Figure 5.
Deletion of GADD45A prevents RSL3-induced ferroptosis and DNA damage through upregulation of FTH1. (A) Flow cytometry dot plots showing the percentage of nonapoptotic or ferroptotic cell death (annexin V negative, SYTOX blue positive; Q3), following treatment of Gadd45a−/− vs Gadd45a+/+ AML LSCs with dimethyl sulfoxide (DMSO) or 3 μM RSL3 for 4 days in methylcellulose. (B) Percentages of viable cells measured by trypan blue exclusion assay in Gadd45a−/− vs Gadd45a+/+ LSCs, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose (n = 3). Data are given as mean ± SD. ∗∗P < .005; NS, not significant (P > .05). Unpaired t-test. (C) In vivo BrdU proliferation assay with dot plots showing the percentage of BrdU+DAPI− leukemic cells engrafted in the mouse bone marrow (BM) at 21 days posttransplantation. Gadd45a−/− vs Gadd45a+/+ LSCs were pretreated ex vivo with DMSO or 3 μM RSL3 for 4 days in methylcellulose, and 1 × 105 GFP+ treated cells were then transplanted into C57BL/6 (BL6) mice for the engraftment of GFP+ LSCs and subsequent in vivo BrdU cell proliferation assay. Data are presented as the mean percentage relative to DMSO ± SD. ∗∗∗P < .0005; NS, not significant (P > .05). Unpaired t-test. (D) Quantitative polymerase chain reaction (qPCR) (n = 3) showing upregulation of Gadd45a and downregulation of Fth1 induced by RSL3 treatment in Gadd45a+/+ LSCs but not in Gadd45a−/− LSCs. Data are given as mean ± SD. ∗∗P < .005; NS, not significant (P > .05), unpaired t-test. (E) qPCR (n = 3) confirming efficient knockdown of Fth1 by shRNA (Fth1-sh) in Gadd45a−/− LSCs and Gadd45a+/+ LSCs. Data are given as mean ± SD. ∗P < .05, ∗∗∗∗P < .0001, unpaired t-test. (F) Flow cytometry histograms illustrating intracellular iron (Fe2+) and ROS levels in Gadd45a−/− vs Gadd45a+/+ LSCs carrying Scr or Fth1-sh, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose. (G) Percentages of viable cells measured by trypan blue exclusion assay in Gadd45a−/− vs Gadd45a+/+ LSCs carrying Scr or Fth1-sh, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose (n = 3). Data are given as mean ± SD. ∗∗P < .005, ∗∗∗∗P < .0001; NS, not significant (P > .05). One-way ANOVA. (H) Western blots showing increased expression of γH2AX induced by RSL3 treatment in Gadd45a+/+ LSCs but not in Gadd45a−/− LSCs. (I) Alkaline comet assay used to quantify the level of DNA-strand breaks illustrating heightened DNA damage (tail moment) in Gadd45a+/+ LSCs treated in methylcellulose with 3 μM RSL3 compared with DMSO. Scatter plots with a bar graph depicting tail moments (a combined measure of tail length and the amount of migrated DNA) calculated using CometScore. Representative fluorescence images showing DAPI-stained single cells after electrophoresis (×20 magnification). During electrophoresis, damaged DNA migrated out of the nucleus toward the anode, forming a comet tail, whereas undamaged DNA remained in the comet head. The comet tail moment represents the extent of DNA damage in individual cells.

Deletion of GADD45A prevents RSL3-induced ferroptosis and DNA damage through upregulation of FTH1. (A) Flow cytometry dot plots showing the percentage of nonapoptotic or ferroptotic cell death (annexin V negative, SYTOX blue positive; Q3), following treatment of Gadd45a−/− vs Gadd45a+/+ AML LSCs with dimethyl sulfoxide (DMSO) or 3 μM RSL3 for 4 days in methylcellulose. (B) Percentages of viable cells measured by trypan blue exclusion assay in Gadd45a−/− vs Gadd45a+/+ LSCs, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose (n = 3). Data are given as mean ± SD. ∗∗P < .005; NS, not significant (P > .05). Unpaired t-test. (C) In vivo BrdU proliferation assay with dot plots showing the percentage of BrdU+DAPI− leukemic cells engrafted in the mouse bone marrow (BM) at 21 days posttransplantation. Gadd45a−/− vs Gadd45a+/+ LSCs were pretreated ex vivo with DMSO or 3 μM RSL3 for 4 days in methylcellulose, and 1 × 105 GFP+ treated cells were then transplanted into C57BL/6 (BL6) mice for the engraftment of GFP+ LSCs and subsequent in vivo BrdU cell proliferation assay. Data are presented as the mean percentage relative to DMSO ± SD. ∗∗∗P < .0005; NS, not significant (P > .05). Unpaired t-test. (D) Quantitative polymerase chain reaction (qPCR) (n = 3) showing upregulation of Gadd45a and downregulation of Fth1 induced by RSL3 treatment in Gadd45a+/+ LSCs but not in Gadd45a−/− LSCs. Data are given as mean ± SD. ∗∗P < .005; NS, not significant (P > .05), unpaired t-test. (E) qPCR (n = 3) confirming efficient knockdown of Fth1 by shRNA (Fth1-sh) in Gadd45a−/− LSCs and Gadd45a+/+ LSCs. Data are given as mean ± SD. ∗P < .05, ∗∗∗∗P < .0001, unpaired t-test. (F) Flow cytometry histograms illustrating intracellular iron (Fe2+) and ROS levels in Gadd45a−/− vs Gadd45a+/+ LSCs carrying Scr or Fth1-sh, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose. (G) Percentages of viable cells measured by trypan blue exclusion assay in Gadd45a−/− vs Gadd45a+/+ LSCs carrying Scr or Fth1-sh, following treatment with DMSO or 3 μM RSL3 for 4 days in methylcellulose (n = 3). Data are given as mean ± SD. ∗∗P < .005, ∗∗∗∗P < .0001; NS, not significant (P > .05). One-way ANOVA. (H) Western blots showing increased expression of γH2AX induced by RSL3 treatment in Gadd45a+/+ LSCs but not in Gadd45a−/− LSCs. (I) Alkaline comet assay used to quantify the level of DNA-strand breaks illustrating heightened DNA damage (tail moment) in Gadd45a+/+ LSCs treated in methylcellulose with 3 μM RSL3 compared with DMSO. Scatter plots with a bar graph depicting tail moments (a combined measure of tail length and the amount of migrated DNA) calculated using CometScore. Representative fluorescence images showing DAPI-stained single cells after electrophoresis (×20 magnification). During electrophoresis, damaged DNA migrated out of the nucleus toward the anode, forming a comet tail, whereas undamaged DNA remained in the comet head. The comet tail moment represents the extent of DNA damage in individual cells.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal