Figure 4.

 

Figure 4. Gain of function experiments with BAX proteins targeted either to the nucleus (NLS-BAX) or excluded from the nucleus (NES-BAX) in A549 cells and primary HLF. (A) Representative immunoblot (n = 3) after subcellular fractionation in primary HLF transfected for 48h with an empty pCDNA 3.1 vector, NES-BAX or NLS-BAX constructs. The upper panel with anti-FLAG (upper panel) and BAX (middle panel) antibodies show that the NES-BAX or NLS-BAX constructs are detected in the cytosolic fraction (cyto.), revealed with GAPDH antibody. In the nuclear fraction (nuc.), revealed with LMN A/C, only NLS-BAX was detected (arrowhead). Note that NES-BAX and NLS-BAX are detected at a higher molecular weight than endogenous BAX (*). (B-C) Effects of NES and NLS-BAX on the proliferation (cell count) of A549 cells (B) and primary HLF (C) compared to cells treated with control plasmid (gray dashed line) at 48h. Note that the increase in cell proliferation was observed only in NLS-BAX transfected A549 cells (41% increase +/− 8, n = 4) and primary HLF (102% increase +/− 67, n = 5) compared to pCDNA 3.1 empty vector and NES-BAX construct (respectively 9% increase +/− 9 compared to control plasmid in A549 cells, n = 4 and 15% decrease +/− 10 in primary HLF, n = 3) compared to control plasmid. (D-E) Expression of CDKN1A mRNA by qPCR (upper part) in (D) A549 cells and (E) primary HLF transfected for 48h with NES-BAX (respectively 19.4% decrease +/− 4 in A549 cells and 27.6% increase +/− 29 in primary HLF, n = 5) or NLS-BAX (respectively 41.6% decrease +/− 7 in A549 cells and 51.54% decrease +/− 16 in primary HLF, n = 5) compared to empty vector (gray dash line). Representative immunoblot (n = 3) of FLAG tagged BAX constructs, CDKN1A, in A549 cells (D) or primary HLF (E) transfected with empty control vector, either NES-BAX or NLS-BAX constructs for 48h. GAPDH was used as loading control. (*p < 0.05, rank t-Test).