![Induction of the IRBC stabilizes p53 and leads to caspase-dependent cell death in Trp53+/+;Eμ-Myc, but not in Trp53–/–;Eμ-Myc lymphoma cells. (A-B) Proliferation assays of control (shRen) and RPL7a and RPL11-depleted Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells treated for 48 hours with doxycycline (dox) as in Figure 1 and of (A) Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells treated for 12 hours with 5 nM ActD or dimethyl sulfoxide (DMSO)-vehicle (NT) (B). Data are shown relative to their respective Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cell controls (n = 3-5). (C) Proportion of apoptotic shRen, shRPL7a, and shRPL11 Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells from panel A determined by annexin V (AnnV) and Zombie Violet (ZV) staining and flow cytometry (n = 2-3). (D) Representative dot plots from panel C. shRen-expressing control and RPL7a- and RPL11-depleted Trp53+/+;Eμ-Myc (top panels) or Trp53–/–;Eμ-Myc (bottom panels) cells are shown. (E) Representative western blot analysis of lysates from shRen and shRPL7a Trp53+/+;Eμ-Myc lymphoma cells treated for 22 hours with doxycycline with or without the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) (Z-VAD-FMK; ZVAD) at 20 μM (n = 3). (F) Proliferation assay of RPL7a-depleted Trp53–/–;Eμ-Myc lymphoma cells in NT, treated under doxycycline or doxycycline and ZVAD conditions at 22 and 48 hours (n = 3). AnnV-AF680, Alexa Fluor 680-conjugated annexin V. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001 (2-way ANOVA test).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/137/24/10.1182_blood.2020007452/1/bloodbld2020007452f2.png?Expires=1738029641&Signature=vN5jJ5WoSHV7MQxgxGwugWLgd4eQYI3Ws49J0LTUihvO4-O--29cLim32maRpADGJHbzPiCiXNtORdDJeXm~mf8VLN9USJ30ixN9bVDKPxq-xjB~ClosKVTsz4loSgxHuHWDsgjD~8fjav5TRZYG-n0wWARwVZcC9FdVZCM4o2TSf0tkOlr7u5n7oV7jA5jDPSGmIrnN~--EWQ1LqeFo909G1Rz6EtVRlz0tl40c1JQ2bMXmnJ6M~azOoDQdQiozrYEfXERvygEi7Y5LGzhMcZIATEU9fPJkyMzBqIs4IEl-phAoi50ER2PFl0GqoWp-DnkmTFzafBbI6DakPvhYlw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Induction of the IRBC stabilizes p53 and leads to caspase-dependent cell death in Trp53+/+;Eμ-Myc, but not in Trp53–/–;Eμ-Myc lymphoma cells. (A-B) Proliferation assays of control (shRen) and RPL7a and RPL11-depleted Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells treated for 48 hours with doxycycline (dox) as in Figure 1 and of (A) Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells treated for 12 hours with 5 nM ActD or dimethyl sulfoxide (DMSO)-vehicle (NT) (B). Data are shown relative to their respective Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cell controls (n = 3-5). (C) Proportion of apoptotic shRen, shRPL7a, and shRPL11 Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells from panel A determined by annexin V (AnnV) and Zombie Violet (ZV) staining and flow cytometry (n = 2-3). (D) Representative dot plots from panel C. shRen-expressing control and RPL7a- and RPL11-depleted Trp53+/+;Eμ-Myc (top panels) or Trp53–/–;Eμ-Myc (bottom panels) cells are shown. (E) Representative western blot analysis of lysates from shRen and shRPL7a Trp53+/+;Eμ-Myc lymphoma cells treated for 22 hours with doxycycline with or without the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) (Z-VAD-FMK; ZVAD) at 20 μM (n = 3). (F) Proliferation assay of RPL7a-depleted Trp53–/–;Eμ-Myc lymphoma cells in NT, treated under doxycycline or doxycycline and ZVAD conditions at 22 and 48 hours (n = 3). AnnV-AF680, Alexa Fluor 680-conjugated annexin V. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001 (2-way ANOVA test).