Figure 1. Efficiency of GALNT2 knockdown. (A) After transfected with 10nM
non-targeting siRNA control (siC) or GALNT2 siRNA (siGALNT2) for 24 hours, cell
lysates were collected and the expression of GALNT2 was confirmed by Western blot
analysis. GALNT2 was significantly lower in siGALNT2 group when compared with
siC group. (B) The relative protein level of GALNT2 was quantified and normalized to
GAPDH. (C) The relative mRNA level of GALNT2 obtained from qPCR was
quantified and normalized to GAPDH. Results are represented as mean ± S.D. from
three independent experiments. *P < 0.05.
Figure 2. Effect of GALNT2 knockdown on EGFR phosphorylation. (A) After
transfection for 24 hours, cells were starved for 6 hours followed by stimulated with
DMSO or EGF (50ng/ml) for 10 min and then cell lysates were collected for Western
blot analysis. Without EGF, there was no difference between groups. However, in the
presence of EGF, phospho-EGFR significantly increased compared with siC group. (B)
The expression of phospho-EGFR was quantified and normalized to GAPDH. Results
are represented as mean ± S.D. from three independent experiments. *P < 0.05.
Figure 3. Effect of GALNT2 knockdown on EGFR glycosylation. (A)After
24h-transfection, lysates were pulled down by Vicia Villosa Lectin (VVA)-conjugated
agarose beads. Total lysates and pulled-down lysates were immunoblotted (IB) with
anti-EGFR antibody. (B) VVA-binding EGFR was quantified and normalized to total
EGFR. GALNT2 knockdown decreased VVA-binding to EGFR. This result indicates
that GALNT2 modifies O-glycosylation of EGFR. The results are represented as mean
± S.D. from three independent experiments. *, P < 0.05.
Figure 4. GALNT2 knockdown or gefitinib did not affect cell viability. After
24h-transfection, cells were incubated with 200µl RPMI medium+ 10% FBS treated
with either DMSO or 1µM gefitinib. Absorbance of sample was measured at day1, 2, 3,
4 and 5. There were no differences during day1-5 between siGALNT2 and siC group
treated with DMSO or gefitinib. The results are represented as mean ± S.D. from three
independent experiments. *, P < 0.05.
Figure 5. GALNT2 knockdown enhanced cell migration through increasing
EGFR phosphorylation. (A) After 24h-transfection, cells (3 ×104) were re-suspended
in 200µl of serum-free RPMI containing EGF(50ng/ml) and treated with/ without 1µΜ
gefitinib. The migrated cells were counted after 24h. (A) GALNT2-knockdown
increased the number of migrated cells compared with siC group, but the addition of
gefitinib would abolish this phenomenon. (B) The number of migrated cells was
calculated at 4 random filelds per experiment. (×100) and expressed as fold change of siC without gefitinib treatment. Results are represented as mean ± S.D. from three independent experiments. *P < 0.05.
Figure 6. GALNT2 knockdown enhanced cell invasion through increasing
EGFR phosphorylation.(A) After 24h-transfection, cells (3 ×104) were re-suspended in 200µl RPMI medium+ 10% FBS of serum-free RPMI containing EGF(50ng/ml) and
treated with/ without 1µΜ gefitinib. The invaded cells were counted after 24h. (A)
GALNT2-knockdown increased the number of invaded cells compared with siC group,
but the addition of gefitinib would abolish this phenomenon. (B) The number of
invaded cells was calculated at 4 random filelds per experiment. (×100) and expressed as fold change of siC without gefitinib treatment. Results are represented as mean ± S.D.
from three independent experiments. *P < 0.05.
Figure 7. Effect of EGFR inhibitor (gefitinib) on Akt and ERK1/2
phosphorylation and total form. (A) After 24h-transfection, cells were starved for 6h
and then treated with 1µM gefitinib followed by 10-min stimulation of EGF.
Subsequently, cell lysates were collected for Western blot analysis. Expression of
phospho-Akt significantly increased in the siGALNT2 group compared to the siC goup,
and this phenomenon would be reversed by the addition of gefitinib. However, total
ERK, Akt and phospho-ERK showed no difference between siC and siGALNT2 treated
with/without gefitinib. (B) Expression of phospho-Akt (left panel) and phospho-ERK
(right panel) were quantified and normalized to GAPDH. Results are represented as
mean ± S.D. from three independent experiments. *P < 0.05.
Figure 8. Inhibition of Akt phosphorylation decreased cell viability. After
24h-transfection, cells were incubated with 200µl RPMI medium+ 10% FBS treated
with either DMSO or 1µM MK2206. Absorbance of sample was measured at day1, 2, 3
and 4. There were no differences between siGALNT2 and siC group. However, cell
viability was significantly lower in groups treated with MK2206 than groups treated
with DMSO. Results are represented as mean ± S.D. from three independent
experiments. *P < 0.05, siG2+DMSO compared to siG2+MK2206; **P < 0.05,
siC+DMSO compared to siC+MK2206
Figure 9. GALNT2 knockdown enhanced cell migration through increasing Akt
phosphorylation. (A) After 24h-transfection, cells (3 ×104) were re-suspended in
200µl of serum-free RPMI containing with/ without 1µΜ MK2206. The migrated cells
were counted after 24h. (A) GALNT2-knockdown increased the number of migrated
cells compared with siC group, but the addition of MK2206 would abolish this
phenomenon. (B) The number of migrated cells was calculated at 4 random filelds per
experiment. (×100) and expressed as fold change of siC without MK2206 treatment.
Results are represented as mean ± S.D. from three independent experiments. *P < 0.05.
Figure 10. GALNT2 knockdown enhanced cell invasion through increasing Akt
phosphorylation. (A) After 24h-transfection, cells (3 ×104) were re-suspended in
200µl of serum-free RPMI containing with/ without 1µΜ MK2206. The migrated cells
were counted after 24h. (A) GALNT2-knockdown increased the number of invaded
cells compared with siC group, but the addition of MK2206 would abolish this
phenomenon. (B) The number of invaded cells was calculated at 4 random filelds per
experiment. (×100) and expressed as fold change of siC without MK2206 treatment.
Results are represented as mean ± S.D. from three independent experiments. *P < 0.05.
Figure 11. Correlation between GALNT2 and p-EGFR expression.
(A) Representative IHC staining of GALNT2 (B) Representative IHC staining of
p-EGFR. ; No staining = 0, weak staining =1, moderate staining = 2, strong staining = 3
(C) The expression of GALNT2/p-EGFR equals IHC intensity multiplies staining
percentage. p-EGFR was positively correlated with GALNT2. The Pearson correlation
coefficients (r) was 0.3281, p=0.0076
R² = 0.10766
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12
GALNT2 expression score
p-EGFR expression score
Figure 12. Kaplan-Meier survival analysis of progression-free survival in gastric cancer patients with p-EGFR(+) or p-EGFR(-). Of the 65 patients, 26
(40%) were p-EGFR positive and 39 (60%) were p-EGFR negative. The 5-year PFS
was 50.4% for p-EGFR positive and 60.3% for p-EGFR negative group, and there was
no difference between two groups. (p=0.2849)
Table 1. Correlation between p-EGFR and clinico-pathological parameters.
VII. References
11. Gomes, J., Marcos, N. T., Berois, N., Osinaga, E., Magalhaes, A., Pinto-de-Sousa,
20. Masuda, H., Zhang, D., Bartholomeusz, C., Doihara, H., Hortobagyi, G. N., &
29. Nieto, Y., Nawaz, F., Jones, R. B., Shpall, E. J., & Nawaz, S. (2007). Prognostic
38. Guo, G., Gong, K., Wohlfeld, B., Hatanpaa, K. J., Zhao, D., & Habib, A. A. (2015).