Figure 4.
Autophagy caused by starvation does not require MAPK8/9 in primary MEFs. (a) (Z)-4-Hydroxytamoxifen-treated primary Rosa-CreERT (WT) MEFs and Rosa-CreERT Mapk8LoxP/LoxP mapk9−/- MEFs were examined by immunoblot analysis by probing with antibodies to MAPK8/9 and TUBA. (b) WT and mapk8−/- mapk9−/- primary MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 25 µm. (c) LC3B and GAPDH expression by WT and mapk8−/- mapk9−/- primary MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test was used for the flux analysis. (d) The amount of SQSTM1 and TUBA in WT and mapk8−/- mapk9−/- primary MEFs after incubation with EBSS containing 5 mM glucose (2 h) was examined by immunoblot analysis. The SQSTM1:TUBA ratio was quantified and normalized to SQSTM1 expression in WT non-starved cells (mean ± SEM; n = 3 independent experiments). (e) MAPK8/9 activation in WT and mapk8−/- mapk9−/- primary MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH after incubation (2 h) with media or EBSS containing 5 mM glucose. WT MEFs exposed to UV (60 J/m2) and mapk8−/- mapk9−/- represent positive and negative controls. (f) RPS6KB1, p-Thr389 RPS6KB1, and TUBA in WT and mapk8−/- mapk9−/- primary MEFs after incubation (2 h) with EBSS containing 5 mM glucose was examined by immunoblot analysis.