The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research

Figures & data

Figure 1. MAPK11-MAPK14/p38-independent effects of SB202190/SB203580 in autophagy. (A) SB202190 and SB203580 (10 µM each) but not the other more specific MAPK11-MAPK14/p38 inhibitors (SB220025, 10 µM; BIRB-796, 1 µM; or VX-745, 10 µM) induce large vacuoles in HT29 cells (24 h treated). (B) The SB202190-induced vacuoles are acidic compartments as shown by strong acridine orange staining in primary HUVECs. (C) Autophagy inhibitor 3-MA suppresses SB-induced vacuolation in HT29 cells. (D) The efficacy of BIRB-796, SB202190, and SB203580 to inhibit MAPK14/p38α-MAPK11/p38β signaling in HeLa cells was compared by monitoring their effect on stress-induced phosphorylation of the direct MAPK14/p38α-MAPK11/p38β substrate MAPKAPK2 at Thr334 (T334) and of the downstream target HSPB1/HSP27 at Ser82 (S82). The membrane was reprobed with MAPKAPK2, HSPB1 and EEF2 (eukaryotic translation elongation factor 2) antibodies as loading controls. Cells were treated with the indicated concentrations of inhibitors (µM) prior to 30 min anisomycin (10 µg/ml) stimulation. (E and F) The off-target effect of SB202190 in autophagy is independent of cell-type specific vacuolation. In both, vacuole-positive HT29 and vacuole-negative HeLa cells (see Table 1), long-term SB202190 treatment (10 µM for 4 or 24 h) leads to the accumulation of autophagy substrates SQSTM1 and lipid conjugated MAP1LC3B (LC3-II) (E). Quantified band intensities for LC3B-II and SQSTM1 normalized to that of the loading control (GAPDH) are shown (F). (G and H) Dose-dependent (10-30 µM) effect of SB203580 on autophagy in HeLa cells demonstrated by monitoring the levels of SQSTM1 and MAP1LC3B (LC3-II) at 24 h treatment (G). Quantified band intensities for lipid conjugated MAP1LC3B (LC3-II) and SQSTM1 normalized to the loading control (EEF2) are shown (H).

Table 1. Cell-type specificity of SB202190-induced vacuole formation.

No	Cell line	Species	Cell type	Vacuoles
1	AGS	Human	gastric adenocarcinoma	+
2	A549	Human	lung carcinoma	+
3	BHK21	Hamster	adult kidney fibroblast	+
4	C2C12	Mouse	myoblast	−
5	Caco-2 Bbe	Human	colorectal adenocarcinoma	+
6	HCT 116	Human	colorectal adenocarcinoma	+
7	HEK293T	Human	embryonic kidney	−
8	HeLa	Human	cervical adenocarcinoma	−
9	hMSC	Human	primary mesenchymal stem cells	+
10	HT29	Human	colorectal adenocarcinoma	+
11	HUVEC	Human	primary endothelial cells	+
12	IEC6	Rat	small intestinal epithelium	+
13	L929	Mouse	fibrosarcoma	+
14	MCF-10A	Human	mammary epithelial	+
15	MEF-T	Mouse	embryonic fibroblast	+
16	NIH 3T3	Mouse	embryonic fibroblast	−
17	NMuMG	Mouse	mammary epithelial	+
18	RAW 264.7	Mouse	monocytic	+
19	RGM1	Rat	gastric epithelium	+
20	Sh-SY5Y	Human	neuroblastoma	−
21	SW480	Human	colorectal adenocarcinoma	+
22	WM1617	Human	melanoma	−
23	WM793	Human	melanoma	−

The table depicts the cell-type specificity of SB202190-induced autophagy-dependent vacuole formation. Cells were treated with 5 µM SB202190 for 12 h. Vacuoles were clearly visible in most of the cell lines after approximately 2 h of SB202190 treatment.