Journal: Cell death & disease

Article Title: Dual inhibition of thioredoxin reductase and proteasome is required for auranofin-induced paraptosis in breast cancer cells.

doi: 10.1038/s41419-023-05586-6

Figure Lengend Snippet: Fig. 6 AF-mediated TrxR1 inhibition enhances Bz-induced ER stress, and ATF4 plays a critical role in the paraptosis induced by TrxR1/ proteasome inhibition. A MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with 5 nM Bz for 12 h, or with the indicated concentrations of Bz and/or AF for 12 h. B, D MDA-MB 435 S cells transfected with siNC, ATF4-targeting siRNA (siATF4), or CHOP-targeting siRNA (siCHOP) were transfected with TrxR1 and further treated with 5 nM Bz for 24 h, or treated with the indicated concentrations of Bz and/ or AF for 24 h. A, B Western blotting of the indicated proteins was performed using β-actin as a loading control. C Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. D Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.

Article Snippet: ATF4 (CREB-2) targeted siRNA was from Santa Cruz: siATF4 (target sequence CCACUCCAGAUCAUUCCUU, GGAUAUCACUGAAGGAGAU, and GUGAGAAACUGGAUAAGAA, sc-35112).

Techniques: Inhibition, Transfection, Western Blot, Control, Microscopy

Journal: Cell death & disease

Article Title: Dual inhibition of thioredoxin reductase and proteasome is required for auranofin-induced paraptosis in breast cancer cells.

doi: 10.1038/s41419-023-05586-6

Figure Lengend Snippet: Fig. 7 ATF4/CHAC1 critically contributes to the paraptosis induced by TrxR1/proteasome inhibition by degrading GSH. A MDA-MB 435 S cells were treated with the indicated concentrations of AF alone (left) or Bz and/or AF (right) for 12 h. MDA-MB 435 S cells transfected with siNC or siTrxR1 were treated with 5 nM Bz for 12 h (middle). MDA-MB 435 S cells transfected with siNC, siATF4, or CHAC1-targeting siRNA (siCHAC1) were cotransfected with TrxR1 and further treated with 5 nM Bz for 24 h (B–E) or 12 h (F), or treated with the indicated concentrations of Bz and/or AF for 24 h (B–E) or 12 h (F). G, H MDA-MB 435 S cells pretreated with 2 μM CHX or 2 mM NAC were treated with 2 μM AF plus 5 nM Bz for 12 h. I Cells were treated with 5 nM Bz and/or 2 μM AF for 12 h. A, C, H, I Western blotting of the indicated proteins was performed using β-actin as a loading control. B The mRNA levels of ATF4 or CHAC1 were assessed by qRT-PCR. D Cellular viability was assessed using IncuCyte, as described in the Materials and Methods. The percentage of live cells was normalized to that of untreated cells (100%). Data represent the means ± SD. (n = 9). One way-ANOVA and Bonferroni’s post hoc test. *p < 0.05. E Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. F, G GSH levels were assessed, as described in Materials and Methods.

Article Snippet: ATF4 (CREB-2) targeted siRNA was from Santa Cruz: siATF4 (target sequence CCACUCCAGAUCAUUCCUU, GGAUAUCACUGAAGGAGAU, and GUGAGAAACUGGAUAAGAA, sc-35112).

Techniques: Inhibition, Transfection, Western Blot, Control, Quantitative RT-PCR, Microscopy

Journal: Nature Communications

Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

doi: 10.1038/s41467-018-03475-7

Figure Lengend Snippet: HDR stimulation by the HE domain takes place at different target genes and can depend on the guide RNA used. a Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 at five different target genes in HEK293 cells using previously published guide RNAs and donor plasmids . Targeted integration of the donor plasmid results in in-frame insertion of E2A - neoR cDNA . G418(neomycin)-resistant colonies were counted after Cresyl violet staining to measure HDR-mediated events and normalized by the number of colonies obtained with Cas9 to give the relative HDR frequencies indicated. Data represented are from three independent experiments for TGIF2 , RAD21 , and CREB genes and from four experiments for ATF4 and GABP genes and are provided in Supplementary Fig. . Error bars indicate standard deviation. b Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 with the indicated guide RNAs, which all cleave to a small 50-bp region of the AAVS1 locus, and a common p84∆ donor plasmid, harboring ~800-bp homology arms. P84∆ was derived from the p84 donor plasmid depicted in Fig. 1a by shortening the homology arms so that they would not be cleaved by any of the guide RNAs. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE to Cas9 in t -test (* P <0.05). Data represented are from five independent experiments and are provided in Supplementary Fig. . Error bars indicate standard deviation

Article Snippet: Guide RNAs and donor plasmids targeting the human ATF4 , GABP , TGIF2 , RAD21 , and CREB genes were from the Mendenhall lab (Addgene #72350, #72351, #64253, and #64254).

Techniques: Plasmid Preparation, Staining, Standard Deviation, Derivative Assay