TAK1 promotes cell survival by TNFAIP3 and IL-8 dependent and NF-κB independent pathway in HeLa cells exposed to heat stress

Figures & data

Figure 1. Decreased cell survival in TAK1 knockdown cells (shTAK1) in comparison to parental cells (shLuc) after HS-exposure. (A) Clonogenic survival assay. Cells were exposed to HS for 15, 30, or 45 min. After 12 days incubation, colonies containing approximately 50 or more cells were counted. Data are presented as mean ± SD (n = 3). *indicates statistical significance compared to shLuc (*p < 0.05, **p < 0.01). (B) Western blots for caspase-3. Control and HS-treated cells were incubated for 24 h before analysis.

Figure 2. HSP induction and NF-κB phosphorylation in TAK1 knockdown cells in comparison to parental cells exposed to HS. (A) Expression level of TAK1 in control TAK1 knockdown versus its expression in control parent cells. (B) Time-dependent induction of Hsp70, 40, and 27 in cells exposed to HS. (C) Phosphorylation levels of NF-κB induced by HS. Band density was quantified and expressed as fold changes over the untreated control using Image J software.

Figure 3. Global gene expression and bioinformatics analyses of TAK1 knockdown cells in comparison to parental cells after HS exposure. (A) The number of differentially expressed genes by a factor of ≥3.0 in HS-treated cells. Venn diagram indicates the commonly and specifically up-regulated genes in each cell line. (B) The identified responsive genetic networks to HS. The up-regulated genes by a factor of ≥3.0 in treated parent cells are included in the networks. Relative gene expression ratios are shown in Tables I and II. (C) Differential induction levels of TNFAIP3 and IL-8 proteins in control cells and TAK1 knockdown cells 6 h after HS exposure. Band density was quantified and expressed as fold changes over the untreated control using Image J software.

Table I. Relative gene expression levels of differentially expressed genes in identified gene network I.

Probe set ID	A. shLuc log	B. shLuc HS log	C. shTAK1 log	D. shTAK1 HS log	B vs A fold	D vs C fold	D vs B fold	Gene symbol	Gene title
213418_at	−5.07	5.07	−5.28	5.38	1126.4	1613.2	0.8	HSPA6	Heat shock 70 kDa protein 6 (HSP70B')
117_at	−4.34	4.34	−5.05	4.72	411.6	869.6	0.8	HSPA6	Heat shock 70 kDa protein 6 (HSP70B')
233694_at	−2.23	2.34	−2.58	2.23	23.8	28.1	1.1	HSPA1L	Heat shock 70 kDa protein 1-like
230031_at	−1.98	2.38	−2.06	1.98	20.6	16.5	1.3	HSPA5	Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)
210189_at	−2.15	2.15	−2.70	2.66	19.6	41.3	0.7	HSPA1L	Heat shock 70 kDa protein 1-like
200664_s_at	−1.84	2.21	−2.39	1.84	16.6	18.7	1.3	DNAJB1	DnaJ (Hsp40) homolog, subfamily B, member 1
202581_at	−1.86	1.86	−2.38	1.98	13.2	20.5	0.9	HSPA1A///HSPA1B	Heat shock 70 kDa protein 1 A///heat shock 70 kDa protein 1B
200666_s_at	−1.50	1.50	−1.98	1.56	8.0	11.6	1.0	DNAJB1	DnaJ (Hsp40) homolog, subfamily B, member 1
203810_at	−1.31	1.31	−1.73	1.55	6.1	9.7	0.8	DNAJB4	DnaJ (Hsp40) homolog, subfamily B, member 4
200800_s_at	−1.22	1.24	−2.39	1.22	5.5	12.2	1.0	HSPA1A///HSPA1B	Heat shock 70 kDa protein 1 A///heat shock 70 kDa protein 1B
1556053_at	−1.06	1.14	−0.93	0.93	4.6	3.6	1.2	DNAJC7	DnaJ (Hsp40) homolog, subfamily C, member 7
221667_s_at	−1.47	0.62	−0.62	1.22	4.3	3.6	0.7	HSPB8	Heat shock 22 kDa protein 8

The italic values indicate expression levels in TAK1 knockdown (shTAK1) or their parental cells (shLuc) with or without HS-exposure. The other three columns indicate the fold changes in shTAK1 or shLuc or both after HS-exposure.

Differentially expressed genes by a factor of 3 or more in either HS-treated TAK1 knockdown (shTAK1) or their parental cells (shLuc) were shown as fold changes.

B vs A fold: the fold changes in shLuc after HS-exposure.

D vs C fold: the fold changes in shTAK1 after HS-exposure.

D vs B fold: the fold changes between two cell lines after HS-exposure.

Table II. Relative gene expression levels of differentially expressed genes in identified gene network II.

Probe Set ID	A. shLuc log	B. shLuc HS log	C. shTAK1 log	D. shTAK1 HS log	B vs A fold	D vs C fold	D vs B fold	Gene symbol	Gene title
211506_s_at	−2.40	4.26	−1.58	1.58	101.2	9.0	6.4	IL8	Interleukin 8
205680_at	−1.24	3.58	−3.49	1.24	28.1	26.5	5.1	MMP10	Matrix metallopeptidase 10 (stromelysin 2)
236561_at	−0.20	1.50	0.20	−0.56	3.2	0.6	4.2	TGFBR1	Transforming growth factor, beta receptor 1
1559529_at	−0.48	2.25	−0.71	0.48	6.6	2.3	3.4	PTK2	PTK2 protein tyrosine kinase 2
202859_x_at	−2.57	2.47	−1.05	1.05	32.8	4.3	2.7	IL8	Interleukin 8
232098_at	−1.59	1.87	−0.60	0.60	11.0	2.3	2.4	DST	Dystonin
202644_s_at	−1.08	1.71	−0.45	0.45	6.9	1.9	2.4	TNFAIP3	Tumour necrosis factor, alpha-induced protein 3
202643_s_at	−0.67	1.41	−0.29	0.29	4.3	1.5	2.2	TNFAIP3	Tumour necrosis factor, alpha-induced protein 3
209774_x_at	−0.84	1.51	−0.47	0.47	5.1	1.9	2.1	CXCL2	Chemokine (C-X-C motif) ligand 2
240221_at	−0.42	1.25	−0.96	0.42	3.2	2.6	1.8	CSNK1A1	Casein kinase 1, alpha 1
222043_at	−0.73	1.27	−0.78	0.73	4.0	2.9	1.5	CLU	Clusterin
205207_at	−1.47	0.73	−0.45	0.45	4.6	1.9	1.2	IL6	Interleukin 6 (interferon, beta 2)
226333_at	−0.96	0.71	−0.44	0.44	3.2	1.8	1.2	IL6R	Interleukin 6 receptor
1556006_s_at	−1.10	0.91	−0.64	0.64	4.0	2.4	1.2	CSNK1A1	Casein kinase 1, alpha 1
211924_s_at	−1.75	0.17	0.00	0.00	3.8	1.0	1.1	PLAUR	Plasminogen activator, urokinase receptor
201110_s_at	−1.39	0.48	−0.42	0.42	3.7	1.8	1.0	THBS1	Thrombospondin 1
204420_at	−1.95	0.70	−0.70	0.85	6.3	2.9	0.9	FOSL1	FOS-like antigen 1
202912_at	−1.18	0.55	−0.55	0.93	3.3	2.8	0.8	ADM	Adrenomedullin

The italic values indicate expression levels in TAK1 knockdown (shTAK1) or their parental cells (shLuc) with or without HS-exposure. The other three columns indicate the fold changes in shTAK1 or shLuc or both after HS-exposure.

Figure 4. Involvement of TNFAIP3 and IL-8 in apoptosis of HeLa cells exposed to HS. (A, B) Densitometric quantification of TNFAIP3 and IL-8 expression analysed by qPCR following knockdown of TNFAIP3 and IL-8 using siRNAs and / or exposure to HS (44 °C, 30 min). Cells were transfected with siRNA 24 h before HS exposure. (C) Promotion of caspase-3 cleavage by knockdown of TNFAIP3 and IL-8 in cells 24 h after HS exposure. Band density was quantified and expressed as fold changes over the untreated control using Image J software. (D) Depression of cell survival by knockdown of TNFAIP3 and IL-8 in cells 48 h after HS-exposure. Cell survival analysis was performed by CCK-8. Data are presented as mean ± SD (n = 3). *Statistical significance from siLuc with or without HS treatment (*p < 0.05, **p < 0.01).