Flexitube sirna

Transfections were carried out using Lipofectamine 2000 and were performed essentially as described [44 (link)]. The following siRNAs were used (all from Qiagen): AllStars Negative Control siRNA (siNC), Hs_MYCN_4 FlexiTube siRNA (siMYCN_1), Hs_MYCN_6 FlexiTube siRNA (siMYCN_2), Hs_TP53_3 FlexiTube siRNA (siTP53_1), and Hs_TP53_9 FlexiTube siRNA (siTP53_2). Final concentrations of the respective siRNAs were 20 nM in all the experiments. The wt-p53 overexpression plasmid was a kind gift from Dr. Ugo Moens, University of Tromsø, Norway, empty vector was pCMV-XL4 (Origene). Final DNA plasmid concentration was 1 μg/mL of media.

Mouse Smg-1 and Smg-7 siRNAs have been described previously^{34 (link)}. Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′), Lat1 (target sequence: 5′-AGUGAAAGAGCAAGCCCAA-3′), Lat3 (target sequence: 5′-UGAAAAAGACCAAACUCAU-3′), and Snat2 (target sequence 5′-UGUUAGCGUCGGCAUUCAA-3′) siRNAs were designed using i-Score Designer^{40 (link)} and asymmetric siRNA^{41 (link)} were synthesized (GeneDesign, Inc.). Other synthetic siRNAs were purchased from Qiagen: mouse Abcc4, FlexiTube siRNA SI02833019; mouse Atf4, FlexiTube siRNA SI00905905; mouse Perk, FlexiTube siRNA SI01319269; and the All Star Negative Control siRNA. Synthetic siRNA transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and the cells were analyzed 48−64 h after transfection. The results were confirmed in more than 3 independent experiments.
The plasmid-expressed human eIF2α-S52A mutant was constructed by site-directed mutagenesis of pSR-strep-HA-eIF2α^{38 (link)}. Subsequently, a puromycin resistance gene expression cassette derived from silentGene-puro (Promega) was inserted into wild-type (WT) and S52A mutant eIF2α expression (SA) plasmids to generate pSR-strep-HA- eIF2α_puro and pSR-strep-HA-eIF2α-S52A_puro. The transfections of plasmid siRNAs were carried out using Lipofectamine 3000 (Thermo Fisher Scientific) and stable cell lines were selected by puromycin (Sigma-Aldrich).

Mouse Smg1 and Smg7 siRNAs have been described previously^{5 (link)}. Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer^{49 (link)} and asymmetric siRNA^{50 (link)} was synthesized (GeneDesign, Inc.). Other siRNAs, including mouse Atf4, FlexiTube siRNA (SI02674427), mouse Perk, FlexiTube siRNA (SI01319269), mouse Mtor, FlexiTube siRNA (SI03099796), and All Star Negative Control siRNA were purchased from Qiagen. Transfections of synthetic siRNAs were carried out with Lipofectamine RNAiMAX (Thermo Fisher Scientific) and the cells were analyzed 48 h after transfection. The results were confirmed in more than three independent experiments.

Cells were transiently transfected with siRNA specifically targeting against Cx37 (Mm_Gja4_1 FlexiTube siRNA), Cx40 (Mm_Gja5_1 FlexiTube siRNA), Cx43 (Mm_Gja 1_2 HP siRNA) and Cx45 (Mm_Gja7_2 FlexiTube siRNA, Qiagen, Japan) or a negative control siRNA (AllStars Negative Control siRNA) at a final concentration of 20 nM using Hyperfect transfection reagent for 24 h [22 (link)]. After transfection, cells were left untreated or exposed to Ca²⁺-free medium for the indicated time. Cellular protein was extracted and subjected to western blot analysis of the targeted proteins.