Fig 2: Puma and its downstream apoptotic signaling were activated in IDD patients, and rhBMPs could block the activation of PUMA-dependent apoptotic signaling. (A) Protein levels of Puma and its downstream apoptotic molecules in IDD specimens. Total cell extracts from the IVDs in one control and different Pfirrmann grades (I-IV) were subjected to immunoblots to examine the protein levels of Puma, Apaf-1, CASP9, CAPS3, and β-Actin (loading control). F: full length; C: cleaved. (B) PUMA mRNA level. The HNPC cells were treated with 0.1 µM RITA for three hours, followed by incubating with different concentrations (0, 10, and 20 ng/mL) of rhBMP2 and rhBMP7 for two hours. Total RNA samples were subjected to RT-qPCR analysis to examine the PUMA mRNA level. **P < 0.01 and ***P < 0.001. (C) Protein levels of Puma and its downstream apoptotic molecules in RITA- and rhBMP-treated cells. Total cell extracts from cells used in (B) were subjected to immunoblots to examine the protein levels of Puma, Apaf-1, CASP9, CAPS3, and β-Actin (loading control).

Fig 3: Deletion of BIM increased clonogenic survival of WEHI7 cells in response to Dex.a One representative WEHI7-derived clone of each genotype (Bax−/−Bak1−/−, Bax−/−Bak1−/−Apaf1−/− and Bax−/−Bak1−/−Bim−/−) was cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed, and plated in soft agar without Dex at a density of 5000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 250 cells per well to generate countable numbers of colonies. After 14 days, the cloning efficiency was calculated by dividing the number of colonies by the number of cells initially plated in each well, and expressed as percentages. The results are from three independently conducted experiments. Photos of one of the three experiments are shown on the right. b Three independent WEHI7 cell clones from each genotype (Bax−/−Bak1−/−, Bim−/−Bmf−/−Puma−/−Bax−/−Bak1−/−Apaf1−/− and Bax−/−Bak1−/−Bim−/−) were cultured for 10 days in the presence or absence of 1 µM Dex. Cells were then washed free of Dex, and plated in soft-agar medium for 14 days. Photos of one of the three experiments are shown on the right. c Four independent ihBIM Bax−/−Bak1−/−Bim−/− WEHI7 cell clones were cultured for 10 days in the presence of 1 µM Dex and/or 1 µg/ml Dox. Cells were then washed free of Dex, and plated in soft-agar medium at a density of 4000 cells per well. Cells without Dex pre-treatment were plated at a lower density of 400 cells per well. Colonies were counted 14 days after plating.

Fig 4: Characterization of clonal lymphoid lines mutant for combinations of pro-apoptotic BCL2 family proteins.a Whole-cell lysates from Bax−/−Bak1−/− and three independent Bax−/−Bak1−/−Bim−/− cell clones treated with 1 µM Dex treatment for 24 hrs were subjected to western blot analysis to detect BIM protein. Upper panel: WEHI7 mutant lines; lower panel: p53−/− T lymphoma mutant lines. b Bax+/+Bak1+/+ WEHI7 cells expressing Cas9 were transduced with sgRNAs targeting mouse Bim, Bmf and Puma. Following treatment with doxycycline to induce sgRNA expression, clones were isolated and validated for absence of BIM, BMF, and PUMA by western blotting after 24 hrs treatment with 1 µM Dex. c Wild type (WT) and Bax−/−Bak1−/− WEHI7 cells were treated for 24 hrs with 1 µM Dex and lysates were run on replicate gels and analyzed by western blot using antibodies specific for the indicated BCL2 family proteins. Roman numerals to the left of blots (i–vi) indicate the membranes probed. d Whole-cell lysates from Bax+/+Bak1+/+ and Bax−/−Bak1−/− p53−/− T lymphoma cells treated with 1 µM Dex for 24 hrs were tested by western for expression of BIM. Note that the absence of protein in the Dex-treated WT p53−/− T lymphoma cells is due to the death of most of the cells by 24 hours.

Fig 5: Dex-induced caspase activation and cytochrome c release in the absence of BAX and BAK1, but this failed to occur if BIM was also deleted.a Bax+/+Bak1+/+ (WT), Bax−/−Bak1−/−, Bax−/−Bak1−/−Casp9−/−, Bax−/−Bak1−/−Apaf1−/−, Bim−/−Bmf−/−Puma−/− and Bax−/−Bak1−/−Bim−/− clonal WEHI7 lines were treated with 1 µM Dex for indicated times. Cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in left panel of Fig. 2a. b Bax−/−Bak1−/− and Bax−/−Bak1−/−Bim−/− p53−/− T lymphoma lines were treated with 1 µM Dex for indicated times. Whole-cell lysates were analyzed by western blot using antibodies specific for cleaved Caspase-3, cleaved Caspase-9, and ACTIN. Note, the first 6 lanes of these blots are also shown in right panel of Fig. 2a. c Bax+/+Bak1+/+, Bax−/−Bak1−/−, Bax−/−Bak1−/−Casp9−/−, Bax−/−Bak1−/−Apaf1−/−, Bim−/−Bmf−/−Puma−/− and Bax−/−Bak1−/−Bim−/− WEHI7 lines were treated with 1 µM Dex for indicated times. Cytoplasmic extracted fractions were subjected to western blot analysis, using antibodies specific for cytochrome c (CYTC), and ACTIN. d Bax−/−Bak1−/− and Bax−/−Bak1−/−Bim−/− p53−/− T lymphoma lines were treated with 1 µM Dex for indicated times. Cytoplasmic fractions were subjected to western blot analysis, using antibodies specific for CYTC, and ACTIN.