago2 antibodies, followed by qPCR analysis for levels of miR-23a-3p, Puma, and Noxa in the RISC. Analysis of a representative experiment (C) n = 3/group in each experiment with 2 technical replicates. Data represent mean ± SD. Significance assigned by one-way ANOVA and Tukey post hoc analysis, miR-23a-3p *** p = 0.0001 vs. control RCNs; ^^ p < 0.0039 vs. 8 Gy + miR-ve mimic; Puma *** p = 0.0004 vs. control RCNs; ^ p < 0.0471 vs. 8 Gy + miR-ve mimic; Noxa *** p = 0.0006 vs. control RCNs; ^ p < 0.0228 vs. 8 Gy + miR-ve mimic. IR causes the down-regulation of miR-23a-3p at the transcription stage. qPCR quantification of pri-miR-23a-3p. Analysis of a representative experiment (D). Data represent the mean ± SD of one-way ANOVA and Tukey post-hoc analysis, n = 6 for the control group, n = 3 for all other groups with 2 technical replicates in each experiment; * p < 0.05, ****p < 0.0001 vs. control RCN. miR-23a-3p mimic attenuates the irradiation-induced elevation of Puma, Noxa, and Bim in primary cortical neurons. RCNs were transfected with miR-23a-3p mimics and negative control mimics before irradiation. Neurons were harvested for RNA isolation at 1 h, 3 h, 6 h, and 24 h after exposure to 8 Gy. qPCR quantification of Puma, Noxa, Bim, and Bax and p21. Analysis of a representative experiment (E). The experiment was repeated three times, n = 6 for control, n = 3 for all other groups in each experiment with 2 technical replicates. Data represent mean ± SD. Statistical significance assigned by one-way ANOVA and Tukey post hoc analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control RCN, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. negative control mimic transfected cells (-ve mim) RCN.