Legends to supplemental figures
Figure S1
KSHV vFLIP causes cell cycle arrest in HeLa cells HeLa cells were
transduced with LV-puro or LV-vFLIP-puro vectors for 48 hours later, and then selected
in medium containing 1 g/ml puromycin for 2 days. Approximately 105 puromycinselected cells were seeded into each well of a 6-well plate (for a total of 4 wells) and
grown in the selection medium for the duration indicated and harvested for counting.
Figure S2 Absence of senescence-associated heterochromatin foci from HeLa-G
cells expressing Tax or vFLIP.
HeLa G cells grown on chamber slides were
transduced by lentiviral vectors, LV- puro, LV-Tax, or LV-vFLIP. Forty-eight hours posttransduction, cells were selected with 1 g/ ml of puromycin for 3 days, and then fixed
by 4% paraformaldehyde and permeabilized by 0.2% Triton X-100 in PBS. To detect
senescence-associated heterochromatin foci (SAHF), cells were stained with 300 ng/ml
Dapi in PBS and examined using a confocal microscope. Fluorescence images from
Dapi staining are shown in the middle column. A selected field (boxed) from each panel
in the middle column was enlarged and represented in the right column. The images in
the left column are either merged between phase contrast (bright field) and Dapi
fluorescence (for LV-puro and LV-vFLIP) or GFP and Dapi (LV-Tax). GFP+ cells are
expressing Tax. Arrowheads denote cells with double nuclei or micro-nuclei.
Figure S3
vCyclin facilitates chronic NF-B activation by vFLIP and Tax. (A)
Immunoblot analysis of HeLa G cell lines stably expressing vCyclin or vCyclin and Tax.
Tax-expressing cell lines were constructed by either transducing Tax into vCyc-G2
using LV-Tax-neo, or transducing bicistronic vCyclin-Tax into HeLa-G using LV-2FlagvCyc-Tax-puro. Cell clones were isolated after G418 or puromycin selection, followed
by limiting selection. Cell lysates from HeLa-G, vCyc-G2, three independent LV-Taxtransduced vCyc-G2 derivatives (lanes 3-5) and two LV-2Flag-vCyc-Tax-purotransduced HeLa G derivatives (lanes 6 and 7) were subjected to immunoblot analysis
with indicated antibodies. (B) Schematic diagram of the lentiviral vectors for bicistronic
vCyclin-vFLIP and vCyclin-Tax cassettes.
(C) Immunoblot analysis of vCyclin- and
vFLIP-expressing cells. The vFLIP gene was transduced into vCyc-G2 cells using LVvFLIP-Flag-neo. G418-resistant clones with stable vFLIP expression were established
by limiting dilution. Cell lysates from parental HeLa-G (lane 1), vCyc-G2 (lane 2), and
two independent vCyc-G2-based vFLIP-Flag-expressing cell clones (lanes 3 and 4),
were analyzed using the indicated antibodies. (D) Immunoblot analysis of vCyclin- and
vFLIP-expressing cell lines derived from transduction of HeLa-G by the lentiviral vector
harboring a bicistronic vCyclin-vFLIP cassette. HeLa-G (lane 1) and vCyc-G2 (lane 2),
and three vCyclin-vFLIP cell clones (lanes 3-5), were probed with the indicated
antibodies.
Figure S4 Rapid loss of KSHV vFLIP or vCyclin expression from BJAB cells. (A)
BJAB human B cells were transduced with lentiviral vectors for vCyclin (LV-2Flag-vCycpuro) and vFLIP (LV-vFLIP-Flag-neo) either individually (vCyclin: lanes 1-3; vFLIP:
lanes 4-6) or in combination (lanes 7-9). Transduced BJAB cells were then grown in
G418 and/or puromycin-containing RPMI medium supplemented with 10% FBS and
split 1:5 once cell density reached one million per ml. Cells were harvested over 3
successive passages (P0 – P2) and subjected to immunoblotting using antibodies for
Flag-epitope and -actin respectively. (B) BJAB cells were transduced with LV-2FlagvCyc-puro and LV-vFLIP-Flag-neo either individually (lanes 2 and 3) or in combination
(lane 4) as in (A). Cells transduced with the empty lentiviral vector, LV-CMV-neo, were
used as a negative control (lane 1). The transduced cells were subjected to G418
and/or puromycin selection 24 hours post-transduction. Five days after antibiotic
selection, cell lysates were immunoblotted using the indicated antibodies.
Supplemental Table 1: Antibodies used for immunoblots.
Antibody
Cyclin B1
p21CIP1/WAF1
p27KIP1
I-B
RelA
RelB
p100/p52
-actin
c-Rel
p105/p50
p-I-B
FLAG
Catalog number
SC-752
SC-397
SC-1641
SC-847
SC-8008
SC-48379
SC-7386
SC-1616
4727
3035
9246
F3165
Vender
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Sigma-Aldrich