KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit anti-PTEN | Cell Signaling Technology | Cat# 9188; RRID: AB_2253290 |
| Mouse anti-AKT (pan) | Cell Signaling Technology | Cat# 2920; RRID: AB_1147620 |
| Rabbit anti-p-AKT (S473) | Cell Signaling Technology | Cat# 4060; RRID: AB_2315049 |
| Mouse anti-β Actin | Sigma-Aldrich | Cat# A2228; RRID: AB_476697 |
| Rabbit anti-PDHK1 | Cell Signaling Technology | Cat# 3820; RRID: AB_1904078 |
| Mouse polyclonal anti-PDK2 | Abcam | Cat# ab172065 |
| Rabbit polyclonal anti-PDK3 | Abcam | Cat# ab154549 |
| Goat polyclonal anti-PDK4 | Santa Cruz Biotechnology | Cat# sc-14495 (discontinued) |
| Rabbit polyclonal anti-p-PDHE1A (S293) | Millipore | Cat# ABS204; RRID: AB_11205754 |
| Mouse anti-cleaved PARP | Cell Signaling Technology | Cat# 9546; RRID: AB_2160593 |
| Mouse anti-GAPDH | Santa Cruz Biotechnology | Cat# sc-365062; RRID: AB_10847862 |
| Rabbit polyclonal anti-p-S6 (S240/S244) | Cell Signaling Technology | Cat# 2215; RRID: AB_331682 |
| Rabbit anti-NFκB p65 | Cell Signaling Technology | Cat# 8242; RRID: AB_10859369 |
| Rabbit anti-p-NFκB p65 (S536) | Cell Signaling Technology | Cat# 3033; RRID: AB_331284 |
| Rabbit anti-Lamin B1 | Cell Signaling Technology | Cat# 12586; RRID: AB_2650517 |
| Rabbit polyclonal anti-HSP90 | Cell Signaling Technology | Cat# 4874; RRID: AB_2121214 |
| Rabbit polyclonal anti-HSP60 | Cell Signaling Technology | Cat# 4870; RRID: AB_2295614 |
| Rabbit polyclonal anti-H3 | Cell Signaling Technology | Cat# 9715; RRID: AB_331563 |
| Rabbit polyclonal anti-NKAP | Abcam | Cat# ab121121; RRID: AB_11139756 |
| Mouse anti-NKAP (K85-80-5) | Burgute et al., 2014 | N/A |
| Rabbit polyclonal anti-V5 | Sigma-Aldrich | Cat# V8137; RRID: AB_261889 |
| Mouse anti-FLAG M2 | Sigma-Aldrich | Cat# F1804; RRID: AB_262044 |
| Mouse anti-GST | Cell Signaling Technology | Cat# 2624; RRID: AB_2189875 |
| Rabbit anti-HA | Cell Signaling Technology | Cat# 3724; RRID: AB_1549585 |
| Mouse anti-Cytochrome c | Abcam | Cat# ab13575; RRID: AB_300470 |
| Rabbit anti-VDAC | Cell Signaling Technology | Cat# 4661; RRID: AB_10557420 |
| Rabbit polyclonal anti-Caspase 9 | Cell Signaling Technology | Cat# 9502; RRID: AB_2068621 |
| Rabbit polyclonal anti-Caspase 7 | Cell Signaling Technology | Cat# 9492; RRID: AB_2228313 |
| Mouse monoclonal anti-Caspase 3 | Cell Signaling Technology | Cat# 9668; RRID: AB_206987 |
| Rabbit polyclonal anti-cleaved Caspase 9 | Cell Signaling Technology | Cat# 9505; RRID: AB_2290727 |
| Rabbit polyclonal anti-cleaved Caspase 7 | Cell Signaling Technology | Cat# 9491; RRID: AB_2068144 |
| Rabbit polyclonal anti-cleaved Caspase 3 | Cell Signaling Technology | Cat# 9661; RRID: AB_2341188 |
| Rabbit anti-BCL-xL | Cell Signaling Technology | Cat# 2764; RRID: AB_2228008 |
| Rabbit anti-BCL2 | Cell Signaling Technology | Cat# 4223; RRID: AB_1903909 |
| Rabbit polyclonal anti-p-GSK-3α/β (S21/9) | Cell Signaling Technology | Cat# 9331; RRID: AB_329830 |
| Rabbit anti-HK2 | Cell Signaling Technology | Cat# 2867; RRID: AB_2232946 |
| Rabbit polyclonal anti-HIF-1α | Cell Signaling Technology | Cat# 3716 |
| Rabbit anti-CA9 | Cell Signaling Technology | Cat# 5649; RRID: AB_10706355 |
| Bacterial and Virus Strains | ||
| PTEN adenovirus (human) | Applied Biological Materials | Cat# 000028A |
| PDK1-HA adenovirus (human) | Applied Biological Materials | Cat# 119140A |
| Biological Samples | ||
| Brain tumor tissue microarray | US Biomax | Cat# GL1921 |
| Prostate adenocarcinoma tissue microarray | US Biomax | Cat# PR803a |
| Lung Carcinoma tissue microarray | US Biomax | Cat# LC801 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| BKM120 (PI3K inhibitor) | Selleckchem | Cat# S2247 |
| GDC-0941 (PI3K inhibitor) | Selleckchem | Cat# S1065 |
| MK-2206 (AKT inhibitor) | Selleckchem | Cat# S1078 |
| DCA (PDHK inhibitor) | Sigma-Aldrich | Cat# 347795 |
| PBS-1086 (NF-κB inhibitor) | Blakely et al., 2015 | N/A |
| Bay 11-7085 (NF-κB inhibitor) | Selleckchem | Cat# S7352 |
| Cisplatin | Selleckchem | Cat# S1166 |
| Docetaxel | Selleckchem | Cat# S1148 |
| NAC | Sigma-Aldrich | Cat# A7250 |
| H2DCFDA | Thermo Fisher Scientific | Cat# D399 |
| Rotenone (ETC complex I inhibitor) | Abcam | Cat# ab143145 |
| Oligomycin (ATP synthase inhibitor) | Abcam | Cat# ab141829 |
| 2-DG | Sigma-Aldrich | Cat# D8375 |
| Human TNF-α | Peprotech | Cat# 300-01A |
| Human NKAP-p-S9 peptide (SGSR-p-SPDREA) | Elim Biopharmaceuticals | This paper |
| Human NKAP-pS149 peptide (VWGL-p-SPKNPE) | Elim Biopharmaceuticals | This paper |
| Human Rab7-p-S72 peptide (ERFQ-p-SLGVAF) | Elim Biopharmaceuticals | This paper |
| Critical Commercial Assays | ||
| DC Protein Assay | Bio-Rad | Cat# 500-0116 |
| Trans-Blot Turbo RTA Midi Nitrocellulose Transfer kit | Bio-Rad | Cat# 170-4271 |
| RNeasy kit | QIAGEN | Cat# 74106 |
| CellTiter-Glo Cell Viability Assay | Promega | Cat# G7573 |
| L-lactate Assay | Cayman | Cat# 700510 |
| SimpleChIP Enzymatic Chromatin IP Kit | Cell Signalling Technology | Cat# 9003 |
| EpiTect ChIP qPCR Primer Assay for human PDK1, NM_002610.3 (−)01Kb | Qiagen | Cat# GPH1007880 (−)01A |
| TaqMan Gene Expression Assay (FAM) for human PDK1, Hs01561850_m1 | Thermo Fisher Scientific | Cat# 4331182 |
| TaqMan Gene Expression Assay (FAM) for human GAPDH, Hs99999905_m1 | Thermo Fisher Scientific | Cat# 4331182 |
| Bright-Glo Luciferase Assay | Promega | Cat# E2610 |
| Malachite Green Assay | Echelon | Cat# K1500 |
| Cytochrome c Release Assay | Abcam | Cat# ab65311 |
| Deposited Data | ||
| Raw and analyzed microarray data | This paper | GEO: GSE121217 |
| Raw and analyzed phospho-proteomics data | This paper | PRIDE: PXD014707 |
| Experimental Models: Cell Lines | ||
| Human lung adenocarcinoma H1650 | Laboratory of William Pao | N/A |
| Human lung Adenocarcinoma H1975 | Laboratory of William Pao | N/A |
| Human lung Adenocarcinoma HCC827 | Laboratory of William Pao1 | N/A |
| Human prostate Adenocarcinoma PC3 | ATCC | Cat# CRL-1435 |
| Human renal clear cell carcinoma 786-O | Laboratory of M. Celeste Simon | N/A |
| Human lung squamous carcinoma H520 | ATCC | Cat# HTB-182 |
| Human skin cutaneous melanoma A2058 | ATCC | Cat# CRL-11147 |
| Human glioblastoma multiforme U87MG | ATCC | Cat# HTB-14 |
| Human embryonic kidney cells 293T | ATCC | Cat# CRL-3216 |
| Human bronchial epithelial cells BEAS2B | ATCC | Cat# CRL-9609 |
| Experimental Models: Organisms/Strains | ||
| Mouse: C.B-Igh-1b/IcrTac-Prkdcscid | Taconic Biosciences | Cat# CB17SC-F |
| Oligonucleotides | ||
| PTEN Mission shRNA (3’-UTR): CCGGCCACAAATGAAGGGATATAAACTCGAGTTTATATCCCTTCATTTGTGGTTTTTG |
Sigma-Aldrich | Cat# TRCN0000230370 |
| PDHK1 Mission shRNA (CDS) #1: CCGGGCTCTGTCAACAGACTCAATACTCGAGTATTGAGTCTGTTGACAGAGCTTTTT |
Sigma-Aldrich | Cat# TRCN0000006261 |
| PDHK1 Mission shRNA (CDS) #2: CCGGCCAGGGTGTGATTGAATACAACTCGAGTTGTATTCAATCACACCCTGGTTTTT |
Sigma-Aldrich | Cat# TRCN0000006263 |
| PDHK1 Mission shRNA (3’-UTR): CCGGGAAGTAGAAGTCTACCATATTCTCGAGAATATGGTAGACTTCTACTT CTTTTTTG |
Sigma-Aldrich | Cat# TRCN0000196728 |
| RELA Mission shRNA (3’-UTR): CCGGGCCTTAATAGTAGGGTAAGTTCTCGAGAACTTACCCTACTATTAAGGCTTTTT |
Sigma-Aldrich | Cat# TRCN0000014683 |
| NKAP Mission shRNA (3’-UTR): CCGGCTGATTGTCCAGAAGACATTTCTCGAGAAATGTCTTCTGGACAATCAGTTTTTTG |
Sigma-Aldrich | Cat# TRCN0000145605 |
| NKAP Mission shRNA (CDS): CCGGGCTGAAGAACCATCAGATTTACTCGAGTAAATCTGATGGTTCTTCAGCTTTTTTG |
Sigma-Aldrich | Cat# TRCN0000145475 |
| ON-TARGETplus Non-targeting Pool siRNA | Dharmacon | Cat# D-001810-10 |
| SMARTpool: ON-TARGETplus PDK1 siRNA | Dharmacon | Cat# L-005019-00-0005 |
| SMARTpool: ON-TARGETplus HIF1A siRNA | Dharmacon | Cat# L-004018-00-0005 |
| Recombinant DNA | ||
| pLKO.1-puro Non-Target shRNA | Sigma-Aldrich | Cat# SHC016 |
| pBABE-Puro-EV | Addgene | Cat# 1764 |
| pBABE-GFP | Addgene | Cat# 10668 |
| pBABE-PTEN | Addgene | Cat# 10785 |
| pBABE-PTENC124S | Addgene | Cat# 10931 |
| pBABE-PTENG129E | Addgene | Cat# 10771 |
| pBABE-PTENY138L | This paper | N/A |
| pCDH-Puro-EV | This paper | N/A |
| pCDH-puro-Bcl2 | Addgene | Cat# 46971 |
| pCDH-puro-Bcl-xL | Addgene | Cat# 46972 |
| pCW22-TRE-Blast-EV | This paper | N/A |
| pCW22-TRE-blast-NKAP-HA-FLAG | This paper | N/A |
| pCW22-TRE-blast-NKAPS9AS149A-HA-FLAG | This paper | N/A |
| pLV-EF1a-IRES-Hygro | Addgene | Cat# 85134 |
| pLV-EF1a-IRES-Hygro-PDHK1 | This paper | N/A |
| pCMV-2xFLAG-PTEN | Addgene | Cat# 28298 |
| pLX304-NKAP-V5 | Dharmacon | Cat# OHS6085-213576668 |
| pEBG-GST-NHERF2 | Addgene | Cat# 28292 |
| pET-30 b-PTEN-6xHis | Addgene | Cat# 20741 |
| pET-30 b-PTENY138L-6xHis | This paper | N/A |
| pGreenFire1-NF-kB (EF1α-Puro) | System Biosciences | Cat# TR012PA-P |
| Plasmid: LightSwitch PDK1 Promoter Reporter | SwitchGear Genomics | Cat# S721832 |
| Software and Algorithms | ||
| ImageJ | Schneider et al., 2012 | https://imagej.nih.gov/ij/ |
| GraphPad Prism 6 | GraphPad | RRID:SCR_002798 |