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Page 4 of 11 Agbo et al. J Cancer Metastasis Treat 2019;5:x I http://dx.doi.org/10.20517/2394-4722.2019.35

°C. Appropriate Alexa Fluor-labeled antibodies (Molecular Probes) were added at 1:500 dilution in blocking
buffer for 30 min at 37 °C. For mice sections, mouse anti-PanCK antibodies conjugated with Alexa 488
(1:100; ThermoFisher Scientific: 53-9003-82) were used. All slides were counterstained with Hoechst 33258
(ThermoFisher Scientific: H3569) and mounted with Fluoromount Aqueous Mounting Medium (Sigma-
Aldrich: F4680). Slides were analyzed using a Nikon eclipse 90i microscope (Nikon Instruments Inc.)
equipped with DS-Qi1Mc and DS-Fi1 CCD cameras (Nikon Instruments Inc.).

Immunofluorescence quantification
For each EMT/KLF4 co-stain, we quantified slides of 4-6 human specimen. For normal adjacent mucosa
and tumor sections, we quantified four separate fields and counted 250-400 cells per each field. Each cell
was labeled as positive or negative for KLF4 and positive or negative for each of the specific EMT markers.
The statistical analysis was performed using a two-tailed Student t test.

Cell culture
SW480 colorectal cancer cell line (ATCC® CCL-228) was maintained in RPMI640 medium supplemented
with 10% FBS and 1% penicillin/streptomycin at 37 °C in atmosphere containing 5% CO . To overexpress
2
Klf4-GFP and GFP-control in SW480 cell line, cells were transiently transfected with 3 μg plasmid DNA
(per well in a six-well plate) using Lipofectamine 2000 reagent (ThermoFisher Scientific) according to
manufacturer’s instructions. The cell lysates were collected using Laemmli buffer and subjected to Western
blot analysis with the following antibodies: rabbit anti-KLF4 (MBL: PM057), rabbit anti-ZEB1 (Cell
Signaling: 3396), rabbit anti-SNAI1 (Cell Signaling: 3879), rabbit anti-SNAI2 (Cell Signaling: 9585), and
mouse anti-actin (SigmaAldrich: A1978). Then, they were developed using secondary antibodies goat anti-
rabbit HRP-conjugated (JacksonImmuno Research: 111-035-144) and goat anti-mouse HRP conjugated
(SigmaAldrich: AP200P), respectively.

Statistical analysis
Student’s paired or unpaired t test was used for statistical analyses. Differences between values were
considered significant when P < 0.05. This analysis was performed using GraphPad Prism version 5.00 for
Windows (GraphPad Software, San Diego, CA).

RESULTS
Expression of KLF4 in human colorectal cancer is negatively correlated with markers of EMT
EMT is a precisely orchestrated multistep process regulated by several transcriptional factors including
[3]
TWIST, SNAI1, SNAI2, and ZEB1 . To determine the correlation between KLF4 expression and EMT in
CRC, we performed immunofluorescence analysis of matched pairs of archived samples from patients after
tumor resections. Firstly, we analyzed the expression pattern of KLF4 and TWIST. As shown in Figure 1A
(SB396N), KLF4 is expressed in the nucleus of epithelial cells in the normal-appearing mucosa adjacent to
the cancer tissues. These cells are positive for PanCK, an epithelial marker, and negative for the nuclear
expression of the biomarker of EMT, TWIST. In contrast, in the tumor samples from the same patient
[Figure 1A, SB396T], the expression of KLF4 is downregulated in the epithelial cells, which is accompanied
by a significant increase in the expression of TWIST. Our statistical analysis showed that there is a
negative correlation between KLF4 and TWIST expression in normal-appearing mucosa and tumor
tissues (P < 0.001). Several common signaling pathways regulate factors involved in EMT including HH,
[19]
WNT, NOTCH, and TGF-β . WNT signaling plays an important role in the homeostasis of the intestinal
epithelium and its deregulation leads to cancer formation which is accompanied by modification of the
pattern and level of expression of its major effector, β-catenin [20-22] . In the normal-appearing mucosa, β-catenin
is predominantly localized to the membrane of the epithelial cells with a modest nuclear staining [Figure 1B,
SB474N]. Upon loss of KLF4 in the colorectal tumor [Figure 1B, SB474T], the levels of the cytoplasmic and