Journal:

Article Title: Upregulation of Twist-1 by NF-?B Blocks Cytotoxicity Induced by Chemotherapeutic Drugs ▿

doi: 10.1128/MCB.01219-06

Figure Lengend Snippet: Twist-1 attenuates TNF-α-induced PCD in NF-κB-deficient cells. (A) The ectopic expression of Twist-1 rescues RelA null fibroblasts from TNF-α-induced PCD. MIGR1- and MIGR1-Twist-1-transduced-RelA−/− cells were seeded onto 60-mm dishes at a density of 0.5 × 106 cells/dish and, 24 h later, treated either with cycloheximide (CHX) alone (0.1 μg/ml) or with CHX (0.1 μg/ml) plus TNF-α (100 U/ml). At the times indicated, cell viability was assessed by determining total cell numbers in both TNF-α-treated and control cultures. For this, adherent cells were recovered by trypsinization followed by centrifugation, resuspended in serum-containing medium, and counted by hemacytometry (36). Fractions of GFP+ cells were assessed by FCM. Values reflect the percentages of live (i.e., adherent) GFP+ cells (determined by combining cell counting and FCM) relative to the numbers observed in cultures treated with CHX alone (left). These percentages represent means ± standard deviations from three independent experiments. Levels of ectopic Flag-Twist were monitored by Western blotting (right). (B) ELISAs of PCD confirming the protective activity of ectopic Twist-1 in RelA null cells. Cell death was monitored in the same MIGR1- and MIGR1-Twist-1 RelA-deficient lines used to obtain the panel A results, following treatment with CHX alone (0.1 μg/ml) or together with a low (100 U/ml) or high (250 U/ml) concentration of TNF-α, as indicated. Treatment times are also shown. Assays were preformed as described previously (33, 36), and cell death is expressed as arbitrary units. Values represent means ± standard deviations from three independent experiments. (C) Survival of representative Hygro- and Twist-1-expressing, 3DO-IκBαM clones (IκBαM-Hygro and IκBαM-Twist-1, respectively) after treatment with TNF-α. For survival assays, cells were plated onto 24-well dishes at 3.5 × 105 cells/well and then exposed to TNF-α (15 U/ml) or left untreated. Viability was assessed at 6 h by performing PI nuclear staining assays. DNA content was determined with FCM analysis and analyzed using the FlowJo software. Percentages of survival reflect fractions of live cells (i.e., of at least G1 DNA content) in the TNF-α-treated cultures relative to the number of live cells in the untreated cultures (top). Clone numbers are shown. Levels of expression of Twist-1, IκBα, and GAPDH mRNAs in each clone were monitored by Northern blotting (bottom).

Article Snippet: Strikingly, however, the expression of Twist-1 in these cells conferred strong protection against TNF-α-induced PCD (Fig. , left; MIGR1-Twist-1 bars).

Techniques: Expressing, Control, Centrifugation, Cell Counting, Western Blot, Activity Assay, Concentration Assay, Clone Assay, Staining, Analysis, Software, Northern Blot

Journal:

Article Title: Upregulation of Twist-1 by NF-?B Blocks Cytotoxicity Induced by Chemotherapeutic Drugs ▿

doi: 10.1128/MCB.01219-06

Figure Lengend Snippet: Twist-1 antagonizes apoptotic signaling induced by TNF-α in RelA−/− cells. (A) Western blots showing that Twist-1 is capable of suppressing TNF-α-induced caspase activation and Bid processing. MIGR1- and MIGR1-Twist-1-transduced RelA−/− cells were seeded as described for Fig. ​Fig.2A2A and stimulated with TNF-α (100 U/ml) plus CHX (0.1 μg/ml). At the times indicated, extracts were prepared from TNF-α-treated and control cultures (i.e., 0-hour time points) and used to assess the kinetics of activation of caspase-8 (p57), caspase-3, and Bid as determined by immunoblotting. Also indicated are the levels of ectopically expressed Twist-1. β-Actin is shown as the loading control. Antibodies and intact and cleaved polypeptide products are indicated (on the left- and right-hand sides, respectively). (B) PI nuclear staining assays showing that TNF-α-induced DNA fragmentation in RelA−/− cells is blocked by the ectopic expression of Twist-1. FCM histogram profiles showing DNA content in MIGR1- and MIGR1-Twist-1-transduced RelA−/− cells which were either left untreated (i.e., CHX alone) (unstimulated [US] panels) or treated with TNF-α (100 U/ml) plus CHX (0.1 μg/ml) (TNFα panels), as indicated. Cells were collected 14 h after these treatments, and DNA fragmentation in these cultures was assessed by performing PI nuclear staining followed by FCM analysis. Values represent percentages of cells manifesting sub-G1 and at least G1 DNA contents, as shown. (C) Twist-1 effectively inhibits MOMP triggered by TNF-α in RelA−/− cells. Mitochondrial depolarization in MIGR1- and Twist-1-transduced RelA−/− cultures treated with CHX alone or together with TNF-α, as described for panel A, was determined by performing JC-1 fluorescence staining. At 4, 8, 12, and 16 h after TNF-α stimulation, detached and adherent cells were harvested, pooled, washed with phosphate-buffered saline (PBS), and then loaded with JC-1 (10 μg/ml in PBS) and incubated at 37°C for 30 min. Cells were then washed three additional times with PBS and analyzed by FCM. Shown are percentages of JC-1+ cells relative to those in cultures treated with CHX alone and reflect means ± standard deviations from three independent experiments. (D) FCM analysis showing comparable levels of surface expression of TNF-R1 in MIGR1- and MIGR1-Twist-1-transduced cells. Cells were stained with a biotin-labeled anti-TNF-R1 antibody (α-TNF-R1) (black line) or a biotin-labeled isotype-matched control (anti-immunoglobulin G2a [α-IgG2a]) (gray line), as indicated, followed by incubation with streptavidin-APC (SA-APC), and FCM was performed using standard methods. FCM histogram profiles depict APC positivity in MIGR1- and MIGR1-Twist-1-infected RelA−/− cultures and were generated using the FlowJo software as described for Fig. ​Fig.2C.2C. (E) Western blots showing levels of the indicated proteins in MIGR1- and MIGR1-Twist-1-transduced cells treated with TNF-α and CHX or left untreated (0 h-time points) as described for panel A. Extracts were the same as those used to obtain the panel A results. Antibodies are indicated.

Article Snippet: Strikingly, however, the expression of Twist-1 in these cells conferred strong protection against TNF-α-induced PCD (Fig. , left; MIGR1-Twist-1 bars).

Techniques: Western Blot, Activation Assay, Control, Staining, Expressing, Analysis, Fluorescence, Saline, Incubation, Labeling, Infection, Generated, Software, FACS