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