Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in human lung adenocarcinoma
- Department of Pharmacology, Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, United States
- Nuventra, United States
- Children’s Research Institute, UT Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
COX6B2 mRNA expression correlates with poor outcome in LUAD.
(A–C) Kaplan-Meier curves for OS and FP in NSCLC (A) LUAD (B) and LUSC patients (C). (D) COX6B2 mRNA expression (RNA-seq RSEM, log2(norm count +1)) from TCGA Lung Cancer dataset. Bars represent …
Figure 1—figure supplement 1
COX6B2 expression in LUAD.
(A) Z-score of caspase3/7 activity screening in indicated cell lines based on data generated in Maxfield et al., 2015. (B) Top: Whole cell lysates from HCC515 depleted or COX6B2-V5 or COX6B1-V5 …
Figure 2
COX6B2 expression enhances mitochondrial respiratory activity.
(A) Left: Whole cell lysates from HCC515 cells + / - COX6B2-V5 were immunoblotted with indicated antibodies. MW markers are indicated. Right: Quantitation of COX6B2 protein expression from blots on …
Figure 3 with 1 supplement
COX6B2 enhances complex IV activity of OXPHOS without increasing ROS production.
(A) Schematic of complex IV activity measurements in isolated mitochondria. Details are described in Material and methods. antA, Antimycin A; Cyt c, Cytochrome c; ATP Syn, ATP synthase. (B) Complex …
Figure 3—figure supplement 1
Measurement of complexes and supercomplexes.
(A) Left: Representative immunofluorescence images of COXIV in HCC515 cells + / - COX6B2-V5. Scale bar, 20 μm. Right: Quantification of staining intensity in indicated cell lines. Bars represent …
Figure 4 with 1 supplement
COX6B2 promotes cell division.
(A) Percentage of cells positive for EdU incorporation in indicated samples. Values represent the mean ± SEM (n ≥ 9). p-Value calculated by Student’s t-test. (B) Population doubling rate in …
Figure 4—figure supplement 1
COX6B2 promotes cell division.
(A) Relative EdU incorporation (%) in indicated samples. Values represent the mean ± SEM (n ≥ 9). p-Value calculated by Student’s t-test. (B) Relative population doubling rate (%) in indicated …
Figure 5 with 1 supplement
Depletion of COX6B2 impairs mitochondrial function.
(A–B) Left: Oxygen consumption rate (OCR) as a function of time in COX6B2 depleted HCC515 cells using an shRNA pool (A) or an siRNA pool (B) following exposure to electron transport chain complex …
Figure 5—figure supplement 1
Depletion of COX6B2 impairs OXPHOS.
(A) Left: Oxygen consumption rate (OCR) as a function of time in siCOX6B2 depleted H2122 cells following exposure to electron transport chain complex inhibitors. Bars represent mean ± SEM (n ≥ 14). …
Figure 6 with 1 supplement
COX6B2 is essential for tumor cell viability and survival.
(A) Left: Representative images of EdU staining in indicated cell lines. (EdU, green; Hoechst, blue). Scale bar, 100 μm. Right: Quantitation of EdU-positive cells. Bars represent the mean ± SEM (n ≥ …
Figure 6—figure supplement 1
Phenotypes following COX6B2 depletion.
(A) The percentage of EdU-positive cells of COX6B2 depleted by three different sgRNA in HCC515 cells. Bars represent mean ± SEM (n = 7). p-Value calculated by Student’s t-test. ***p<0.001, ****p<0.00…
Figure 7
COX6B2 supports tumor growth in vivo.
(A and C) Xenograft experiments in indicated cell lines. Tumor volumes were measured using calipers on indicated days. Each data point represents mean ± SEM (n = 5). p-Value calculated by Student’s t…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, female) | Athymic nude mice | Envigo | Hsd:Athymic Nude-Foxn1nu | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216; RRID:CVCL_0063 | |
| Cell line (Homo sapiens) | H1395 | John Minna | RRID:CVCL_1467 | UT Southwestern |
| Cell line (Homo sapiens) | H1373 | John Minna | RRID:CVCL_1465 | UT Southwestern |
| Cell line (Homo sapiens) | H2030 | John Minna | RRID:CVCL_1517 | UT Southwestern |
| Cell line (Homo sapiens) | H2122 | John Minna | RRID:CVCL_1531 | UT Southwestern |
| Cell line (Homo sapiens) | H2126 | John Minna | RRID:CVCL_1532 | UT Southwestern |
| Cell line (Homo sapiens) | H1944 | John Minna | RRID:CVCL_1508 | UT Southwestern |
| Cell line (Homo sapiens) | HCC515 | John Minna | RRID:CVCL_5136 | UT Southwestern |
| Cell line (Homo sapiens) | HCC1195 | John Minna | RRID:CVCL_5127 | UT Southwestern |
| Antibody | Anti-COX6B2 (Rabbit polyclonal) | MilliporeSigma | Cat# SAB1401983, RRID:AB_10609550 | IB (1:1000) IF (1:50) IHC (1:150) |
| Antibody | Anti-COX6B1 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-393233, RRID:AB_2814984 | IB (1:1000) |
| Antibody | Anti-COXIV (Rabbit monoclonal) | Cell Signaling Technology | Cat# 4850, RRID:AB_2085424 | IB (1:5000) IF (1:200) |
| Antibody | Anti-β-Actin (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-47778 | IB (1:10,000) |
| Antibody | Anti-V5 (Mouse monoclonal) | Thermo Fisher Scientific | R960-25; RRID:AB_2556564 | IB (1:5000) |
| Antibody | Anti-HIF-1α (Mouse monoclonal) | BD Biosciences | Cat# 610959, RRID:AB_398272 | IB (1:1000) |
| Antibody | Anti-Cleaved Caspase-3 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 9661, RRID:AB_2341188 | IB (1:500) |
| Antibody | Anti-PARP (Rabbit monoclonal) | Cell Signaling Technology | Cat# 9532, RRID:AB_659884 | IB (1:1000) |
| Antibody | Anti-ERK (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-135900, RRID:AB_2141283 | IB (1:3000) |
| Antibody | Anti-NDUFA9 (Mouse monoclonal) | Thermo Fisher Scientific | Cat# 459100, RRID:AB_2532223 | IB (1:1000) |
| Antibody | Anti-UQCRC2 (Mouse monoclonal) | Abcam | Cat# ab14745, RRID:AB_2213640 | IB (1:1000) |
| Antibody | Anti-Tom20 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-17764, RRID:AB_628381 | IF (1:200) |
| Commercial assay or kit | MitoTracker Green | Thermo Fisher Scientific | M7514 | |
| Commercial assay or kit | MitoSOX Red | Thermo Fisher Scientific | M36008 | |
| Commercial assay or kit | MitoProbe JC-1 | Thermo Fisher Scientific | M34152 | |
| Commercial assay or kit | Cell-Titer Glo | Promega | PR-G7572 | |
| Commercial assay or kit | NAD+/NADH-Glo Assay | Promega | G9071 | |
| Commercial assay or kit | Click-iT EdU Alexa Fluor 488 Imaging Kit | Thermo Fisher Scientific | C10037 | |
| Commercial assay or kit | Senescence Beta-Galactosidase Staining Kit | Cell Signaling Technology | 9860 | |
| Commercial assay or kit | Colloidal Blue Staining Kit | Thermo Fisher Scientific | ||
| Chemical compound, drug | oligomycin | MilliporeSigma | O4876 | |
| Chemical compound, drug | CCCP | MilliporeSigma | C2759 | |
| Chemical compound, drug | Antimycin A | MilliporeSigma | A8674 | |
| Chemical compound, drug | Seahorse XF Plasma Membrane Permeabilize | Agilent | 102504–100 | |
| Chemical compound, drug | NAC | MilliporeSigma | N7250 | |
| Chemical compound, drug | Saponin | MilliporeSigma | 47036 | |
| Software, algorithm | FlowJo | FlowJo | RRID:SCR_008520 | ver 10 |
| Software, algorithm | ImageJ (Fiji) | Schindelin et al., 2012 | RRID:SCR_002285 | ver 2.0.0 |
| Software, algorithm | Integrative Genomics Viewer | Robinson et al., 2011 | RRID:SCR_011793 | ver 2.3.93 |
