Journal: Scientific Reports

Article Title: Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca 2+ -dependent mechanism

doi: 10.1038/s41598-019-56675-6

Figure Lengend Snippet: Gene regulation of neferine-mediated autophagy induction. ( A ) RT 2 profiler autophagy PCR array analysis of neferine. HeLa cells were with treated with 10 μM of neferine for 24 h. The total RNA was extracted and reverse-transcripted as cDNA. Real-time PCR reactions were performed using the RT2 SYBR® Green qPCR Mastermix and data analysis was determined using the Qiagen’s integrated web-based software package for the PCR Array System. Scatter plot highlighted the up-regulation and down-regulation of genes in response to neferine treatment. Inner panel: quantification of PCR array analysis. ( B ) Neferine-mediated genes regulation was confirmed by western blotting. Upper panel, HeLa cells were treated with neferine (10 μM) for the indicated time. Cell lysates were analyzed with antibodies against CXCR4, P-PERK, PERK, p62, ULK-1 and actin respectively. Lower panel, HeLa cells were treated with neferine (10 μM) for the indicated time and rapamycin (300 nM) for 24 h. Cell lysates were analyzed with antibodies against P-eIF-2α, eIF-2α and actin respectively. The quantification graphs and full-length blots/gels are presented in Supplementary Figs. and ( A ), respectively. ( C ) Activation of PERK and ULK-1 is required for neferine-induced autophagy. HeLa cells were transfected with control si RNA, PERK or ULK-1 si RNA together with EGFP-LC3 plasmid for 48 h, cells were treated with neferine (10 μM) for 4 h and then fixed for fluorescence imaging and cells counting. Western blot images indicate the gene knock down efficiency. Bar chart represents the quantitation of autophagic cells. The full-length blots/gels are presented in Supplementary Fig. ( B ). ( D ) Effect of CXCR4 in Nef-induced autophagy. EGFP-LC3 transfected HeLa cells were treated with 10 μM neferine in the presence or absence of CXCR4 specific inhibitor, AMD 3100 (25 mg/mL) for 4 h. The cells were then fixed for fluorescence imaging and cells counting. Bar chart represents the quantitation of autophagic cells. Western blot image indicates the LC3-II conversion in HeLa cells in response to neferine and AMD 3100 treatment. The full-length blots/gels are presented in Supplementary Fig. ( C ). Percentages of autophagic cells demonstrated by the increased number of cells with EGFP-LC3 dots signal (≥10 dots/cell) over the total number of EGFP-positive cells in the same field. More than 1000 EGFP-positive cells were scored for each treatment. Error bars, S.D. **P < 0.01, ***P < 0.001 for neferine-treated HeLa cells with or without PERK/ULK-1 si RNA knockdown.

Article Snippet: Real-time PCR reactions were performed using the RT2 SYBR® Green qPCR Mastermix and data analysis was determined using the Qiagen’s integrated web-based software package for the PCR Array System.

Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Software, Western Blot, Activation Assay, Transfection, Control, Plasmid Preparation, Fluorescence, Imaging, Knockdown, Quantitation Assay