. Author manuscript; available in PMC: 2012 Dec 1.

Published in final edited form as: Cytoskeleton (Hoboken). 2011 Nov 29;68(12):671–693. doi: 10.1002/cm.20544

Table 3.

Detection of IL-1β and IL-8 gene expression and protein production in MDA-MB-231 cells.

(A) Measurement of IL-1β and IL-8 gene expression and protein production in HA-treated MDA-MB-231 cells:
Treatment	(a) Amount of Pro-inflammatory Cytokine & Chemokine Gene Expression (Relative Abundance)^*		(b) Amount of Pro-inflammatory Cytokine & Chemokine Protein Production^**
Treatment	IL-1β Gene Expression (% of control)	IL-8 Gene Expression (% of control)	IL-1β Protein Production (pg/ml) (% of control)	IL-8 Protein Production (pg/ml) (% of control)
No HA-treated (Control)	100 ± 3.0	100 ± 2.2	85 ± 4.1 (100%)	88 ± 1.8 (100%)
LMW-HA-treated	268 ± 15^a	295 ± 5.6^b	232 ± 10.8 (273%)^c	233 ± 8.4 (265%)^d
LMW-HA +anti-CD44-reated	98 ± 2.4^a	92 ± 1.2^b	70 ± 3.2 (82%)^c	73 ± 1.5 (83%)^d
HMW-HA-treated	102 ± 0.8^a	107 ± 1.6^b	84 ± 3.2 (99%)^c	91 ± 1.8 (102%)^d
Scrambled siRNA-treated (No HA)	105 ± 1.2^a	102 ± 1.0^b	86 ± 4.2 (101%)^c	90 ± 2.9 (102%)^d
Scrambled siRNA-Treated (+LMW-HA)	222 ± 9.0^a	238 ± 6.0^b	238 ± 12.2 (280%)^c	241 ± 14.2 (274%)^d
CD44 siRNA-treated (+LMW-HA)	44 ± 0.4^a	50 ± 0.3^b	44 ± 2.5 (52%)^c	42 ± 1.1 (47%)^d
TLR2 siRNA-treated (+LMW-HA)	210 ± 10.0^a	202 ± 16.0^b	207 ± 10.4 (243%)^c	199 ± 12 (226%)^d
TLR4 siRNA-treated (+LMW-HA)	217 ± 12.0^a	220 ± 18.0^b	221 ± 8.9 (261%)^c	200 ± 18 (228%)^d
TLR2 siRNA+TLR siRNA-treated (+LMW-HA)	62 ± 0.6^a	69 ± 0.7^b	47 ± 1.7 (55%)^c	56 ± 2.1 (63%)^d
Normal IgG + no HA-treated	102 ± 0.8^a	107 ± 1.6^b	84 ± 2.2 (99%)^c	91 ± 1.4 (103%)^d
Normal IgG + LMW-HA-treated	222 ± 0.9^a	238 ± 0.6^b	231 ± 3.2 (272%)^c	241 ± 2.8 (274%)^d
HA fragment (2–3 disaccharide)-treated	102 ± 0.8^a	107 ± 1.6^b	80 ± 2.4 (94%)^c	87 ± 1.5 (99%)^d

(B) Effects of MyD88 siRNA treatment on IL-1β and IL-8 gene expression & protein production in HA-treated MDA-MB-231 cells:
	(a) Amount of Pro-inflammatory Cytokine & Chemokine Gene Expression (Relative Abundance)^*				(b) Amount of Pro-inflammatory Cytokine & Chemokine Protein Production^**
	IL-1β Gene Expression (% of control)		IL-8 Gene Expression (% of control)		IL-1β Protein Production (pg/ml) (% of control)		IL-8 Protein Production (pg/ml) (% of control)
Treatment	Scrambled siRNA-treated	MyD88 siRNA-treated	Scrambled siRNA-treated	MyD88 siRNA-treated	Scrambled siRNA- treated	MyD88 siRNA-treated	Scrambled siRNA-treated	MyD88 siRNA-treated
No HA (control)	100 ± 5	85± 2^e	100 ± 2	82 ± 2^f	84 ± 1.0 (100%)	72 ± 0.2 (86%)^g	85 ± 1.1 (100%)	70 ± 0.5 (82%)^h
LMW-HA Treatment	285 ± 10^e	80± 3^e	294 ± 12^f	84 ± 1^f	235 ± 4.8 (280%)^g	74 ± 0.3 (88%)^g	221 ± 2.2 (260%)^h	72 ± 0.6 (85%)^h
HMW-HA Treatment	105 ± 4^e	88± 2^e	104 ± 1^f	86 ± 2^f	88 ± 0.8 (105%)^g	71 ± 0.2 (84%)^g	92 ± 0.7 (108%)^h	75 ± 0.2 (88%)^h
HA fragment (2–3 disaccharide) treatment	98 ± 3^e	83± 3^e	96 ± 2^f	80 ± 3^f	85 ± 0.4 (101%)^g	68 ± 0.1 (81%)^g	82 ± 0.5 (97%)^h	77 ± 0.2 (91%)^h

(C) Effects of AFAP-110 siRNA treatment on IL-1β and IL-8 gene expression & protein production in HA-treated MDA-MB-231 cells:
	(a) Amount of Pro-inflammatory Cytokine & Chemokine Gene Expression (Relative Abundance)^*				(b) Amount of Pro-inflammatory Cytokine & Chemokine Protein Production^**
	IL-1β Gene Expression (% of control)		IL-8 Gene Expression (% of control)		IL-1β Protein Production (pg/ml) (% of control)		IL-8 Protein Production (pg/ml) (% of control)
Treatment	Scrambled siRNA-treated	AFAP-110 siRNA-treated	Scrambled siRNA-treated	AFAP-110 siRNA-treated	Scrambled siRNA-treated	AFAP-110 siRNA-treated	Scrambled siRNA-treated	AFAP-110 siRNA-treated
No Treatment (Control)	100 ± 3	83 ± 3ⁱ	100 ± 2	88 ± 2^j	84 ± 1.2 (100%)	70 ± 0.3 (83%)^k	82 ± 1.2 (100%)	72 ± 0.2 (86%)^l
LMW-HA Treatment	289 ± 13ⁱ	89 ± 2ⁱ	300 ± 16^j	85 ± 3^j	235 ± 1.8 (280%)^k	67 ± 0.2 (80%)^k	238 ± 1.6 (290%)^l	71 ± 0.1 (87%)^l
HMW-HA Treatment	104 ± 3ⁱ	82 ± 2ⁱ	106 ± 7^j	82 ± 2^j	90 ± 1.3 (107%)^k	69 ± 0.4 (82%)^k	96 ± 1.7 (117%)^l	68 ± 0.2 (83%)^l
HA fragment (2–3 disaccharide) treatment	98 ± 2ⁱ	84 ± 1ⁱ	96 ± 5^j	85 ± 3^j	86 ± 1.0 (102%)^k	71 ± 0.4 (85%)^k	86 ± 1.4 (105%)^l	65 ± 0.1 (79%)^l

The expression of NF-κB target genes (e.g., IL-1β and IL-8) was measured using specific primers and Q-PCR in MDA-MB-231 cells according to the procedures described in the Materials and Methods. Relative mRNA expression levels of IL-1β or IL-8 in various treatments were calculated after normalization with 36B4 mRNA levels as determined by Q-PCR. IL-1β or IL-8 gene expression from untreated cells (Table 3A-control) or scrambled siRNA-treated cells (Table 3B-control or Table 3C-control) is designated as 100%.

^**

For the measurement of IL-1β and IL-8 protein production, MDA-MB-231 cells [untreated or pretreated with anti-CD44 or pretreated with various siRNAs (e.g., CD44siRNA or TLR2 siRNA or TLR4 siRNA or MyD88 siRNA or AFAP-110 siRNA or scrambled siRNA] were washed three times with serum free (SF)-DMEM and incubated in 3 ml of serum free-DMEM containing various reagents [e.g., LMW-HA or HMW-HA or HA fragment (2–3 disaccharide) no HA treatment] for 24 hr. Subsequently, concentrations of IL-1β or IL-8 secreted in the medium from these cells were determined using the Quantikine immunoassay for IL-1β or IL-8 (R & D Systems). IL-1β or IL-8 protein production in the medium from untreated cells (Table 3A-control) or scrambled siRNA-treated cells (Table 3B-control or Table 3C-control) is designated as 100%. All data represent mean ± SEM of the amount of IL-1β or IL-8 gene expression and protein production detected in each sample. The value represents an average of triplicate determinations of six experiments with an S.D. value less than ±5%.

^{a, b, c & d}

Statistically significant (p<0.001; analysis of variance; n=6) as compared with control samples [e.g., untreated cells (control)].

^{e, f, g, h, i, j & l}

Statistically significant (p<0.005; analysis of variance; n=6) as compared with control samples [e.g., scrambled siRNA-treated cells (control) with no HA].