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Figure legends
Figure 1. TM6SF2 regulates αSMA expression in LX-2 cells
A) The cloned TM6SF2 expression plasmid (p3FLAG/TM6SF2-WT) and empty vector
(Mock) were transiently transfected into LX-2 cells followed by 24 hours of incubation.
Intracellular αSMA expressions were measured by quantitative PCR. The expression of
GAPDH served as a control. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD.
B) Non-treated and TM6SF2 knocked-down LX-2 lysates were transferred onto an automated
capillary western blot. Anti-TM6SF2 antibody or anti-GAPDH antibody were applied,
followed by anti-rabbit immunoglobulin. Signal intensity was corrected by GAPDH and is
shown in the bar graph. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD (*P < 0.05).
C) Intracellular αSMA expression, measured by quantitative PCR, was compared in
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non-treated and TM6SF2 knocked-down LX-2 cells. GAPDH expression was used as a
control. Experiments were performed in triplicate wells. All data are expressed as the mean ±
SD (*P < 0.05).
D) Non-treated and TM6SF2 knocked-down LX-2 lysates were transferred onto an automated
capillary western blotting system. Anti-αSMA antibody or anti-GAPDH antibody were
applied, followed by anti-rabbit immunoglobulin. Signal intensities were corrected by
GAPDH and are shown in the bar graph. Experiments were performed in duplicate wells. All
data are expressed as the mean ± SD (*P < 0.05).
Figure 2. TM6SF2 suppresses αSMA induction by TGFβ1 in LX-2 cells
A) The cloned TM6SF2 expression plasmid (p3FLAG/TM6SF2-MT) and empty vector
(Mock) were transiently transfected into LX-2 cells followed by 24 hours of incubation. LX-2
cells were stimulated with or without 10 ng/ml of TGFβ1 for 48 hours. Intracellular αSMA
expression was measured by quantitative PCR. The expression of GAPDH served as a control.
Experiments were performed in triplicate wells. All data are expressed as the mean ± SD (*P
< 0.05).
B) Non-treated and TM6SF2 knocked-down LX-2 cells were stimulated with or without
10ng/mL of TGFβ1 for 48 hours and intracellular αSMA expression was compared via
quantitative PCR. The expression of GAPDH served as a control. Experiments were
performed in triplicate wells. All data are expressed as the mean ± SD (*P < 0.05).
Figure 3. The impact of TM6SF2 phenotype on αSMA induction in LX-2 cells
A) The cloned TM6SF2 expression plasmid consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. Intracellular αSMA expression was measured by
quantitative PCR, with the expression of GAPDH serving as a control. Experiments were
17
performed in triplicate wells. All data are expressed as the mean ± SD.
B) Cloned TM6SF2 expression plasmids consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. LX-2 lysates were transferred onto an automated
capillary western blotting system. Anti-TM6SF2 antibody or anti-GAPDH antibody were
applied, followed by anti-rabbit immunoglobulin. Signal intensity was corrected by GAPDH,
as shown in the bar graph. Experiments were performed in triplicate wells. All data are
expressed as the mean ± SD.
C) Cloned TM6SF2 expression plasmids consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT and empty vector (Mock) were transiently transfected into LX-2 cells,
followed by 24 hours of incubation. LX-2 cells were stimulated with or without 10 ng/ml of
TGFβ1 for 48 hours. Intracellular αSMA expression was measured by quantitative PCR, with
GAPDH as a control. Experiments were performed in triplicate wells. All data are expressed
as the mean ± SD.
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Supplementary Figure 1. Fluorescence intensities of TM6SF2 in LX-2 cells
Non-treated and TM6SF2 knockdown LX-2 were stained with anti-TM6SF2 monoclonal
antibody. The bound antibodies were detected with an Alexa 594-conjugated antibody against
rabbit IgG. Fluorescence intensities of TM6SF2 were shown in box-whisker plot (*P < 0.05).
Supplementary Figure 2. Immunostaining of TM6SF2 LX-2 cells
Non-treated and TM6SF2 knockdown LX-2 were stained with anti-TM6SF2 monoclonal
antibody. The bound antibodies were detected with an Alexa 594-conjugated antibody (red)
against rabbit IgG. Nuclei were counterstained with bisbenzimide H 33258 (blue).
Supplementary Figure 3. TM6SF2 regulates COL1A1 expression in LX-2 cells
Intracellular COL1A1 (collagen type 1 alpha 1) expression, measured by quantitative PCR,
was compared in non-treated and TM6SF2 knocked-down LX-2 cells. GAPDH expression
was used as a control. Experiments were performed in triplicate wells. All data are expressed
as the mean ± SD (*P < 0.05).
Supplementary Figure 4. The impact of TM6SF2 phenotype on COL1A1 induction in
LX-2 cells
The cloned TM6SF2 expression plasmid consisting of p3FLAG/TM6SF2-WT and
p3FLAG/TM6SF2-MT were transiently transfected into LX-2 cells, followed by 24 hours of
incubation. TM6SF2 overexpressing or non-treated LX-2 cells were stimulated with or
without 10ng/mL of TGFB for 48 hours and compared by intracellular COL1A1 expressions
measured by quantitative PCR. The expression of GAPDH served as a control. Experiments
were performed in triplicate wells. All data are expressed as the mean ± SD.
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