cc2650 simplelinktm multistandard wireless mcu  (Texas Instruments)

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

Article Snippet: All chemicals, unless otherwise stated, were purchased from Sigma–Aldrich with respective CAS number: thapsigargin (67526-95-8), tunicamycin (11089-65-9), 5-bromo-2′-deoxyuridine (BrdU) (59-14-3), 5-ethynyl-2′-deoxyuridine (EdU) (61135-33-9), EdU Click-it ® Thermo Fisher ( C10337 ), MCU-i4 (HY-138620, MedChemExpress), MnCl 2 (13446-34-9), cyclohexamide (66-81-9), actinonin (13434-13-4), HPG Click-it ® Thermo Fisher ( C10186 ).

Techniques: Inhibition, Disruption, Control, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

Article Snippet: All chemicals, unless otherwise stated, were purchased from Sigma–Aldrich with respective CAS number: thapsigargin (67526-95-8), tunicamycin (11089-65-9), 5-bromo-2′-deoxyuridine (BrdU) (59-14-3), 5-ethynyl-2′-deoxyuridine (EdU) (61135-33-9), EdU Click-it ® Thermo Fisher ( C10337 ), MCU-i4 (HY-138620, MedChemExpress), MnCl 2 (13446-34-9), cyclohexamide (66-81-9), actinonin (13434-13-4), HPG Click-it ® Thermo Fisher ( C10186 ).

Techniques: Inhibition, Cell Culture