RELT induces cellular death in HEK 293 epithelial cells

Abstract

RELT is a recently identified Tumor Necrosis Factor Receptor that possess two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway.

Fig. 1. Expression of RELT family members induces cell death in HEK 293 cells. HEK 293

cells (≈1 × 10⁵) were plated in 6 well plates and transfected with a mixed solution of 2 µg of the indicated expression plasmid plus 0.2 µg of an expression plasmid for β-galactosidase. Cells were fixed and stained with X-gal as described in Materials and Methods. Fig. 1A; morphology of cells fixed and stained 48 hours cells after transfection. Rounded cells are observed in cells transfected with RELT family members, consistent of cell death by an apoptotic pathway. Fig. 1B; experiment performed as in Fig. 1A, except a time course was performed with cells fixed and stained with X-gal at the indicated times. Data is expressed as a percentage of cells containing both β-galactosidase activity and apoptotic-like morphology versus cells that possess only β-galactosidase activity. A minimum of 5 viewing fields containing 20 cells each was quantified for each individual data point. A representative of one of three time course experiments is shown. Fig. 1C; Representative Western Blot demonstrating RELT family member overexpression from transient transfections in HEK 293 cells. Western blot was performed on protein lysates with anti-Flag and anti-HA antibodies to examine expression of RELL2 (Flag-tagged), RELT (HA-tagged) and RELL1 (HA-tagged). Fig. 1D; Western blot of HEK 293 cells using anti-RELT polyclonal antibody (R&D Systems #AF1385). Expression of endogenous RELT was compared between untransfected HEK 293 cells and HEK 293 cells transfected with RELT expression plasmid.

Fig. 1. Expression of RELT family members induces cell death in HEK 293 cells. HEK 293

cells (≈1 × 10⁵) were plated in 6 well plates and transfected with a mixed solution of 2 µg of the indicated expression plasmid plus 0.2 µg of an expression plasmid for β-galactosidase. Cells were fixed and stained with X-gal as described in Materials and Methods. Fig. 1A; morphology of cells fixed and stained 48 hours cells after transfection. Rounded cells are observed in cells transfected with RELT family members, consistent of cell death by an apoptotic pathway. Fig. 1B; experiment performed as in Fig. 1A, except a time course was performed with cells fixed and stained with X-gal at the indicated times. Data is expressed as a percentage of cells containing both β-galactosidase activity and apoptotic-like morphology versus cells that possess only β-galactosidase activity. A minimum of 5 viewing fields containing 20 cells each was quantified for each individual data point. A representative of one of three time course experiments is shown. Fig. 1C; Representative Western Blot demonstrating RELT family member overexpression from transient transfections in HEK 293 cells. Western blot was performed on protein lysates with anti-Flag and anti-HA antibodies to examine expression of RELL2 (Flag-tagged), RELT (HA-tagged) and RELL1 (HA-tagged). Fig. 1D; Western blot of HEK 293 cells using anti-RELT polyclonal antibody (R&D Systems #AF1385). Expression of endogenous RELT was compared between untransfected HEK 293 cells and HEK 293 cells transfected with RELT expression plasmid.

Figure 2. Increased TUNEL staining in HEK 293 cells expressing RELT family members

293 cells (≈ 5×10⁴) were grown on coverslips in 12 well plates and fixed for TUNEL staining as described in Materials and Methods. Fig. 2A; Representative images of TUNEL staining 24 hours after transfection of HEK 293 cells with either empty vector control or a RELT expression plasmid. Fig. 2B; Dose dependence of TUNEL staining as a function of amount of RELT expression plasmid used for transfection. A total of 1 µg of DNA was utilized for each data point. At least five viewing fields were utilized to obtain each data point, and a representative of three individual dose dependent experiments is shown. Data is expressed as the percentage of GFP positive cells that are also TUNEL positive. Fig. 2C; Time course of TUNEL staining produced by transfecting 0.5 µg of GFP with 0.5 µg of Vector, RELT or TNFR1. At least five viewing fields were utilized to obtain each data point, and a representative of three individual time course experiments is shown. Data is expressed as the percentage of GFP positive cells that are also TUNEL positive. Figure 2D; Cells were cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining 24 hours after transfection. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields under 40 X magnification were utilized to obtain each data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control. Figure 2E; 1.0 µg of GFP plasmid was combined with 0.5µg of each of the indicated expression plasmids for a total of 2.0 µg of DNA, and TUNEL staining was performed after 24 hours as described in Materials and Methods. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields containing at least 20 cells were utilized to obtain one data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control.

Figure 2. Increased TUNEL staining in HEK 293 cells expressing RELT family members

293 cells (≈ 5×10⁴) were grown on coverslips in 12 well plates and fixed for TUNEL staining as described in Materials and Methods. Fig. 2A; Representative images of TUNEL staining 24 hours after transfection of HEK 293 cells with either empty vector control or a RELT expression plasmid. Fig. 2B; Dose dependence of TUNEL staining as a function of amount of RELT expression plasmid used for transfection. A total of 1 µg of DNA was utilized for each data point. At least five viewing fields were utilized to obtain each data point, and a representative of three individual dose dependent experiments is shown. Data is expressed as the percentage of GFP positive cells that are also TUNEL positive. Fig. 2C; Time course of TUNEL staining produced by transfecting 0.5 µg of GFP with 0.5 µg of Vector, RELT or TNFR1. At least five viewing fields were utilized to obtain each data point, and a representative of three individual time course experiments is shown. Data is expressed as the percentage of GFP positive cells that are also TUNEL positive. Figure 2D; Cells were cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining 24 hours after transfection. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields under 40 X magnification were utilized to obtain each data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control. Figure 2E; 1.0 µg of GFP plasmid was combined with 0.5µg of each of the indicated expression plasmids for a total of 2.0 µg of DNA, and TUNEL staining was performed after 24 hours as described in Materials and Methods. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields containing at least 20 cells were utilized to obtain one data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control.

Figure 3. RELL2, but not other RELT family members binds TRAF2

Fig. 3A: RELL2, but not RELL1 or OSR1, binds to TRAF2. 293 cells (≈5×10⁶) were transfected with 10 mg of an expression plasmid for HA-tagged TRAF2 together with 10 mg of Flag tags of RELL1, RELL2 or OSR1 as indicated. Co-immunoprecipitations were performed with anti-HA (αHA) antibody or control mouse IgG (C) antibody as indicated. Western blot of co-immunoprecipitations was performed with anti Flag antibody to visualize proteins pulled down with HA tagged TRAF2. Expression of proteins in the whole cell lysate was visualized using a western blot for both anti- Flag and anti-HA antibodies. Figure 3B. RELT does not interact with TRAF2. Experiments performed as in Fig. 3A. 293 cells transfected with 10 mg Flag-tagged TRAF2 together with 10 mg of HA-tagged RELT and co-immunoprecipitated with anti-Flag antibody or control mouse IgG (C) antibody as indicated. Western blot of co-immunoprecipitations was performed with anti HA antibody and expression of proteins in the whole cell lysate was visualized using both anti- Flag and anti-HA antibodies.

Figure 4. Effect of overexpressing RELT in COS-7 cells

Figure 4A and 4B; effect of RELT family member overexpression on morphology of COS-7 cells. COS-7 cells (≈1×10⁵) were plated in 6 well plates and transfected with a mixed solution of 2 µg of the indicated expression plasmid plus 0.2 µg of an expression plasmid for β-galactosidase. Cells were fixed and stained with X-gal as described in Materials and Methods. Fig. 4A: morphology of COS-7 cells fixed and stained 48 hours cells after transfection. Fig. 4B; experiment performed as in Fig. 4A, except a time course was performed with cells fixed and stained with x-gal at the indicated times. Data is expressed as the percentage of cells containing both β-galactosidase activity and apoptotic-like morphology versus cells that possess only β-galactosidase activity. A minimum of 5 viewing fields under 20 X magnification was quantified for each individual data point. A representative of one of three separate time course experiments is shown. Fig. 4C and 4D; effect of RELT overexpression on TUNEL staining in COS-7 cells. COS-7 cells (≈5 ×10⁴) were grown on coverslips in 12 well plates and cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining as described in Materials and methods. Fig. 4C; representative images of COS-7 cell TUNEL staining 18 hours after cells were transfected with Vector, RELT or Caspase-8. Fig. 4D; Cells were cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining at either 18 or 24 hours after transfection. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields under 40 X magnification were utilized to obtain one data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control. Figure 4E; Expression of RELT in COS-7 cells. Untransfected and RELT transfected COS-7 cell lysates were analyzed by Western blotting utilizing an anti-RELT polyclonal antibody (R&D Systems #AF1385).

Figure 4. Effect of overexpressing RELT in COS-7 cells

Figure 4A and 4B; effect of RELT family member overexpression on morphology of COS-7 cells. COS-7 cells (≈1×10⁵) were plated in 6 well plates and transfected with a mixed solution of 2 µg of the indicated expression plasmid plus 0.2 µg of an expression plasmid for β-galactosidase. Cells were fixed and stained with X-gal as described in Materials and Methods. Fig. 4A: morphology of COS-7 cells fixed and stained 48 hours cells after transfection. Fig. 4B; experiment performed as in Fig. 4A, except a time course was performed with cells fixed and stained with x-gal at the indicated times. Data is expressed as the percentage of cells containing both β-galactosidase activity and apoptotic-like morphology versus cells that possess only β-galactosidase activity. A minimum of 5 viewing fields under 20 X magnification was quantified for each individual data point. A representative of one of three separate time course experiments is shown. Fig. 4C and 4D; effect of RELT overexpression on TUNEL staining in COS-7 cells. COS-7 cells (≈5 ×10⁴) were grown on coverslips in 12 well plates and cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining as described in Materials and methods. Fig. 4C; representative images of COS-7 cell TUNEL staining 18 hours after cells were transfected with Vector, RELT or Caspase-8. Fig. 4D; Cells were cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining at either 18 or 24 hours after transfection. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields under 40 X magnification were utilized to obtain one data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control. Figure 4E; Expression of RELT in COS-7 cells. Untransfected and RELT transfected COS-7 cell lysates were analyzed by Western blotting utilizing an anti-RELT polyclonal antibody (R&D Systems #AF1385).

Figure 4. Effect of overexpressing RELT in COS-7 cells

Figure 4A and 4B; effect of RELT family member overexpression on morphology of COS-7 cells. COS-7 cells (≈1×10⁵) were plated in 6 well plates and transfected with a mixed solution of 2 µg of the indicated expression plasmid plus 0.2 µg of an expression plasmid for β-galactosidase. Cells were fixed and stained with X-gal as described in Materials and Methods. Fig. 4A: morphology of COS-7 cells fixed and stained 48 hours cells after transfection. Fig. 4B; experiment performed as in Fig. 4A, except a time course was performed with cells fixed and stained with x-gal at the indicated times. Data is expressed as the percentage of cells containing both β-galactosidase activity and apoptotic-like morphology versus cells that possess only β-galactosidase activity. A minimum of 5 viewing fields under 20 X magnification was quantified for each individual data point. A representative of one of three separate time course experiments is shown. Fig. 4C and 4D; effect of RELT overexpression on TUNEL staining in COS-7 cells. COS-7 cells (≈5 ×10⁴) were grown on coverslips in 12 well plates and cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining as described in Materials and methods. Fig. 4C; representative images of COS-7 cell TUNEL staining 18 hours after cells were transfected with Vector, RELT or Caspase-8. Fig. 4D; Cells were cotransfected with 0.5 µg of a GFP expression vector and 0.5 µg of the indicated expression vector and assayed for TUNEL staining at either 18 or 24 hours after transfection. Data is expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are only GFP positive. At least five viewing fields under 40 X magnification were utilized to obtain one data point, and the results of three separate experiments were averaged and normalized to the TUNEL staining produced by empty vector control. Figure 4E; Expression of RELT in COS-7 cells. Untransfected and RELT transfected COS-7 cell lysates were analyzed by Western blotting utilizing an anti-RELT polyclonal antibody (R&D Systems #AF1385).