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. 2016 Dec 19;18(1):150–168. doi: 10.15252/embr.201642360

Figure 7. ATF3 and NOXA mediate low‐dose PI‐induced apoptosis in GSCs.

Figure 7

  1. HEK293 cells were transfected with ATF3 shRNA or vector (pLKO1), treated with 50 nM MG132, and then subjected to immunoblotting.
  2. Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in the Materials and Methods. After 4 days, individualized GSCs were treated with 50 nM MG132 and subjected to immunoblotting.
  3. XO8 GSCs were transfected with control or CHOP siRNA for 48 h and then treated with 50 nM MG132 for 24 h. Cell viability was determined using the trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments (*P < 0.05, n = 3). For statistical analysis, Student's t‐test (two‐sided, paired) was used.
  4. Immunoblotting analysis of (C). The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies.
  5. Wild‐type and CHOP −/− MEF cells were treated with MG132 for 24 h. Cell viability was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from three separate experiments (*P < 0.05, n = 3). For statistical analysis, Student t‐test (two‐sided, paired) was used.
  6. Cell lysates obtained in (E) were subjected to immunoblot analysis with anti‐PARP‐1, anti‐CHOP, or anti‐ATF3 antibody.
  7. The expression of NOXA was assessed with real‐time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress‐driven apoptosis (salubrinal; 15 μM, STK064652; 20 μM, STK047915; 20 μM). The mean + SD values from the three independent experiments are shown after normalization to those of control cells treated with DMSO. For statistical analysis, Student's t‐test (two‐sided, paired) was used. *P‐values: salubrinal, 0.0003; STK064650, 0.0036; STK047915, 0.0019.
  8. Similar to (C) except that XO8 GSCs transduced with lentiviral shRNAs were treated with MG132. The mean + SD values from the three independent experiments are shown after normalization to those of control cells treated with DMSO. For statistical analysis, Student's t‐test (two‐sided, paired) was used. *P‐values: #1, 0.026; #5, 0.0033; #6, 0.0482.
  9. XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in (C) and subjected to immunoblotting. Sal., salubrinal.

Source data are available online for this figure.