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SUPPLEMENTAL FIGURES
Figure S1.
(A) Localization and activation of PTK6 is not appreciably different in
SW480 and SW620 cells. Immunoblotting was performed with lysates from fractionated
parental SW480 and SW620 cell lines as well as SW480 PTK6 knockdown cell lines (M
= membrane, C = cytoplasmic, N = nuclear). PTK6 was primarily in the cytoplasm, but
a small fraction was also detected at the membrane in both cell lines. Active PY342
PTK6 was not detected. Loading controls: ERBB2 was used for membrane fraction and
SP-1 was used for the nuclear fraction. The ProteoExtract Subcellular Proteome
Extraction Kit was purchased from EMD Millipore and cell fractionations were performed
using the kit according to manufacturers instructions. (B) Knockdown of PTK6 leads to
reduced E-cadherin and increased ZEB1 expression in HCT116 cells. Immunoblotting
for PTK6, E-Cadherin, and ZEB1 expression in HCT116 cells with stable knockdown of
PTK6 by shRNA49 and shRNA52 or empty vector (Vec). (C) PTK6 levels increase
during differentiation of Caco-2 cells. Immunoblotting was performed with total Caco-2
cell lysates harvested at 2, 7, 14, and 21 days post-plating. β-Actin was used as a
loading control.
Figure S2. Differential activation of p38 in breast and colon cancer cell lines. The
breast cancer cell line T47D and the colon cancer cell lines SW480 and HCT116
express comparable levels of EGFR, ERBB2, ERBB3 and respond to the ligand
heregulin (Perekatt unpublished data). Previously, heregulin was shown to activate
PTK6 and PTK6-mediated activation of p38 mitogen-activated protein kinase in breast
cancer cells (30). (A) Phosphorylation and activation of p38 (P-p38) was detected in
T47D cells but not SW480 or HCT116 cells. (B and C) Stable knockdown of PTK6 in
colon cancer cell lines enhances p38 activation.
T47D and HCT116 cells were
purchased from American Type Culture Collection (Manassas, VA) and cultured as per
their instructions.
For heregulin treatment, cells were grown to seventy percent
confluence in normal media after which they were serum starved for 48 hours in media
containing 0.1% serum and treated with 25 ng/ml heregulin in serum-starvation media
for indicated times. Total and phospho-p38 antibodies were purchased from Cell
Signaling Technology.
Figure S3. Decreased PTK6 Expression in Tumors in Oncomine Datasets
(A) Expression of PTK6 mRNA in human colon cell lines (43). Supporting the data
presented here, PTK6 expression was significantly reduced in SW620 cells (n = 3)
compared with SW480 cells (n = 3); p-value < 0.01. (B) Expression of PTK6 mRNA in
early onset colorectal cancer (44). Gene expression was examined in tumor specimens
and adjacent normal epithelia from young (<50 years of age) Chinese colon cancer
patients (non-HNPCC). PTK6 transcription is significantly reduced in colorectal cancer
(n = 12) compared with normal colorectal mucosa (n = 10); p-value < 0.01.
(C)
Expression of PTK6 mRNA in human colorectal cancer (45). PTK6 expression is
significantly reduced in colorectal cancer (n = 34) compared with normal colorectal
mucosa (n = 15); p-value < 0.01. (D) Expression of PTK6 mRNA in human colorectal
cancer (46). PTK6 expression is significantly reduced in colorectal cancer (n = 215)
compared with normal colorectal mucosa (n = 22); p-value < 0.01. (E) Expression of
PTK6 mRNA in Head and Neck Squamous Cell Carcinoma (HNSCC) (47). PTK6
expression is significantly reduced in HNSCC (n = 22) compared with normal tissue (n =
22); p-value < 0.01. (F) Expression of PTK6 mRNA in Oral Squamous Cell Carcinoma
(OSCC) (48). PTK6 expression is significantly reduced in OSCC (n = 16) compared with
normal oral mucosa (n = 4); p-value < 0.01. (G) Expression of PTK6 mRNA in human
cervical SCC (49). PTK6 expression is significantly reduced in both cervical SCC (n =
21, p-value < 0.01) and in high grade squamous intraepithelial lesions of the cervix (n =
7, p-value < 0.01) compared with normal cervical tissue (n = 10). (H) Expression of
PTK6 mRNA in human Esophageal Squamous Cell Carcinoma (ESCC) (50). PTK6
expression is significantly reduced in ESCC (n = 17) compared with normal esophageal
mucosa (n = 17); p-value < 0.01. For statistical analysis of Oncomine™ (Compendia
Bioscience, Ann Arbor, MI) datasets (43-50), p-values were determined using a twotailed Student’s t-test (Microsoft Excel 2011).