Table 1.
Lung epithelial cell lines used.
Figure 1.
Mitochondrial length of lung epithelial cells.
(A) Representative mito-YFP expression in cells. Numbered boxes (1–4) are magnifications (10×) of the corresponding numbered boxed regions in the original images of NL20, NL20TA, Calu1 and A549. Scale bar is 2 µm. (B) Mean mitochondrial length (y-axis; ± SEM) measurements (n = 35–50) from three independent experiments for NL20, NL20TA, Calu1, and A549 cells. 1-way ANOVA analysis with Tukey post-tests (P<0.0001).
Figure 2.
Mitochondrial mass of lung epithelial cells.
(A) Mitotracker green fluorescence relative fluorescence units (y-axis, RFU) represents mitochondrial mass (mean and SEM); measurements (n = 8 wells) from three independent experiments. 1-way ANOVA analysis with Tukey post-tests (P<0.0001). (B) Representative immunoblots demonstrating expression of endogenous proteins complex IV subunit I, TIM23, frataxin, and VDAC in NL20 (lane 1), NL20TA (lane 2), Calu1 (lane 3), and A549 (lane 4) cells. β-actin is shown for loading. Markers in kilodaltons (kDa). (C) Immunoblots from two independent experiments were quantified to show relative complex IV subunit I protein expression normalized to TIM23. Mean and SEM shown. 1-way ANOVA analysis with Tukey post-tests compared to NL20. (D) Representative mitochondrial biogenesis dipstick ELISA assay showing frataxin and complex IV expression. (E) Three independent mitochondrial biogenesis ELISA assays were quantified to show relative mitochondrial-encoded complex IV protein expression normalized to the nuclear-encoded frataxin protein. Mean and SEM shown. 1-way ANOVA analysis with Tukey post-tests compared to NL20.
Figure 3.
Resistance to mitochondrial uncoupling.
TMRE staining from two independent experiments (n = 8). TMRE expression is plotted as a relative ratio ΔF/F0 showing mean and SEM, where F indicates fluorescence intensity and F0 indicates baseline values before stimulation following background subtraction. Cell lines NL20 (blue), NL20 (orange), Calu1 (pink1) and A549 (green) were treated with oligomycin (6 µM; 0 minutes) and CCCP (10 µM; 18 minutes) to induce hyperpolarization and depolarization, respectively. 2-way ANOVA analysis with Bonferroni post-tests.
Figure 4.
Decreased Drp1 protein expression and Drp1-dependent mitochondrial fission.
(A) Mitochondrial morphology of NL20 (left) and A549 (right) cells following mito-DsRED transfection. Representative images shown: basal (upper) and following Drp1 RNAi (middle) or Drp1-YFP transfection (lower). The scale bar indicates 2 µm. Numbered boxes (1–6) are magnifications (10×) of the corresponding numbered boxed regions in the original images. (B) Immunoblot of endogenous Drp1 expression before (lanes 1,3) and after (lanes 2,4) Drp1 RNAi transfection in NL20 and A549 cells. (C) Immunoblot of endogenous (lanes 1,2; arrow) and ectopic (lanes 3,4; arrowhead) Drp1 expression before and after Drp1-YFP transfection in NL20 and A549 cells. (B,C) β-actin reprobe to show loading; markers in kDa. (D) Relative Drp1 protein expression normalized to β-actin before and after Drp1 RNAi transfection in NL20 (white bar) and A549 (black bar) cells (two independent experiments). 2-way ANOVA analysis with Bonferroni post-tests. (E) Representative images from FRAP analysis of NL20 (left) and A549 (right) cells transfected with mito-YFP. Cells imaged under basal (top), Drp1 K38A-myc downregulation of Drp1 (middle) and Drp1-myc overexpression (bottom) conditions. Numbered boxes (1–6) are magnifications (10×) of the corresponding numbered boxed regions in the original images. Scale bar is 1 µm. (F–H) Mobile fraction of mito-YFP values occurring within a single subcellular region of interest (n = 60 cells). Mean and SEM shown from two independent experiments. 1-way ANOVA analysis with Tukey post-tests. Additional FRAP statistics are shown in Figure S2.
Figure 5.
Impaired release of cytochrome c following apoptotic stimulus with STS.
(A,B, E) Representative immunoblots of three independent experiments of (A) untreated, (B) 1 µM STS treatment for 3 h to induce apoptosis, and (E) 1 µM STS treatment for 3 h after Drp1-myc transfection in NL20 and A549 cells. Endogenous Drp1 and cytochrome c protein expression were evaluated in total (lanes 1,2), mitochondrial (lanes 3,4) and cytosolic (lanes 5,6) fractions. Mitochondrial (VDAC), cytosol (GAPDH) and β-actin (total) are included as fractionation and loading controls. Myc expression after Drp1-myc transfection is shown in (E). Markers in kDa. (C, D, F) Cytochrome c protein expression normalized to β-actin in NL20 (white bars) and A549 (black bars) cell fractions under untreated conditions (C), 1 µM STS treatment for 3 h (D) or 1 µM STS treatment for 3 h with ectopic expression of Drp1-myc (F). Mean and SEM shown from two independent experiments. 2-way ANOVA analysis with Bonferroni post-tests. Mitochondrial morphology under these conditions is shown in Figure S3C and S5.
Figure 6.
Resistance to STS-induced caspase-3 and PARP cleavage.
(A, B) NL20 and A549 cells treated with (lanes 2,4) and without (lanes 1,3) 1 µM STS for 3 hours to induce apoptosis. β-actin reprobe to show loading. Immunoblots show (A) endogenous caspase-3 protein expression; full length (arrow) and cleaved products (open or closed arrowhead). (B) Endogenous PARP protein expression; full length (arrow) and cleaved product (arrowhead). (C) Endogenous PARP protein expression; full length (arrow) and cleaved product (arrowhead) in NL20 and A549 cells treated with 50 µM zVAD-FMK (lanes 3,7) for 4 h to inhibit caspase activity in non-transfected (lanes 1–3, 5–7) or Drp1-myc transfected (lanes 4,8) cells or co-treatment with 1 µM STS for 3 h (lanes 2–4,6–8). (A–C) Markers in kDa.
Figure 7.
Impaired turnover of mitochondria prior to degradation by the lysosome.
(A) Representative immunoblot of LC3-I (arrow) to LC3-II (arrowhead) conversion in total protein from untreated (UT; lanes 1,5), 10 nM 24 h Bafilomycin A1 treated (BafA1; lanes 2,6) 24 h serum starved (EBSS; lanes 3,7) or combined 24 h BafA1/EBSS treated (B/E; lanes 4,8) NL20 and A549 cells. β-actin reprobe to show loading. (B) LC3-II quantification (normalized to β-actin) from two independent experiments. Cells conditions were as in (A) to examine differences between (B) cell lines. Mean and SEM shown, 2-way ANOVA analysis with Bonferroni post-test. (C) Mito-YFP transfection (left) and LC3 immunostaining (middle) in A549 cells show mitophagy by colocalization of LC3 with the mitochondria (merged, right). A549 cells treated with 10 µM CCCP for 30 minutes (middle) or transfected with Drp1-myc for 24 h and CCCP treated (bottom). Representative images of mitophagy by colocalization of mito-YFP and LC3 punctae (yellow expression) in CCCP and CCCP/Drp1-myc conditioned A549 cells. Scale bar is 2 µm. (D) Quantification of mitophagy as in (C). Box insets show enlarged microscopy sections to illustrate a selection of mitophagy events occurring (white open arrowheads) which are labeled 1–3 corresponding to original images as shown in (E); A549 (1), A549+CCCP (2), and A549+CCCP/Drp1-myc (3). Percent cell counts (>200) from three independent experiments were based on colocalization of LC3 punctae staining with mito-YFP (values represent mean number of punctae counts per cell binned with SEM). 2-way ANOVA analysis with Bonferroni post-tests.