supplementary figure legends; sup. figure 1: (a-b) chip was
TRANSCRIPT
Supplementary Figure Legends; Sup. Figure 1: (A-B) ChIP was performed in U251 and T98G cells with -146C>T mutant
Tert promoter and HCT116 with -146C wild type Tert promoter against active histone marks
(H3K4Me3 and H3K9Ac) followed by qPCR with primers specific for Tert promoter region
proximal to TSS. Graph shows qPCR results with % input method as indicated. (C) Gene
expression analysis performed by RT-qPCR for Tert gene and data normalized to actin levels.
n=2 for all the experiments, error bars indicate mean±SD of 2 independent experiments.
Sup. Figure 2: (A) Graph shows qPCR analysis of Tert expression normalized to actin levels
in BLM cells. (B) Graph shows telomerase activity (TRAP) in BLM6 and BLM14 cells. (C)
Telomere length analysis using southern hybridization method in BLM6 and BL14 in passage
6 (P6). (D) Telomere length analysis using southern hybridization method in BLM6 and
BLM14 in passage 10 (P10), P18 and P28. (E) Representative confocal microscopy images
of ALT associated PML bodies. BLM6 and BLM14 cells were stained with TRF2 and PML
antibodies and ALT associated PML bodies were shown as yellow color. ALT+ U2OS cells
were used as a positive control that TRF2 and PML co-localization in nucleus was shown by
arrows in the merged image.
Sup. Figure 3: (A) Jaspar transcription factor prediction tool detected 11 GABPA motifs in
chr5:1,556,087-1,558,758 region (T-INT1). Score and relative score of GABPA sites along
with start and end positions are shown. GABPA sites are present in sense (1) or anti-sense
strand (-1). Predicted GABPA site sequence in T-INT1 region is also shown. (B) ChIP was
performed against GABPA followed by qPCR with primers specific to 5’ end of
chr5:1,556,087-1,558,758 (T-INT1) in BLM6 (-146C>T) and BLM14 (-146C) cells. ChIP q-
PCR primers were targeting ~290 to 450 nucleotides of chr5:1,556,087-1,558,758 (T-INT1).
Error bars indicate the mean ± SD of the two independent experiments. P values were
calculated by two tailed Student’s t test method.
Sup. Figure 4: (A) BLM cells were transfected with siControl (siCont), siGabpa or siBRD4.
48h post transfection TRAP assay was performed. Graph shows telomerase activity (TRAP)
of BLM cells upon siRNA transfection. (B) GABPA and BRD4 depletion in BLM6 cells with
mutant Tert promoter decreases colony formation and this is not as significant as the effect of
these depletions in BLM14 cells with WT Tert promoter. (C) Quantification of the colonies
in B. (D) Tert and (E) BRD4 expression is shown upon siCont and siBRD4 transfection of
Fadu cells that harbor wild type Tert promoter. (F) Graph shows expression of Tert isofoms
as indicated on x-axis. Actin was used for normalizing the data. Error bars indicate mean±SD
of 3 independent experiments. P values were calculated by student’s t-test method.
Sup. Figure 5: ChIP was performed against GABPA followed by qPCR with primers
specific to 5’ end of chr5:1,556,087-1,558,758 in HCT116 -146C and HCT116 -146C>T
cells. ChIP q-PCR primers were targeting ~290 to 450 nucleotides of chr5:1,556,087-
1,558,758. Error bars indicate the mean ± SD of the two independent experiments. P values
were calculated by two tailed Student’s t test method.
Sup. Figure 6: (A) Genotyping PCR was performed from genomic DNAs of T-INT1 WT
and KO cells. Agarose gel electrophoresis for the PCR products from indicated cell types is
shown. WT PCR product was of 4530bp and is indicated in red arrow while KO PCR product
was of 550bp and is indicated in orange arrow. DNA ladder is shown in lane 1 and 8. (B)
Graph shows qPCR analysis of GABPA expression normalized to actin levels in BLM T-
INT1 WT and BLM T-INT1 KO cells. (C) ChIP was performed against BRD4 in BLM T-
INT1 WT and BLM T-INT1 KO cells with primers specific to TSS region of Tert promoter.
Error bars indicate the mean ± SD of the two independent experiments. P values were
calculated by two tailed Student’s t test method.
A B
Sup. Fig1
α-H3K4Me3
α-H3K9Ac
α-H3K4Me3
α-H3K9Ac
CTert Expression
Mutant Tert Promoter Wild Type Tert Promoter
ATert Expression
BTRAP Assay
E MergedTRF2PML
U2O
SB
LM
6B
LM
14
Sup. Fig2
C D
1 2 3 4 5 6
BL
M14
BL
M6
1 2
BL
M14
BL
M6
BL
M14
BL
M6
BL
M14
BL
M6
P10 P18 P28
M (kb)
P6
3
4
5
6
8
9
10
12
7
3
4
5
6
8910
12
7
2
1
M (kb)
Chr5:1,556,087-1,558,758
A
B
Fo
ld r
ecru
itm
ent
Anti-GABPAof chr5:1,556,087-1,558,758
Sup. Fig3
BLM Cells TRAP Assay
A
FFadu CellsTert Expression
D Fadu CellsBRD4 Expression E
Sup. Fig4
BLM6
BLM14
si-Cont si-GABPA si-BRD4
BLM6 BLM14
B
C
Anti-GABPAof chr5:1,556,087-1,558,758
HCT116-146C>T
HCT116-146C
Sup. Fig5
GABPA Expression
BLMT-INT1
KO
BLMT-INT1
WT
Sup. Fig6
B CAnti-BRD4
BLMT-INT1
KO
BLMT-INT1
WT
2 31 4 5 6 7 8
BL
M T
-IN
T1
WT
BL
M T
-IN
T1
KO
A37
5 T
-IN
T1
WT
A37
5 T
-IN
T1
KO
U25
1 T
-IN
T1
WT
U25
1 T
-IN
T1
KO
DN
A L
AD
DE
R
DN
A L
AD
DE
R
4530bp
550bp 500bp
1000bp
3000bp
A