1. Proapoptotic BAX and BAK control multiple initiator caspases
Lab meeting; Antonio Ruiz-Vela
9/23/03

2. Bax and Bak Double-Knockout MEFs
#1 Resistant to BH3-only-induced apoptosis
.BIM
.t-Bid
.BAD
.Noxa
#2 Resistant to DNA-damaging agents
.Etoposide
.UV light
.Cisplatinum
.Doxorubicin
#3 Resistant to chronic UPR-induced apoptosis
.Tunicamycin
.Brefeldin A

3. BAX and BAK gateway? ? ? ER stress-induced apoptosis
ER membrane
“BH3-only”
CED-4 like
CASP-12 ?
Misfolded proteins
ER stress-induced apoptosis
“BH3-only”
CED-4 like
CASP-2 ?
Mito. membrane
DSS
DNA damage-induced apoptosis
CASP-3, -7
IRE-1
TRAF2
CASP-12
CASP-3, -7
RAIDD
CASP-2
BAX and BAK gateway?

4. #1 BCL-2 function upstream of BAX and BAK gateway
#2 Caspases upstream of BAX and BAK gateway
#3 Caspase activation always requires APAF-1/ CASP-9

5. “DNA damage-induced apoptosis” “ER stress-induced apoptosis”
DKO/ GFP MEFs
“DNA damage-induced apoptosis”
CED-4 like
CASP -2 ?
WT/ GFP
DKO/ GFP
WT/ hBCL-2
DKO/ hBCL-2
hBCL-2
Actin
“ER stress-induced apoptosis”
CED-4 like
CASP-12 ?

6. “DNA damage-induced apoptosis” “ER stress-induced apoptosis”
WT/ GFP
DKO/ GFP
WT/ hBCL-2
DKO/ hBCL-2
hBCL-2
Actin
DKO/ BCL-2 MEFs
“DNA damage-induced apoptosis”
CED-4 like
CASP -2 ?
“ER stress-induced apoptosis”
CASP-12

9. Etoposide (5 mM) proCASP-7 p20 p17 CASP-3 WT DKO Etoposide (mM) WT
DKO
Etoposide (mM)
12h 24h

10. WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7)
ETO-24
ETO-24
P19 (Cleaved CASP-3)
proCASP-7
p2O (Cleaved CASP-7)
proCASP-9
P37 (Cleaved CASP-9)

11. WT
DKO
FL5.12
ETO-24
ETO-24
+IL-3
-IL-3
proCASP-2
p32 (Cleaved CASP-2)
P19 (Cleaved CASP-2)

12. - WT DKO - 4h 12h 24h - 4h 12h 24h CHOP/ GADD153 28S 18S
P17 (Cleaved CASP-3)

13. Brefeldin A (0.5 mg/ml) proCASP-7 p20 p17 CASP-3 WT DKO WT
DKO
Brefeldin A (mg/ml)
12h 24h

14. WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 proCASP-9
BA-24
BA-24
P19 (Cleaved CASP-3)
proCASP-7
proCASP-9
P37 (Cleaved CASP-9)

15. WT DKO Untreated BA-24h Untreated BA-24h proCASP-12
HM LM S-100 HM LM S HM LM S HM LM S-100
proCASP-12
P42 (Cleaved CASP-12)
p35 (Cleaved CASP-12)
proCASP-9
P37 (Cleaved CASP-9)
COX IV

16. Calpain
CASP-12
CASP-7 and CASP-9
CASP-12

17. Late cell death Early cell death
(CASP-9- APAF-1- and BAX-BAK-independent)
Early cell death
(CASP-9- APAF-1- and BAX-BAK-dependent)

19. WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7)
UV-24
UV-24
P19 (Cleaved CASP-3)
proCASP-7
p2O (Cleaved CASP-7)
proCASP-9
P37 (Cleaved CASP-9)

20. DKO/ UV-light (100 mJ/cm2)
Annexin V (%)
VEID-ase activity (a.u)
DEVD-ase activity (a.u)
Time (h)

21. DKO Time (h) proCASP-7 P19 (Cleaved CASP-3) p16 (Cleaved CASP-6)
UV-light 100 mJ/cm2
P19 (Cleaved CASP-3)
DKO
proCASP-7
p16 (Cleaved CASP-6)
Time (h)
proCASP-9

22. RNAi Caspase-2 RS-CASP-2 #101 RS-CASP-2 #102 RS-CASP-2 #103
TGGGAGGCTGGGGGAGGGATGCGGGGGCTGGCGCGGCCCAGCGTTTGTTTGTTTTGTGGG
TGGTGCGTGTGCAGCCCGGGAAAAGCTGGAAATGGCGGCGCCGAGCGGGAGGTCGCAGTC
CTCCCTGCACAGGAAGGGGCTGATGGCGGCTGACAGGAGGAGCAGGATTTTGGCAGTGTG
TGGAATGCATCCTGACCACCAGGAAACACTGAAAAAGAATCGAGTGGTGCTGGCCAAGCA
GCTGCTGCTGAGCGAGCTGTTAGAACACCTCCTAGAGAAGGACATTATCACTTTGGAAAT
GAGGGAGCTCATCCAGGCCAAAGGGGGCAGTTTCAGCCAGAATGTGGAACTCCTCAACCT
GCTGCCAAAGAGAGGACCCCAGGCTTTTGATGCCTTCTGTGAAGCCCTGCGGGAGACCAG
GCAGGGTCACTTGGAAGACTTACTGCTCACAACCCTCTCAGATATTCAGCACGTACTCCC
ACCGTTGAGCTGTGACTATGACACAAGTCTCCCTTTCTCGGTGTGTGAGTCCTGCCCTCC
TCACAAGCAGCTCCGCCTATCCACAGATGCTACGGAACACTCCTTAGATAATGGTGATGG
TCCTCCCTGTCTTCTGGTGAAGCCATGCACTCCTGAGTTTTACCAGGCACACTACCAGCT
GGCCTATAGGTTGCAATCTCAGCCCCGTGGCTTGGCACTGGTGCTGAGCAATGTGCACTT
CACTGGAGAGAAAGACCTGGAATTCCGCTCTGGAGGGGATGTGGACCACACTACTCTAGT
CACCCTCTTCAAGCTTTTGGGCTACAATGTCCATGTGCTACATGACCAGACCGCACAGGA
AATGCAAGAGAAACTTCAGAATTTTGCACAGTTACCTGCACACCGGGTCACAGACTCCTG
CGTAGTGGCACTCCTCTCACATGGTGTGGAAGGTGGCATCTATGGTGTAGATGGCAAACT
GCTTCAGCTCCAAGAGGTTTTTCGACTTTTTGACAATGCTAACTGTCCAAGTCTACAGAA
CAAGCCAAAAATGTTCTTCATCCAAGCATGTCGTGGAGATGAGACAGATAGAGGTGTCGA
CCAGCAAGATGGAAAGAACCACACACAATCCCCTGGATGTGAGGAGAGTGATGCTGGCAA
AGAGGAGTTGATGAAGATGAGACTGCCTACTCGCTCAGACATGATATGTGGCTATGCTTG
RS-CASP-2 #101
RS-CASP-2 #102
RS-CASP-2 #103
Retro-Super
CASP-2
Actin
DKO

23. proCASP-3 P19 (Cleaved CASP-3) Annexin V (%) Retro-S RS-C2#101
UV-48
UV-48
UV-48
UV-48
proCASP-3
P19 (Cleaved CASP-3)
Annexin V (%)

24. RNAi Caspase-6 Retro-Super Actin CASP-6 DKO APAF-1 null CASP-9 null
CGGAGGCCTCAGGGCTAGGACACCGGTGGGAGCTCGCGGGCAGGTGAAAGTAAAACATGA
CAGAAACCGATGGCTTCTACAAAAGTAGGGAAGTGTTCGATCCAGCCGAGCAGTACAAGA
TGGACCACAAGAGGAGAGGAGTTGCCCTCATCTTCAATCACGAGAGGTTCTTTTGGCACC
TGACCCTCCCAGAGAGGCGGGGCACCAACGCAGACAGAGACAACCTGACTCGCAGGTTTT
CAGATCTAGGATTTGAAGTGAAATGCTTTAACGACCTCAGAGCAGAAGAACTCCTGCTCA
AAATTCACGAGGTGTCGACTTCAAGCCACATAGATGCCGATTGCTTCATCTGTGTCTTCC
TGAGCCATGGGGAAGGCAACCACGTTTACGCATACGACGCCAAAATTGAAATTCAGACGT
TGACTGGCTTGTTCAAAGGAGACAAGTGTCAGAGCCTGGTTGGAAAACCCAAGATATTTA
TCATCCAGGCCTGTCGGGGTAGCCAGCATGACGTACCCGTGGTTCCCCTGGACATGGTGG
ATCACCAGACAGACAAGCTGGACAACGTGACCCAGGTGGATGCTGCGTCCGTGTACACGC
TGCCGGCAGGGGCAGACTTCCTCATGTGCTACTCTGTCGCAGAAGGGTATTACTCTCACC
GAGAAACTGTGAATGGCTCCTGGTACATTCAGGATTTGTGTGAGATGCTGGCGAGGTACG
GCAGTTCCCTGGAGTTCACGGAGCTGCTCACGCTGGTGAACAGAAAGGTCTCTCAGCGCC
GCGTGGACTTCTGCAAAGACCCGGATGCAATCGGCAAGAAGCAGGTGCCCTGCTTCGCCT
CAATGCTGACCAAAAAGCTGCATTTCTGTCCCAAACCTAGCAAGTAGGGCCATCTGTCTT
GCTACATATATTTCACACACATTTCCCTTTTTATACAAAGGCCAGCTGGTGGAAGACTTT
Retro-Super
Actin
CASP-6
DKO
APAF-1 null
CASP-9 null
RS-CASP-6 #104

25. proCASP-3 P19 (Cleaved CASP-3) UV-light (10 mJ/cm2) CASP-9 null UV-48
Retro-S
proCASP-3
RS-C6#104
CASP-9 null
Annexin V (%)
RS-CASP-6 #104
Retro-Super

26. UV-light (10 mJ/cm2) APAF-1 null 0 24 36 48 Annexin V (%) DKO
RS-CASP-6 #104
Retro-Super
Annexin V (%)
DKO
Retro-Super
RS-CASP-6 #104
Annexin V (%)

27. Ac-Tyr-Val-Lys-(Biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone
(Potent and irreversible inhibitor of caspases)
UV-light 100mJ/cm2
72h
24h M M
p35
p17
DKO Wt

28. p17

29. p35

30. Acknowledgements
Stan Korsmeyer
Joe Opferman