Fig 2: Neurotoxicity of primary cultured SN dopaminergic neurons caused by miR-204-5p is mediated by the increased level of DYRK1A protein. (A) Cotransfection of shRNAs of DYRK1A or treatment of DYRK1A inhibitor harmine (1µM) attenuated miR-204-5p mimic-induced upregulation of a-Syn, phospho-a-Syn, tau, or phospho-tau in primary cultured dopaminergic neurons. (B) In the presence of DYRK1A shRNAs and DYRK1A inhibitor harmine, miR-204-5p mimic failed to upregulate the expression of ER stress-related proteins, including Grp78, IRE1a, CHOP, and active caspase-12, in primary cultured dopaminergic neurons. (C) The miR-204-5p mimic-induced decrease in protein expression of autophagy markers, including Beclin-1, Atg7, Atg16L1, and LC3-II/I ratio, in primary cultured dopaminergic neurons was prevented by cotransfection of shRNA of DYRK1A or treatment of DYRK1A inhibitor harmine. (D) Knockdown of DYRK1A expression or treatment of harmine in primary cultured dopaminergic neurons attenuated miR-204-5p mimic-induced activation of JNK, c-Jun, caspase-9, and caspase-3 in primary cultured SN dopaminergic neurons. (E) In the presence of DYRK1A shRNA or DYRK1A inhibitor harmine, the miR-204-5p mimic failed to significantly upregulate the mRNA level of Bim in primary cultured dopaminergic neurons. Each qRT-PCR experiment was performed in triplicate. (F) Cotransfection of DYRK1A shRNA or treatment of DYRK1A inhibitor harmine reversed miR-204-5p mimic-induced cell death of primary cultured dopaminergic neurons. For the assays of cell viability, each experiment was performed in triplicate. Each bar indicates the mean ± SEM value of four independent experiments. *p < 0.05, **p < 0.01 compared to scramble miR control (SC)-transfected primary cultured dopaminergic neurons.

Fig 3: a-Mangostin increases intracellular ROS level in 143B and Saos-2 cells. (A) 143B and Saos-2 cells were treated with different concentrations of a-mangostin (0, 10, 20 and 30 µM) for 24 h, and the ROS production was detected by DCF-DA staining. 143B and Saos-2 cells were pre-treated by NAC (2 mM) for 2 h, then co-treated with a-mangostin (30 µM) for 24 h. (B) The ROS production was measured by DCF-DA staining. (C) Cell apoptosis was analyzed with flow cytometry. (D, E) The protein levels of cleaved-caspase-3/8, CHOP and ATF6 were determined. (F, G) The expression of Wnt3a, p-Gsk3ß and nuclear ß-catenin was analyzed. N = 5, *P < 0.05, **P < 0.01.

Fig 4: Effect of 17ß-HSD12 knockdown on CHOP and ERp44 expression. a The protein levels of CHOP were assessed by western blot 48 h after 17ß-HSD12 silencing in SUM159 cells. mRNA expression of the CHOP target GADD34 in MDA-MB-231 and SUM159 cells 48 h following 17ß-HSD12 downregulation (mean ± SD, n = 4, *p < 0.05, **p < 0.01). b Expression of RXRa protein 48 h after 17ß-HSD12 knockdown in MDA-MB-231 and SUM159 cells (mean ± SD, n = 4, *p < 0.05). c SUM159 cells were transfected with mock-, RXRa-, 17ß-HSD12- or RXRa- and 17ß-HSD12-siRNA, and CHOP or 17ß-HSD12 mRNA expression was assessed by qPCR (mean ± SD, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant). d The levels of the folding protein ERp44 were analyzed by western blot following 17ß-HSD12 knockdown in MDA-MB-231 and SUM159 cells (mean ± SD, n = 5, **p < 0.01, ***p < 0.001). e Summary of the most prominent cellular changes observed in MDA-MB-231 and SUM159 cells after 17ß-HSD12 knockdown. (Left panel) In SUM159 cells, 17ß-HSD12 downregulation leads to decreased proliferation and migration, which can be restored by EPA supplementation. Aberration in synthesis of other FAs could additionally lead to the observed phenotype. In MDA-MB-231 cells, knockdown of 17ß-HSD12 leads to increased AA metabolism through the COX and CYPB1 pathways that promote proliferation and migration. The augmented proliferation after 17ß-HSD12 knockdown is supported by energy and/or building blocks provided by ß-oxidation. (Right panel) Comparison of the alterations in UPR/folding and energy metabolism in SUM159 and MDA-MB-231 cells following 17ß-HSD12 silencing

Fig 5: CNOT2 knockdown increases ER stress and partially induces apoptotic signals. A549 and H1299 cells were stably transfected with plasmid containing shRNA against CNOT2 or control. Cell lysates were analyzed according to the expression of protein to ER stress-related molecules, such as GRP78, CHOP, DR4 and DR5 by western blot analysis. CNOT2, CCR4-NOT transcription complex subunit 2; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; DR4, death receptor 4; DR5, death receptor 5; CHOP, C/EBP homologous protein.