Figure 2.

Autophagic flux in WT and bs MEFs. Immunostaining of WT (A) and bs (B) MEFs for SQSTM1. (C) Quantification analysis revealed a significantly greater (P = 0.0008; n = 30) percentage area of SQSTM1 per cell in bs MEFs when compared to WT MEFs. (D) Western blot for SQSTM1 of lysates from WT and bs MEFs; immunoblotting for ACTB served as a loading control. (E) Quantification analyses revealed a significantly greater (P = 0.0002; n = 3) levels of SQSTM1:ACTB in lysates from bs MEFs when compared to WT MEFs. (F) Immunoblotting of WT and bs MEF cell lysates for SQSTM1 following 2-h treatment with DMSO as vehicle control (lanes 1 and 5), serum-free and amino-acid free media (ST) (lanes 2 and 6), 100 nM bafilomycin A₁ (BF) in complete medium (lanes 3 and 7), and combined treatment with bafilomycin A₁ and serum-free and amino-acid free media (BF+ST) (lanes 4 and 8); immunoblotting for ACTB served as a loading control. (G) Quantification analysis revealed significantly greater levels (P = 0.0027; n = 3) of SQSTM1:ACTB in lysates from bs MEFs when compared to WT following treatment with DMSO (P = 0.0027; n = 3) or ST (P = 0.0001; n = 3). No significant differences were identified between WT and bs MEFs following treatments with BF (P = 0.0969; n = 3), and BF+ST (P = 0.097; n = 3). In WT MEFs ST significantly (P = 0.0049; n = 3) lowered SQSTM1 levels whereas BF and BF+ST significantly increased (P = 0.0013 and P = 0.0007 respectively; n = 3) levels of SQSTM1 when compared to DMSO treatment; by contrast SQSTM1 levels in bs MEFs did not significantly change following DMSO treatment and ST (P = 0.31; n = 3), BF (P = 0.851; n = 3) and ST+BF (P = 0.3234; n = 3).