C/EBPβ mediates TNF-α-induced cancer cell migration by inducing MMP
expression dependent on p38 MAPK
Peiyi Xia, Rui Zhang and Gaoxiang Ge
The supplementary material contains 4 supplementary figures and 1
supplementary table.
Figure S1. C/EBPβ is required for TNF-α-induced cell migration. (A) Cell
migration of 10 ng/mL TNF-α-treated MDA-MB-231 cells expressing Scramble or
C/EBPβ shRNAs was determined using scratching assays. (B) Cell migration of
MDA-MB-231 cells treated with 10 ng/mL TNF-α and 10 M SB203580 was
determined using scratching assays. (C) Cell migration of C/EBPβ-expressing
MDA-MB-231 cells treated with 10 M SB203580 was determined using scratching
assays. Data are presented as mean ± SD of six experiments. **P < 0.01, ***P <
0.001.
1
Figure 2. Activation of NF-B or JNK is not required for TNF-α-induced
C/EBPβ expression. (A) MDA-MB-231 cells (3×104 cells/well in a 24-well
Transwell chamber) were treated with 10 ng/mL TNF-α in Transwell assays along
with 10 M IKK inhibitor TPCA-1 or 1 M JNK inhibitor SP600125 for 20 hr. Cell
migration was quantified by counting the number of cells that migrated to the lower
side of the filter within 5 randomly selected microscope fields. The experiments were
performed in triplicates, and repeated three times. Data are presented as mean ± SD of
three independent experiments. **P < 0.01. NS: not significant. (B) Western blot of
C/EBPβ, pIKB and IKB in MDA-MB-231 cells treated with 10 ng/mL TNF-α and
10 M TPCA-1 for 24 hr. (C) Western blot of C/EBPβ, pJNK and JNK in
MDA-MB-231 cells treated with 10 ng/mL TNF-α and 1 M SP600125 for 24 hr.
2
Figure S3. Cell viability was not affected by TNF-α treatment or altered C/EBPβ
expression. (A) Cell viability of MDA-MB-231 cells expressing Scramble or C/EBPβ
shRNAs treated with or without 10 ng/mL TNF-α was determined using MTT assays.
(B) Cell viability of MDA-MB-231 cells expressing C/EBPβ or individual isoforms
was determined using MTT assays. Data are presented as mean ± SD of four
experiments.
3
Figure S4. Significantly enriched Gene Ontology (GO) terms associated with
C/EBPβ expression in cancer cells. The primary functions of the sorted differentially
expressed genes in C/EBPβ-expressing MDA-MB-231 cells compared with control
cells were assessed by GO analysis. Ten significantly enriched GO terms are shown.
The vertical axis is the GO category and the horizontal axis is the enrichment value of
GO.
4
Table S1. Genes significantly upregulated in C/EBPβ-expressing MDA-MB-231
cells
Probe
A_23_P161698
A_23_P79518
A_23_P1691
A_23_P91230
A_24_P347378
A_23_P85209
A_24_P190472
A_23_P2793
A_23_P501754
A_32_P87013
A_23_P202978
A_23_P7144
A_23_P127584
A_24_P250922
A_24_P11506
A_24_P183150
A_23_P56898
Gene
Symbol
MMP3
IL1B
MMP1
SLPI
ALOX5AP
IL13RA2
SLPI
ALOX5AP
CSF3
IL8
CASP1
CXCL1
NNMT
PTGS2
KYNU
CXCL3
KYNU
Description
matrix metallopeptidase 3
interleukin 1, beta
matrix metallopeptidase 1
secretory leukocyte peptidase inhibitor
arachidonate 5-lipoxygenase-activating protein
interleukin 13 receptor, alpha 2
secretory leukocyte peptidase inhibitor
arachidonate 5-lipoxygenase-activating protein
colony stimulating factor 3
interleukin 8
caspase 1, apoptosis-related cysteine peptidase
chemokine (C-X-C motif) ligand 1
nicotinamide N-methyltransferase (NNMT)
prostaglandin-endoperoxide synthase 2
kynureninase
chemokine (C-X-C motif) ligand 3
kynureninase
5
Fold change
(Log2 transformed)
8.44
8.14
7.96
7.86
7.07
6.73
6.61
6.48
6.39
6.39
5.97
5.51
5.20
5.00
4.90
4.89
4.82