Supplemental Figure S1: a) NB1 NB3 NB4 NB8 NB15 pPKB(Ser473) - - PDF document

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Supplemental Figure S1: a) NB1 NB3 NB4 NB8 NB15 pPKB(Ser473) - - PDF document

Jan 29, 2023 333 likes •439 views

Supplemental Figure S1: a) NB1 NB3 NB4 NB8 NB15 pPKB(Ser473) PKB(total) GAPDH b) NB1 NB3 Infect. - + - + FOXO3(A3)ER TM ectopic FOXO3 endogenous 1.3 7.8 ratio ectopic/endogenous GAPDH c) Magnification Merge

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SLIDE 1

Infect. FOXO3(A3)ERTM ectopic FOXO3 endogenous GAPDH

  • NB1

+

  • NB3

+

ratio ectopic/endogenous 1.3 7.8

Supplemental Figure S1:

a)

pPKB(Ser473) PKB(total) GAPDH NB1 NB3 NB4 NB8 NB15

b) c) Hoechst FOXO3 Merge Magnification FOXO3 Merge Merge (neg Ctr) NB1 NB3

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SLIDE 2

Supplemental Figure S2:

a)

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 24h

NB4/Ctr NB4/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 48h

NB4/Ctr NB4/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 72h

NB4/Ctr NB4/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 24h

NB8/Ctr NB8/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 48h

NB8/Ctr NB8/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 72h

NB8/Ctr NB8/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 72h

NB15/Ctr NB15/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 48h

NB15/Ctr NB15/FOXO3

** *** *** *** ***

### ##

** ** *** *** ***

### 10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 24h

NB15/Ctr NB15/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 72h

NB1/Ctr NB1/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 48h

NB1/Ctr NB1/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 24h

NB1/Ctr NB1/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 24h

NB3/Ctr NB3/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 48h

NB3/Ctr NB3/FOXO3

10 20 30 40 50 60 0 nM 25 nM 50 nM 100 nM 250 nM 500 nM % cell death 4OHT 72h

NB3/Ctr NB3/FOXO3

** *** ** *** *** ** ** ** **

NB1 NB3 NB4 NB8 NB15

slide-3
SLIDE 3

b) NB15/FOXO3

34.6% 7.0% 8.1% 34.6% 7.0% 8.1% 61.7% 6.2% 13.8% 61.7% 6.2% 13.8% 67.6% 12.5% 9.9% 67.6% 12.5% 9.9% 54.0% 21.8% 10.4% 54.0% 21.8% 10.4% 51.7% 26.0% 12.1% 51.7% 26.0% 12.1% 49.7% 23.2% 14.3% 49.7% 23.2% 14.3%

+ 4OHT

  • 4OHT

24 h 48 h 72 h NB4/FOXO3 NB8/FOXO3

59.3% 12.8% 8.8% 59.3% 12.8% 8.8% 56.7% 14.3% 8.7% 56.7% 14.3% 8.7% 62.6% 10.6% 11.2% 62.6% 10.6% 11.2% 60.5% 12.6% 8.8% 60.5% 12.6% 8.8% 57.0% 15.4% 11.3% 57.0% 15.4% 11.3% 63.0% 11.7% 9.7% 63.0% 11.7% 9.7%

+ 4OHT

  • 4OHT

24 h 48 h 72 h

57.1% 13.4% 11.3% 57.1% 13.4% 11.3% 57.8% 12.9% 14.1% 57.8% 12.9% 14.1% 57.7% 13.2% 12.9% 57.7% 13.2% 12.9% 55.1% 13.9% 14.6% 55.1% 13.9% 14.6%

+ 4OHT

  • 4OHT

48 h

52.0% 14.4% 12.5% 52.0% 14.4% 12.5% 53.2% 13.7% 12.9% 53.2% 13.7% 12.9%

24 h 72 h

slide-4
SLIDE 4

Supplemental Figure S3:

20 40 60 80 100 120 140 160 180 200 0h 3h 6h 9h BCLXL mRNA expression (fold over control)

NB4/FOXO3 NB8/FOXO3 NB15/FOXO3

*** *** *** ***

20 40 60 80 100 120 140 0h 3h 6h 9h BIRC5 mRNA expression (fold over control)

NB4/FOXO3 NB8/FOXO3 NB15/FOXO3

*** *** **

slide-5
SLIDE 5

Supplemental Figure S4:

10 20 30 40 50 60

untreated 4OHT 5-azadC

BIM_02 methylation (PMR %)

NB4/FOXO3 NB8/FOXO3 NB15/FOXO3 ** *

NB15 NB4 NB8 NB15 NB4 NB8 NB15 Input IP:FOXO3 IP:IgG FOXO3 Tubulin Ac-FOXO3

1.02 0.98 1.43 0.0 % Ac-FOXO3/IP-FOXO3

a) b)

slide-6
SLIDE 6

Supplemental Figure S5:

Mutation site codon 72 Pro → Arg (C→G)

TP53 (homo sapiens) 393 aa

TAD DNA-binding domain TET

1 44 102 292 325 356

NB4 NB8 NB15

*

NB3 NB1

TP53 wt TP53 NB1 TP53 NB3 TP53 NB4 TP53 NB8 TP53 NB15

slide-7
SLIDE 7

Supplemental Figure S6:

1 2 3 4 5 BIM NOXA SESN3 mRNA expression (fold over control)

NB4 0h Etop NB4 2h Etop NB4 4h Etop

** * 1 2 3 4 BIM NOXA SESN3 mRNA expression (fold over control)

NB8 0h Etop NB8 2h Etop NB8 4h Etop

# # *** ** *** *

1 2 3 BIM NOXA SESN3 mRNA expression (fold over control)

NB15 0h Etop NB15 2h Etop NB15 4h Etop * * * *

slide-8
SLIDE 8

Supplemental Figure S7:

  • 4OHT

+ 4OHT control Etop Doxo NB1/FOXO3 control Etop Doxo NB1/Ctr

20 40 60 80 100 120 untreated Etop Doxo colonies [%] NB1/Ctr

without 4OHT with 4OHT

20 40 60 80 100 120 untreated Etop Doxo colonies [%] NB1/FOXO3

without 4OHT with 4OHT

** * ## ###

slide-9
SLIDE 9

Supplemental Figure S8:

a)

20 40 60 80 100 120 NB8/FOXO3-shCtr NB8/FOXO3-shSESN3 SESN3 mRNA expression (fold over control)

20 40 60 80 100 120 NB4/FOXO3-shCtr NB4/FOXO3-shSESN3 SESN3 mRNA expression (fold over control)

*** ***

50 100 150 200 250 300 NB8/FOXO3-shCtr NB8/FOXO3-shSESN3 ROS % of untreated Ctr

untreated 2h Etop

50 100 150 200 250 300 NB4/FOXO3-shCtr NB4/FOXO3-shSESN3 ROS % of untreated Ctr

untreated 2h Etop

c) FOXO3 GAPDH NB8 shCtr shFOXO3-17

100 44 % FOXO3/GAPDH

b)

NB8/FOXO3-shCtr untreated Etop NB8/FOXO3-shSESN3

*

NB4/FOXO3-shCtr NB4/FOXO3-shSESN3

* Bright field CM-H2XROS Bright field CM-H2XROS

untreated Etop untreated Etop untreated Etop