Figure 1.
Twist proteins lower intracellular ROS levels.
(A–C) HDF, (D–F) MEF and (G–I) REF52 cells were transduced with retroviruses encoding either Twist1 (T–1) or Twist2 (T–2) tagged with a Myc epitope. The empty vector (pMSCV) was used as a control. (A, D and G) Expression of the transgenes was verified by western-blotting (CT:pMSCV; T-1:Twist1, T-2:Twist2). (B, E and H) ROS levels were analyzed by flow cytometry after DHE labeling. (C, F, and I) Quantitative analysis of ROS levels in HDF (C), MEF (F) and REF52 (I) cells with data representing the mean values ± SEM of the fluorescence geometric means (HDF: n = 10; MEF: n = 8; REF: n = 3, *: compared to control).
Figure 2.
Twist2 inhibition increases oxidative stress.
(A) HDF cells were transfected with a siRNA control or a siRNA directed against Twist2 and the silencing efficiency was measured by RT-qPCR normalized to GAPDH. (B) The level of ROS in control and Twist2-inhibited HDF cells was monitored by labeling with DHE and a representative FACS histogram is shown. (C) ROS levels are presented as mean geometric fluorescence intensities (± SEM) in cells transfected with a siRNA against Twist2 relative to a control siRNA (*: n = 6, relative to control). (D) HDF were infected with a retroviral vector (pRetro-SUPER-puromycin) containing a shRNA sequence directed against Twist2 or an irrelevant sequence as a control, selected and analyzed 3 to 5 days post-selection. Knockdown efficiency was measured by RT-qPCR relative to GAPDH. (E) DHE fluorescence was analyzed as in panel B. (F) Quantitative analysis of ROS was determined as in C (*:n = 6, relative to control).
Figure 3.
Twist inhibits c-Myc-induced ROS accumulation and apoptosis.
(A) REF52 cells expressing a MycER construct were cultured for 24 hours in 0.1% serum (dark bars) in the presence or absence of 4-hydroxytamoxifen (OHT), as indicated, to induce c-Myc activity. Control cells were cultured in 10% serum (white bars). Cells were collected, labelled with Cy3-Annexin-V and apoptosis was quantified by flow cytometry. Error bars represent mean ± SEM of three independent experiments. (B) REF52-MycER cells were treated for 8 hours as in A and DHE fluorescence was measured as in Figure 1B. (C) Represents the quantitative analysis of B (*: n = 7, compared to control). (D) REF52-MycER cells were cultured in the presence of 10% FBS and absence of OHT (white bars) or in 0.1% serum with OHT (dark bars) in the presence or absence of 2 mM Tiron, as indicated. Apoptosis was quantified as in A. (E) Cells were cultured as in D and ROS levels were monitored as in B. (F) Quantification of E (*: n = 6, compared to black bar. #: n = 6, compared to green bar). (G) Quantification of apoptosis in REF52-MycER cells expressing Twist1 or Twist2. Black bars: cells are grown under pro-apoptotic conditions (active MycER, 0.1% FBS), white bars: control cultures. Cell death was quantified by Cy3-Annexin-V staining as in A. (H) Cells expressing Twist1 or Twist2 were treated as in G and ROS levels were determined as in D. (I), quantification of ROS levels from H (*: n = 6, compared to black bar. #: n = 6, compared to blue bar).
Figure 4.
Twist antioxidant activity is associated with modulation of apoptosis in primary mouse embryonic fibroblasts.
(A) MEF cells expressing the MycER construct were cultured in the presence of 0.5% or 10% FBS, with or without OHT, and in the presence or absence of Vitamin C, used as an antioxidant. Apoptosis was quantified by Annexin-V staining and is presented as means ± SEM of three independent experiments. (B) MEF-MycER cells were treated as in A and ROS was measured by DHE staining by flow cytometry. (C) Quantification of B (*: n = 5, compared to black bar. #: n = 5, compared to grey bar). (D) MEF-MycER cells were infected with either Twist1, Twist2 or pMSCV vectors and selected with puromycin. Cells were then cultured for 24 hr. in 10% FBS in the absence of OHT (white bars) or in 0.5% FBS supplemented with OHT (dark bars). Apoptosis was determined as in A. (E) ROS levels in all cell lines was monitored as in B. (F) Quantitative analysis of E (*: n = 6, compared to black bar. #: n = 6, compared to grey bar).
Figure 5.
Molecular mechanisms regulating Twist antioxidant activity.
(A) Microarray results for genes modulated by Twist1 and involved in the regulation of oxidative stress. Data show expression mRNA level of MEF-empty vector and MEF-Twist1. (B) mRNA levels for Cdo1, Mgst3 ApoD, Dhxr24, Aox1 and Osgin1 were assessed in MEF cells infected with empty pMSCV vector, pMSCV-Twist1 or pMSCV-Twist2. RNA levels were monitored by RT-qPCR, normalized with Hprt and adjusted relative to levels in pMSCV-transduced control cells.
Figure 6.
Role of Twist target genes in the control of ROS levels and apoptosis.
(A, B) MEF cells were transduced with retroviruses encoding either Twist1 tagged with a Myc epitope (A), or with Mgst3 tagged with an HA epitope (B). Empty vector was used as a control. Expression of the transgenes was verified by western-blotting (1: Empty vector; 2: Transgene). (C) MEF cells were transfected with a siRNA control or a siRNA directed against Osgin1 and the silencing efficiency was measured by RT-qPCR normalized to HPRT. (D) Quantitative analysis of ROS levels of cell lines from A, B and C (*: n = 6, compared to control). (E) MEF-MycER cells were infected with either control (pMSCV), Twist1, or Mgst3 or were transfected with corresponding siRNA as in C (n = 3). Apoptosis was induced and determined as in Figure 4. (F) Alternative presentation of data from D and E, allowing better visualization of partial effects on ROS (*: n = 6, compared to red bar) and apoptosis (*: n = 3, compared to red bar) exercised by Mgst3 and Osgin-1. These graphs show the percentage of inhibition compared to control (pMSCV of si-CT).