Kip2 WB: a-p57 WB: a-CD31 PB Cs M Activ ated PBM s C Sti ul te d M m a PB Cs None WB: a-CD31 Kip1 WB: a-p27 WB: a- INK4 p16 WB: a-CD31 WB: a-CD31 WB: a- 8 INK4 p1 PBM Cs c i a P A t v ted BMCs Stim ulate d P MCs B None PB Cs M Activ ated PBM Cs Sti ul te d MC m a PB s n No e PBM Cs Aci a P t v te d BM Cs d Stim ulate P MCs B None Figure S1 siCt l PKC si d c WB: a-PKCd WB: a-actin siCt l Ci p1/W af1 p si 21 a Cip1/Waf1 WB: a-p21 WB: a-actin siCt l S si TA T1 b WB: a-STAT1 WB: a-actin WB: a-p38 siC l t p38 si d WB: a-actin Cip1/Waf1
Fig. S1 Effect of siRNA targeting p21 (a), STAT1 (b), PKCd (c) and p38 (d) in ECs. Human ECs were transfected twice with either the specific oligonucleotides or with control non-targeting siRNA. Western blot analysis was performed 24 h after the second transfection. The data shown are exemplificative of at least five experiments
Figure S2
Fig. S2 Expression of CDK inhibitors in human ECs cultures alone or with PBMCs, activated PBMCs or stimulated PBMCs for
48 h. At the end of incubation EC lysates were divided in four aliquots, separated and immunoblotted as indicated. This picture is representative of one experiment out of three performed
Angiogenesis
Supplementary Material
Cip1/Waf1
IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21 -mediated mechanism
Lucia Napione, Marina Strasly, Claudia Meda, Stefania Mitola, Maria Alvaro, Gabriella Doronzo, Serena Marchiò, Enrico Giraudo, Luca Primo, Marco Arese and Federico Bussolino
Corresponding author: Dr. Lucia Napione, Institute for Cancer Research and Treatment, Department of Oncological Sciences, University of Torino, 10060 Candiolo, Torino, Italy; e-mail: [email protected]
Figure S3
Cip1/Waf1
Fig. S3 Densitometric quantification of co-culture induced changes in total and/or phosphorylated protein level of p21 , p53, STAT1 and p38. Panel a, b, c, d and e are the densitometric analysis of the results shown in Fig. 2a, Fig. 2b, Fig. 4a, Fig. 5a and Fig. 7a, respectively. Measurement of band intensity was performed as described in Material and Methods (main text). Results are expressed as fold-increase over the basal value in control cells, after normalization for the values of CD31 (a, b, c and d) or p38 (e). Values shown are the means ± SD of three indipendent experiments
C i 1 /W a f1 p p 2 1 ( fo ld in c re s e ) -a p 5 3 ( fo ld -i n c re a s e ) a c h s h 3 -n r a p o p o p 8 ( fo ld i c e s e ) e 0 0.2 0.4 0.6 0.8 1 1.2 1.4 B P MC s ctv te A ia d P MC B s Stim ula ed t PB MC s CD 4+ CD 8+ NK 0 20 40 60 80 100 120 140 160 180 200 STAT1 phospho STAT1-Y701 phospho STAT1-S727 d c t A t v ed ia PB MC s Stim ula ed t PB MC s F ld c e a e o -i n r s 0 2 4 6 8 10 12 14 16 18 20 cv t A tia ed PB MC s t ul ed Sim at PB MC s 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 B P MC s ctv t A ia ed P M B Cs Stim ul ed at PB MC s C i 1 /W a f1 p p 2 1 ( fo ld in c re s e ) -a b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2 B P MC s B P MC s
Table S1 Densitometric quantification of STAT1 protein level in Fig. 5b
Measurement of band intensity was performed as described in Materials and Methods (main text) and calculated as percentage of all the band intensities in each blot. GAPDH , nuclear matrix p84 or actin were then used as normalizers. Values are the mean SD of three independent experiments
a b c
±
3
Co-culture condition Cytosola Nucleusb Whole cell lysatec
None 0.14 ± 0.02 0.06 ± 0.02 0.20 ± 0.02
PBMCs 0.33 ± 0.04 0.28 ± 0.04 0.61 ± 0.05
Activated PBMCs 0.86 ± 0.18 0.56 ± 0.06 0.68 ± 0.07
Stimulated PBMCs 2.50 ± 0.29 3.07 ± 0.19 2.52 ± 0.26
Table S2 Densitometric quantification of PKC protein level in Fig. 6bd
Measurement of band intensity was performed as described in Materials and Methods (main text) and calculated as percentage of
all the band intensities in each blot. GAPDH , C 31 or actin were then used as normalizers. Values are the mean SD of three
independent experiments
a b c
D ±
Co-culture condition Cytosola Membraneb Whole cell lysatec
None 1.47 ± 0.01 0.21 ± 0.04 0.99 ± 0.04
Activated PBMCs 0.97 ± 0.12 0.59 ± 0.03 1.04 ± 0.07
