Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: Role of cytokines

Table 1 List of human primers sequences used for polymerase chain reactions

Primer	5’→3’ forward	5’→3’ reverse
Human β-actin	CTG GCACCCAGCACAATG	CCGATCCACACGGAGTACTTG
Human CXCL1 (GROα)	GTGTGAACGTGAAGTCCCCC	GCTGCAGAAATCAGGAAGGC
Human CXCL8 (IL-8)	ATGACTTCCAAGCTGGCCG	GCTGCAGAAATCAGGAAGGC
Human CXCL10 (IP-10)	CCAGAATCGAAGGCCATCAA	CATTTCCTTGCTAACTGCTTTCAG
Human TNFα	CCCAGGCAGTCAGATCATCTTC	AGCTGCCCCTCAGCTTGA
Human IFNγ	CCAACGCAAAGCAATACATGA	TTTTCGCTTCCCTGTTTTAGC
Human IL-1β	AATTTGAGTCTGCCCAGTTCCC	AGTCAGTTATATCCTGGCCGCC
Human TNFα Rec1	AGGAAGAACCAGTACCGGCAT	TCTGTTTCTCCTGGCAGGAGA
Human IL1β Rec1	TGTTCAGGAGCTGAAGCCCAT	AATTCACACAGCAGGACAG
Human IFNγ Rec1	AAGAGCCGTTGTCTCCAGCAA	TAAAGCGATGCTGCCAGGTTC
Human KRAS Codon 12 and 13	RAS A (Forward): 5' ACTGAATATAAACTTGTGGTCCATGGAGCT 3'
	RAS B (Reverse): 5' TTATCTGTATCAAAGAATGGTCCTGCACCA 3'
	RAS C (Reverse): 5' GGATGGTCCTCCACCAGTAATATGGATATT 3'
Human BRAF V600E	A-Allele (Forward): 5' AAAAATAGGTGATTTTGGTCTAGCTACTGTA 3'
	T-Allele (Forward): 5' AAAAATAGGTGATTTTGGTCTAGCTACTGT 3'
	(Reverse): 5' ACACTGATTTTTGTGAATACTGGGAACT 3'

TNFα:Tumor necrosis factor α; INFγ: Interferon-γ; IL-1β: Interleukin-1β.

Table 2 List of KRAS siRNA sequences for knockdown

KRAS siRNA sequence	5’→3’ forward	5’→3’ reverse
Sequence 1	GCAAGUAGUAAUUGAUGGA	UCCAUCAAUUACUACUUGC
Sequence 2	ACAGGCUCAGGACUUAGCA	UGCUAAGUCCUGAGCCUGU
Sequence 3	GCAAGAAGUUAUGGAAUUCUA	GAAUUCCAUAACUUCUUGCUC

Table 3 List of antibodies used in Western blotting analysis

Primary antibody	Origin	Dilutions primary Ab	Provider
β-actin	Mouse	1:5000	Sigma aldrich
CXCL1/GROα	Goat	1:500	R and D system
CXCL10/IP-10	Goat	1:500	R and D system
CXCL8/IL-8	Goat	1:500	R and D system
MAPK1 (ERK1/2)	Rabbit	1:1000	Cell signalling
IκBα	Rabbit	1:10000	Abcam
KRAS	Rabbit	1:1000	ABBIO technology