Fig 2: Methylation analysis of miR-224/-340 and expression of DPYSL2 in human gastric cancer tissues.Representative results of miR-224 (A) and miR-340 (B) MSP analyses in primary GC tissues. The bands in the ‘Mt’ lanes are PCR products obtained with methylation-specific primers, and those in the ‘Un’ lanes were obtained with unmethylation-specific primers. ‘Ca’ and ‘N’ denote primary GCs and paired non-cancerous tissues, respectively. ‘F’ and ‘M’ denote female and male. Black stars indicate samples in which the aberrant hypermethylation of CpG islands was detected. (C) Comparison of the miR-224 methylation status and miR-224 levels in gastric tissues. The relative levels of miR-224 expression in gastric tissues were determined by real-time RT-PCR and then compared with the miR-224 methylation status in gastric tissues: N miR-224 Un (non-cancerous without miR-224 methylation; n = 20), Ca miR-224 Un (GC tissues without miR-224 methylation; n = 10), and Ca miR-224 Mt (GC tissues with miR-224 methylation; n = 15). The two-sample t-test was performed to examine the difference between two groups. (D) Comparison of the miR-224 methylation status and DPYSL2 mRNA levels in GC tissues. The relative levels of DPYSL2 expression in gastric tissues were determined by real-time RT-PCR, normalized as to GAPDH, and then compared with the miR-224 methylation status in gastric tissues: N miR-224 Un (non-cancerous without miR-224 methylation, n = 6), Ca miR-224 Un (GC tissues without miR-224 methylation, n = 8), and Ca miR-224 Mt (GC tissue with miR-224 methylation, n = 7). The boxes indicate the 25th and 75th percentiles, the horizontal lines inside the boxes indicate the medians and the bars indicate the 10th and 90th percentiles. NS, not significant.

Fig 3: Effects of CRMP2 overexpression on axonal degeneration after axotomy of cortical neurons in vitro.(a) The microfluidic chamber system consists of 4 wells and two channels. The two channels are connected by microgrooves. Rat cortical neurons are plated in the channel named ‘soma compartment’. Only axons can grow across the microgrooves to enter the other channel called ‘axonal compartment’. (b) Quantification of newly formed bulbs in the axons in the given areas proximal to the lesion site at the indicated time points after axotomy after transfection of p.CMV-EGFP. Error bars represent the standard error of the mean (SEM). N = 19 axons. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s test. (c) Representative Western blot with anti-flag antibody in cortical neurons after transfection with p.CMV-EGFP or p.CMV-CRMP2-flag confirming the overexpression of the CRMP2-flag transcript from the plasmid. (d) Immunostaining with anti-flag antibody confirming co-transfection of p.CMV-CRMP2-flag and p.CMV-EGFP in the cortical neurons. (e) Representative axons proximal to the lesion site at 480 min after axotomy and previous transfection with p.CMV-EGFP as control or co-transfection with p.CMV-CRMP2-flag and p.CMV-EGFP. The number of bulbs was decreased in the axon overexpressing CRMP2 compared to control. (f) Quantification of the number of newly formed axonal bulbs within 400 µm proximal to the lesion site at the indicated time points after axotomy and previous transfection with p.CMV-EGFP or both p.CMV-CRMP2-flag and p.CMV-EGFP. N = 19 axons per group. Error bars represent the standard error of the mean (SEM). *P < 0.05, **P < 0.01 by independent samples t-test.

Fig 4: Live imaging of mitochondrial transport after axotomy in cortical neurons in vitro.(a) Mitochondria labeled with AAV.mito-RFP (arrows) are visualized along the axon. (b) Representative kymographs of mitochondrial movements along the axons within 100 µm proximal to the lesion site before and after axotomy during a recording time of 37 s (y-axis). (c,d) Quantification of mitochondrial movements in the axon segment 100 µm proximal to the lesion site before and after axotomy. 18–20 axons from >3 independent experiments are included. Error bars represent the standard error of the mean (SEM). ***P < 0.001 by one-way ANOVA and Dunnett’s test. (e) Representative kymographs of mitochondrial movements within 100 µm proximal to the lesion site before and after axotomy after transduction with AAV.mcherry or AAV.CRMP2 during a recording time of 37 s (y-axis). (f,g) Quantification of mitochondrial movements in the axon 100 µm proximal to the lesion site before and after axotomy after transduction of AAV.mcherry or AAV.CRMP2. N = 14 axons per group. Error bars represent the standard error of the mean (SEM). **P < 0.01 by independent samples t-test.

Fig 5: Protein expression levels of Nefm, Lasp1, Dpysl2 and Dlg4 in the medial prefrontal cortex of MIA-induced mice. (A) Protein expression levels were determined by western blot analysis; Actb was used as an internal control. Nefm, Lasp1, Dpysl2 and Dlg4 expressions were decreased in the MIA group compared with CON, and treatment with PG inhibited this decrease. (B-E) Quantitative analysis of (B) Dpysl2, (C) Lasp1, (D) Nefm and (E) Dlg4 expression. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. CON; #P<0.05 vs. MIA. Actb, ß-actin; CON, control group; Dlg4, discs large MAGUK scaffold protein 4; Dpysl2, dihydropyrimidinase like 2; Lasp1, LIM and SH3 protein; MIA, maternal induced activation group; Nefm, neurofilament M; PG, Panax ginseng-treated MIA group.