Fig 2: Optimization of viral transduction conditions for human SW579 cells: (a) the purified and concentrated PLJM1-GFP lentivirus was measured as virus copy number by Q-PCR analysis. SW579 cells were seeded in a 6 cm dish and infected with 1000-, 3000-, 9000-, 15,000-, 30,000-, and 90,000-fold concentrations of virus to SW579 cell number for three days. The GFP-positive (infected) cell population was assessed using flow cytometry. (b) The image shows the linear curve comparison of virus input and the GFP-positive cell population. (c) The image shows the western blot analysis of EGFR protein expression on SC sgRNA control SW579 cells; EGFR levels were significantly reduced in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The downstream signaling proteins such as phosphor-AKT and phosphor-ERK were also analyzed in EGFR sgRNA_1- and EGFR sgRNA_2-transduced SW579 cells. The tumor-suppressive proteins such as P53 and P21 were found to be induced in EGFR gene-edited cells. GAPDH served as internal control. (d) TIDE algorithm analysis of the EGFR gene-edited sequence (indels, insertions, and deletions) showed a high editing efficiency in SW579 cells. The pie charts show the percentages of indels in the EGFR gene edited by EGFR sgRNA_2. The gene-editing efficiency of the two sgRNAs is presented in green, while the two most common -1 and other mutations are presented in purple and pink, respectively. (e) The panels illustrate the aberrant sequence signal in the scrambled (green versus black). (f) The EGFR gene in SW579 cells was analyzed with the RGEN-RFLP assay to measure the gene-editing efficiency. The agarose image of EGFR gene cleavage with specific EGFR sgRNA_2 and Cas9 addition represents the indel percentage in the gene-editing pool. The fragments of cleavage DNA were highlighted with an asterisk. (g) The CRISPR Design website was used to predict off-target candidate genes for both the EGFR sgRNA_1 and EGFR sgRNA_2 viruses. To note, no potential off-target candidate gene was predicted for EGFR sgRNA_1 (mismatch < 4). Similarities are presented as dots, and mismatch sites are indicated by nucleotide substitution. (h) Sanger sequencing of SW579 cells infected with SC and EGFR sgRNA_2 virus was used to examine potential indels in off-target candidate genes.

Fig 3: EA reduced MPP+-induced dopaminergic neuronal apoptosis by increasing BDNF (brain-derived neurotrophic factor) expression and further Akt phosphorylation in the rat substantia nigra. (A) Eight days after MPP+ administration, our Western blot results show that MPP+ treatment reduced tyrosine hydroxylase and Bcl-2 expression in the ipsilateral side of the rat substantia nigra (SN), but not in the contralateral side. EA stimulation (50 Hz) enhanced mature BDNF, tyrosine hydroxylase, and Bcl-2 expression in the MPP+-treated ipsilateral side; (B) EA enhanced the phosphorylation of Akt (phospho-Akt) but had no effect on Erk1/2 phosphorylation (phospho-Erk1/2) in the MPP+-treated ipsilateral side (Figure 4B). The data suggest that the way in which EA exerts dopaminergic neuroprotection in the unilateral MPP+-lesion model is by activating Akt phosphorylation and enhancing BDNF and Bcl-2 expression in the rat SN. * p < 0.05 compared with contralateral side of EA+MPP+ or ipsilateral side of EA+MPP+ group.

Fig 4: Celastrol activates autophagy and inhibits MPP+-induced neurotoxicity in dopaminergic neurons. The dopaminergic cell line SH-SY5Y was treated with celastrol (0.1–3 µM) for 2 to 24 h. (A) MTT assay results show that celastrol did not affect cell viability in 24 h. (B) Western blot data show that celastrol treatment for 4 h dose-dependently increased autophagosome formation (LC3-I? and LC3-II?) and enhanced autophagic flux/substrate degradation (p62?) as compared to control. (C) Celastrol dose-dependently enhanced p38, ERK1/2, Akt1/2/3, NF?Bp65, and JNK1/2/3 phosphorylation 2 h after treatment. (D) 1-methyl-4-phenylpyridinium (MPP+; 1 mM) but not celastrol treatment for 24 h caused about 50% neuronal apoptosis induced by mitochondrial dysfunction. Celastrol cotreatment dose-dependently suppressed MPP+-induced neurotoxicity as compared to MPP+. (E) Pretreatment with the autophagy inhibitor Ba (100 nM bafilomycin A1) for 30 min before MPP+ treatment for 24 h enhanced MPP+-induced neuronal death but pretreatment with the autophagy activator Ra (200 nM rapamycin) reversed it. Pretreatment of cells with autophagy inhibitors Ba or Wo (20 nM wortmannin) antagonized the neuroprotection of celastrol against MPP+ as compared to MPP++celastrol; pretreatment with the autophagy activator Ra reversed it. Celastrol, Ba, Wo, and Ra did not affect cell viability as compared to control. (F) MPP+ but not celastrol induced mitochondrial depolarization after treatment for 24 h. Celastrol (1 µM) cotreatment with MPP+ suppressed mitochondrial depolarization as compared to MPP+. (G) MPP+ but not celastrol treatment decreased the ATP production after treatment for 24 h. Celastrol (1 µM) cotreatment dose-dependently reversed the ATP production as compared to MPP+. Data are given as the mean ± SEM (n = 3–8). p-value was determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test. * p < 0.05 compared to control.

Fig 5: Contributions of ASK1 and AKT in gold(III)-induced apoptotic signaling. A2780 cells were treated for 24 h with Au2phen, Auoxo6, and AuL12 72 h IC50 doses. The phosphorylation levels and total protein amounts of (A) ASK1 and (B) AKT were analyzed through Western blotting. Histograms report the percentages of normalized mean values ± SD of the phospho-ASK1/total ASK1 ratio and phospho-AKT/total AKT ratio compared to controls. The phospho-ASK1/total ASK1 and phospho-AKT/total AKT ratios were obtained from the normalized relative integrated density values of immunostained bands of three independent Western blots. Representative immunoblots are also shown together with the matching Coomassie-stained PVDF membranes, which were used as loading control. Original Western blot images of ASK1 and AKT phosphorylation level and of their total amount are reported in Figures S6 and S7. The statistical analysis was carried out using one-way ANOVA test followed by Tukey’s multiple comparisons test using GraphPad Prism software v 6.0 (* p < 0.05, ** p < 0.01).