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

Journal: Cell Death & Disease

Article Title: Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNF α )-induced apoptosis through SIRT1 inhibition

doi: 10.1038/cddis.2012.10

Figure Lengend Snippet: CK2 inhibitor affects p53 target genes associated with cell cycle regulation and apoptosis. ( a ) CK2-I increases p21 expression in a p53-dependent manner. Representative blot is shown from three independent experiments with identical results. Blots were re-probed with c23 to establish equivalent loading. ( b ) CK2-I increases mRNA levels of pro-apoptotic molecules Noxa and GADD45 β in a p53-dependent manner. Total RNA was isolated from cells treated with different combinations of TNF α and CK2-I, and the mRNA levels for Noxa, GADD45 β and constitutive enzyme GAPDH were determined by RT-PCR. ( c ) Pifithrin- α reverses CK2-I-mediated G2/M phase arrest in A172 cells. FACS analysis was performed on A172 cells treated with different combinations of TNF α , CK2-Is and Pifithrin- α . Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, Pf and A denote control, TNF α , Pifithrin- α and Apigenin, respectively

Article Snippet: Results were analyzed as per user manual guidelines using integrated web-based software package for the PCR Array System (SuperArray Biosciences RT2 Profiler PCR Array Human Apoptosis PAHS-012–A-12).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Control