Fig 2: BI 2536 induced cell apoptosis in neuroblastoma cells. (A) Apoptosis analysis by Annexin V/PI staining in NB cells treated with BI 2536 for 24h. SH-SY5Y cells were harvested after 24h of incubation with 5 or 10nM BI 2536. SK-N-BE(2) cells were treated with BI 2536 at 10 or 50nM. (B) Proportion of apoptotic cells increased significantly in both SH-SY5Y and SK-N-BE(2) cells after treated with BI 2536 at indicated concentrations compared with DMSO control group. (C) Western blot analysis of PLK1, cleavage of PARP and Caspase-3 in lysates from SH-SY5Y and SK-N-BE(2) cells treated with serial concentrations of BI 2536 for 24h. * presents the cleaved band of PARP. *P<0.05, **P < 0.01 and ***P < 0.001. P values were determined by two-tailed t tests. All data are representative of three independent experiments with n = 3-6 per group and are means ± s.e.m.

Fig 3: Role of oxidative stress in PA-induced endoplasmic reticulum stress and apoptosis in podocytes. (A and C) Representative immunofluorescence images of intracellular ROS in podocytes (magnification, ×200) following treatment with different concentrations of PA, or PA (150 µmol/l) for 24 h after pretreatment with or without NAC (150 µmol/l) for 2 h. (B) Mean fluorescence intensities of ROS shown in (A). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. PA (0 µmol/l) group, #P<0.05 vs. PA (50 µmol/l) group, ?P<0.05 vs. PA (150 µmol/l) group. (D) Mean fluorescence intensities of ROS shown in (C). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ?P<0.05 vs. NAC group. (E and M) Representative images of BIP, PERK, ATF4, CHOP, ATF6, IRE1a, caspase 12 and cleaved-caspase 3 expression, as detected by western blotting after treatment with PA (150 µmol/l) or NAC (150 µmol/l). ß-actin was used as a loading control. Densitometric analysis of the data shown in (E and M) for (F) BIP, (G) PERK, (H) ATF4, (I) CHOP, (J) ATF6, (K) IRE1a, (L) caspase 12 and (N) cleaved-caspase 3. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ?P<0.05 vs. NAC group. (O) Representative plots of podocyte apoptosis evaluated by flow cytometry after treatment with PA (150 µmol/l) and NAC (150 µmol/l) for 24 h. (P) Quantification of podocyte apoptosis shown in (O). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ?P<0.05 vs. NAC group. PA, palmitic acid; ROS, reactive oxygen species; NAC, N-acetylcysteine; BIP, binding immunoglobulin protein; PERK, protein kinase RNA-like endoplasmic reticulum kinase; ATF, activating transcription factor; CHOP, C/EBP homologous protein; IRE1a, inositol-requiring enzyme 1a; SEM, standard error of the mean.

Fig 4: BIND inhibits sCAG RNA generation, CAG RNA foci formation, and polyQ protein aggregation in SCA2CAG104-expressing SK-N-MC cells(A) BIND treatment rescued SCA2CAG104-induced cell death in a dose-dependent manner. (B) RT-PCR analysis showed that BIND treatment did not alter the transcript level of SCA2CAG104. (C) The elevated levels of sCAG RNA, p-ATR, p-CHK1, p-ATM, p-CHK2, and cleaved caspase 3 proteins were suppressed upon the treatment of BIND in SCA2CAG104-expressing SK-N-MC cells. BIND treatment restored NUDT16 at both mRNA and protein levels. (D) Fluorescence microscopic analysis showed that the abundance of both CAG RNA foci and polyQ protein aggregates were reduced upon the treatment of BIND in a dose-dependent manner. (E) Filter-trap assay showed that BIND reduced the level of SDS-insoluble polyQ protein in SCA2CAG104-expressing SK-N-MC cells. (F) BIND did not co-localize with CAG RNA foci nor polyQ protein aggregates. Scale bars: 10 μm. Error bars represent ± SD. Statistical analysis was performed using one-way ANOVA. NS indicates no significance, ∗∗∗p < 0.001. RNU66, beta-actin, and beta-tubulin were used as loading controls. Experiments were independently repeated for three times. Only representative images, gels, and blots are shown.

Fig 5: IFN-α attenuates anti-tumor activity of M1 virus in vivo subcutaneous Hep-3B tumors(A) Nude mice (NU/NU) bearing subcutaneous Hep-3B tumors were treated with vehicle, ETV (75 μg/kg/day, i.p.), DAC (15 mg/kg/day, i.p.), RBV (15 mg/kg/day, i.p.) IFN-α (35μg/kg/week, s.c.), M1 virus (2.7 × 106 PFU/day, i.v.), M1 virus and anti-hepatitis virus drugs. i.p. intraperitoneal injection, i.v., intravenously injection (tail vein), s.c. subcutaneous injection, PFU, plaque forming unit. (B and C) Body weight (B) and Tumor growth (C) of tumor-bearing mice. Data are shown in means ± SDs. N.S. Not significant. *P < 0.05, compared with the combination group. (D) At experimental endpoints, mice were anesthetized and sacrificed. Tumors were subsequently dissected and photographed. (E) Intratumoral expression of Ki-67 and Cleaved-Caspase-3. (F) Immunohistochemistry was performed to analyze the expression of Ki-67 and Cleaved-Caspase-3. Relative protein expressions were quantified with Image-Pro Plus 6.0 (IPP 6.0) N.S., not significant. *P < 0.05.