Supplementary Data
Ju et al.
Cytokeratin19 Induced by HER2/ERK Binds and Stabilizes HER2 on Cell Membranes
Supplementary Table & Figure Legends
Supplementary Table 1 LC/MS-MS identification of differentially expressed KRTs between
MCF-7 vec and MCF-7 HER2 cells.
Supplementary Figure 1 HER2-overexpression increases KRT19 in both detergentextractable and non-extractable fractions. MCF-7 HER2 cells were lysed in Tris-Triton buffer
and both soluble fraction (supernatant) and cell debris (pellet) were analyzed for KRT19
expression.
Supplementary Figure 2 KRT19 remodeling is dependent on Ser35 phosphorylation by Akt.
(a) Ectopic KRT19 shape in 293T cells was monitored by immunostaining. KRT19 constructs
were co-transfected with Akt1-CA or Akt1-KD in 293T cells. Monitoring of KRT19 shape was
conducted by immunostaining using KRT19 antibody. (b) KRT19 constructs were cotransfected with pcDNA, HER2-WT, HER2-CA, HER2-KD, MEK1-CA, MEK2-CA, ERK1-WT,
ERK2-WT, p38 MAPK-WT or p38 MAPK-KD in 293T cells. KRT19 remodeling was
monitored by KRT19 immunostaining using KRT19 antibody and anti-mouse IgG-Cy3
secondary antibody.
Supplementary Figure 3 KRT19 is physically associated with HER2 in the plasma
membrane and HER2/Akt induced phosphorylation of KRT19 at Ser35 can induce
membrane localization of KRT19. (a) BT-474 cells were stained with HER2 antibody (green)
or KRT19 antibody (red). The samples were then subjected to serial Z-coordinate optical
sectioning using Z-sectioning (2 μm/section) with an Olympus laser scanning confocal
microscope. Serial sections were shown to indicate the spatial organization of KRT19 and
association of KRT19 and HER2. Rightmost panels are the zoom-in images of boxed
regions in 8 µm sections. (b) KRT19 constructs were co-transfected with pcDNA, HER2-WT,
HER2-CA, HER2-KD, Akt1-CA and Akt1-KD in 293T cells. Cellular relocalization of KRT19
was monitored by cell surface immunostaining and subcellular fractionation into cytosol
membrane and cytosol fractions.
Supplementary Figure 4 Photographs of each xenograft group.
Supplementary Figure 5 HER2-induced release of KRT19 to culture medium. (a) A
conditioned media from both MCF-7 vec and MCF-7 HER2 cells were collected and
concentrated using various methods as indicated (C: centricon precipitation, CM:
chloroform/methanol precipitation, TA: TCA/acetone precipitation, A: ammonium sulfate
precipitation). (b) MCF-7 HER2 cells were treated with dimethyl amiloride (DMA) as an
exosome inhibitor for 48 h to see its effect on exosome-based secretion mechanism of
KRT19. (c) MCF-7 HER2 cells were treated with Akt inhibitor or DMA for 1 h. The cell lysates
incubated with HER2 antibody, and the immunoprecipitates were subjected to western blot
analyses with KRT19 antibody. (d) Kinase inhibitors were treated to see their effects on
KRT19 release in MCF-7 HER2 cells. Cell medium was concentrated by TCA/acetone
precipitation method.
Supplementary Figure 6 Biphasic effect of KRT19 on cancer cell proliferation and its
downstream signaling. (a) BT474, SKBR3 and MCF7 HER2 cells were transfected with
varying amount of KRT19 shRNA. Cell lysates from each cell lines were subjected to
western blot analyses using KRT19 antibody to detect endogenous KRT19 protein
expression. (b) The lysates were divided into nuclear and cytosolic fractions. Each fraction
subjected to the indicated western blot analyses with indicated antibodies. (c) mRNA from
transfected MCF7 HER2 cells with KRT19 shRNA was analyzed by RT-PCR using primers
specific for PTEN and GAPDH (as loading control). (d) MCF7 HER2 cells were seeded at a
density of 5 × 103 cells per well in 96-well dishes and transfected with varying amount of
KRT19 shRNA. Each group was analyzed by MTT assays for 72 h.