Acridine orange (Sigma, 158550), anisomycin (Sigma, A9789) and 3-methyladenine (Calbiochem, 189490) were purchased as indicated

Acridine orange (Sigma, 158550), anisomycin (Sigma, A9789) and 3-methyladenine (Calbiochem, 189490) were purchased as indicated. discussed to preserve the high standards of em Autophagy /em . Our major point concerns the analysis of the role of the MAPK11-MAPK14/p38 pathway in the regulation of autophagy by the pyridinyl imidazole class MAPK14/p38-MAPK11/p38-inhibitor SB203580. Several technically robust publications in the past decade have conclusively established a context-dependent role for the stress-activated MAPK11-MAPK14/p38 pathway in the regulation of MTOR signaling and autophagy.2-4 Furthermore, a connection between the MAPK14/p38-MAPK11/p38-activated protein kinase MAPKAPK2/MK2 and autophagy was established recently via demonstrating phosphorylation of BECN1/Beclin-1 at serine 90, using a dominant-negative mutant of MAPK14/p38 instead of MAPK11-MAPK14/p38 inhibitors.5 However, we are deeply concerned about the use of a class of pyridinyl imidazole inhibitors, such as SB203580 and SB202190, in monitoring the role of MAPK14/p38-MAPK11/p38 signaling in autophagy, because we had previously reported that these compounds alter autophagic flux and pro-autophagic gene expression in a cell type-specific, MAPK14/p38-MAPK11/p38-independent manner.6 In the figure panels (Fig.?1A-H), we provide additional data to support our claims that: Open in a separate window Figure 1. MAPK11-MAPK14/p38-independent effects of SB202190/SB203580 in autophagy. (A) SB202190 and SB203580 (10?M each) but not the other more specific MAPK11-MAPK14/p38 inhibitors (SB220025, 10?M; BIRB-796, 1?M; or VX-745, 10?M) 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/p38-MAPK11/p38 signaling in HeLa cells was compared by monitoring their effect on stress-induced phosphorylation of the direct MAPK14/p38-MAPK11/p38 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 (M) prior to 30?min anisomycin (10?g/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?M 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?M) 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). 1. SB202190 and SB203580, but not the structurally nonrelated and more potent MAPK11-MAPK14/p38 inhibitor BIRB-796,7 induce vacuoles (Fig.?1A) characterized as acidic compartments (Fig.?1B) in HT29 cells in a 3-methyladenine (3MA)-sensitive manner (Fig.?1C) indicating a compound-specific, MAPK11-MAPK14/p38-independent autophagic response.6,8,9 2. SB202190 does induce vacuole GW843682X formation in about 70% of the cell lines analyzed when used at very low concentrations (Table?1), but induces accumulation of the autophagy substrate SQSTM1/p62 and lipid-conjugated MAP1LC3B (LC3-II) also in cells, FAAP24 which display no vacuole formation, in a compound-specific, MAPK11-MAPK14/p38-independent manner (Fig.?1E and F). As expected from the structural similarity, SB203580 gave results very similar to SB202190 albeit with less potency (Fig.?1G and H). In contrast, BIRB-796 did not affect the levels of autophagy substrates (Fig.?1ECH), although it effectively blocked MAPK14/p38-MAPK11/p38 signaling as GW843682X monitored by stress-induced downstream phosphorylation events (Fig.?1D) already at low concentrations. Table 1. Cell-type specificity of SB202190-induced vacuole formation. thead th align=”left” rowspan=”1″ colspan=”1″ No /th th align=”center” rowspan=”1″ colspan=”1″ Cell line /th th align=”center” rowspan=”1″ colspan=”1″ Species /th th align=”center” rowspan=”1″ colspan=”1″ Cell type /th th align=”center” rowspan=”1″ colspan=”1″ Vacuoles /th /thead 1AGSHumangastric adenocarcinoma+2A549Humanlung carcinoma+3BHK21Hamsteradult kidney fibroblast+4C2C12Mousemyoblast?5Caco-2 BbeHumancolorectal adenocarcinoma+6HCT 116Humancolorectal adenocarcinoma+7HEK293THumanembryonic kidney?8HeLaHumancervical adenocarcinoma?9hMSCHumanprimary mesenchymal GW843682X stem cells+10HT29Humancolorectal adenocarcinoma+11HUVECHumanprimary endothelial cells+12IEC6Ratsmall intestinal epithelium+13L929Mousefibrosarcoma+14MCF-10AHumanmammary epithelial+15MEF-TMouseembryonic fibroblast+16NIH GW843682X 3T3Mouseembryonic fibroblast?17NMuMGMousemammary epithelial+18RAW 264.7Mousemonocytic+19RGM1Ratgastric epithelium+20Sh-SY5YHumanneuroblastoma?21SW480Humancolorectal adenocarcinoma+22WM1617Humanmelanoma?23WM793Humanmelanoma? Open in a separate window The table depicts the cell-type specificity of SB202190-induced autophagy-dependent vacuole formation. Cells were treated with 5?M SB202190 for 12?h. Vacuoles were clearly visible in most of the cell lines after approximately 2?h of SB202190 treatment. Because of the MAPK11-MAPK14/p38-independent interference with autophagy, the SB-compounds should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. Another concern regards the title and results from the paper, the latter which explicitly areas that MAPK11/12/13/14 get excited about the transcriptional response induced from the copper complicated. These conclusions derive from the usage of the inhibitor exclusively.