Key Resources Table.
| Reagent Or Resource | Source | Identifier |
|---|---|---|
| Antibodies | ||
| Rabbit monoclonal AKT1 antibody | Cell Signaling Technology | Cat# 2938S; Clone# C73H10; RRID: AB_915788. |
| Rabbit monoclonal P-AKT (S473) antibody | Cell Signaling Technology | Cat# 4060S; Clone# D9E; RRID: AB_2315049. |
| Rabbit monoclonal P-AKT (T308) antibody | Cell Signaling Technology | Cat# 2965S; Clone# C31E5E; RRID: AB_2255933. |
| Rabbit monoclonal P-MEK1/2 (S217/221) antibody | Cell Signaling Technology | Cat# 9154S; Clone# 41G9; RRID: AB_2138017. |
| Mouse monoclonal MEK1 antibody | Cell Signaling Technology | Cat# 2352S; Clone# 61B12; RRID: AB_10693788. |
| Rabbit polyclonal p44/42 MAP kinase (phosphorylated Erk1/2) antibody | Cell Signaling Technology | Cat# 9101L; Clone# N/A; RRID: AB_331646. |
| Rabbit polyclonal p44/42 MAPK (Erk1/2) antibody | Cell Signaling Technology | Cat# 9102S; Clone# N/A; RRID: AB_330744. |
| Rabbit monoclonal PTEN, P-Ser380/Thr382/Thr383 antibody | Cell Signaling Technology | Cat# 9549S; Clone# 44A7; RRID: AB_659891. |
| Rabbit monoclonal PI3 Kinase p85 antibody | Cell Signaling Technology | Cat# 4257S; Clone# 19H8; RRID: AB_10695255. |
| Rabbit monoclonal P-Bad (S136) antibody | Cell Signaling Technology | Cat# 4366S; Clone# D25H8; RRID: AB_10547878. |
| Rabbit monoclonal Bad antibody | Cell Signaling Technology | Cat# 9239S; Clone# D24A9; RRID: AB_2062127. |
| Rabbit monoclonal P-GSK-3-beta (S9) antibody | Cell Signaling Technology | Cat# 9322S; Clone# D3A4; RRID:AB_2115196. |
| Rabbit monoclonal GSK-3-beta XP antibody | Cell Signaling Technology | Cat# 12456S; Clone# D5C5Z; RRID: N/A. |
| Rabbit polyclonal CSGLCAT antibody | Abcam | Cat# ab75050; Clone# N/A; RRID: AB_1523416. |
| Rabbit polyclonal Ki67 antibody | Abcam | Cat# ab15580; Clone#N/A; RRID: AB_443209. |
| Rabbit polyclonal Anti-Phospho - (Ser/Thr) Phe antibody | Abcam | Cat# ab17464; Clone# N/A; RRID: AB_443891. |
| Rat monoclonal anti-mouse IgG VeriBlot for IP secondary antibody (HRP) | Abcam | Cat# ab131368; Clone# N/A; RRID: N/A. |
| Mouse monoclonal PTEN antibody | Santa Cruz Biotechnology | Cat# sc-7974; Clone# A2B1; RRID: AB_628187. |
| Mouse monoclonal casein kinase II alpha antibody | Santa Cruz Biotechnology | Cat# sc-373894; Clone# E-7; RRID: AB_10947405. |
| Rabbit monoclonal Anti-HA-probe Polyclonal antibody, Unconjugated | Santa Cruz Biotechnology | Cat# sc-805; Clone# Y-11; RRID: AB_631618. |
| Mouse monoclonal anti-Chondroitin 4 Sulfate antibody | Millipore | Cat# MAB2030; Clone# be-123; RRID: AB_94510. |
| Mouse monoclonal ANTI-FLAG M2 antibody | Sigma-Aldrich | Cat# F3165; Clone# M2; RRID: AB_259529. |
| Mouse monoclonal Anti-beta-Actin antibody | Sigma-Aldrich | Cat# A1978; Clone# AC-15; RRID: AB_476692. |
| Bacterial and Virus Strains | ||
| N/A | ||
| Biological Samples | ||
| Patient-derived xenografts (PDX) TM00943; TM01612; TM01386; TM01149. |
The Jackson Laboratory | Mouse Tumor Biology Database (MTB), Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org/) |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Myelin Basic Protein bovine | Sigma-Aldrich | Cat# M1891 |
| Vemurafenib (PLX4032, RG7204) | Selleck Chemicals | Cat# S1267 CAS: 918504-65-1 |
| Dabrafenib (GSK2118436) | Selleck Chemicals | Cat# S2807 CAS: 1195765-45-7 |
| PTEN phospholipid phosphatase | Echelon Biosciences | Cat# S1105 |
| LY294002 | Selleck Chemicals | Cat# E3000 |
| Casein Kinase 2 Protein | Millipore | Cat# 14-197 Product# 6QQ199 |
| Chondroitin sulfate A sodium salt from bovine trachea | Sigma-Aldrich | Cat# C9819 CAS: 39455-18-0 |
| Chondroitinase ABC from Proteus vulgaris | Sigma-Aldrich | Cat# C3367 CAS: 9024-13-9 |
| Chondroitin disaccharide Δdi-4S sodium salt | Sigma-Aldrich | Cat# C4045 CAS: 136144-56-4 |
| Phosphatidylinositol 3,4,5-trisphosphate diC8 (PI(3,4,5)P3 diC8) | Echelon Biosciences | Cat# P-3908 CAS: 163563-77-7 |
| Chondroitin Sulfate Fluorescein | Creatvie PEGWorks | Cat# CS-101 |
| ProLong™ Gold AntifadeMountant with DAPI | Invitrogen | Cat# P36931 |
| Adenosine 5′-triphosphate (ATP) disodium salt hydrate | Sigma-Aldrich | Cat# A1852 CAS: 34369-07-8 |
| Hygromycin B | Gibco | Cat# 10687010 CAS: 31282-04-9 |
| Puromycin dihydrochloride from Streptomyces alboniger | Sigma-Aldrich | Cat# P8833 CAS: 58-58-2 |
| 3,3′-Diaminobenzidine | Sigma-Aldrich | Cat# D8001 CAS: 91-95-2 |
| Hematoxylin | Sigma-Aldrich | Cat# H3136 CAS: 517-28-2 |
| Crystal Violet | Sigma-Aldrich | Cat# C3886 CAS: 548-62-9 |
| Critical Commercial Assays | ||
| PfuTurbo DNA polymerase | Agilent Technologies | Cat# 600250 |
| Two-Step RT-PCR kit | TaKaRa/Clontech | Cat# RR019A |
| BEGM™ Bronchial Epithelial Cell Growth Medium | LONZA | Cat# CC-3170 |
| TRIzol® Reagent RNA extraction | Invitrogen | Cat# 15596026 |
| FuGENE® 6 Transfection Reagent | Promega | Cat# E2691 |
| TransIT®-LT1 Transfection Reagent | Mirus | Cat# MIR 2305 |
| Lipofectamine™ 3000 Transfection Reagent | Invitrogen | Cat# L3000008 |
| PowerUp™ SYBR™ Green Master Mix | Applied Biosystems | Cat# A25742 |
| cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail | ROCHE | Cat# 4693159001 |
| RIPA Buffer | Sigma-Aldrich | Cat# R0278 |
| PI(3)P Mass ELISA | Echelon Biosciences | Cat# K-3300 |
| Malachite Green Assay Kit | Echelon Biosciences | Cat# K-1500 |
| PI3-Kinase Activity ELISA: Pico | Echelon Biosciences | Cat# K-1000s |
| DakoEnVision+ System, HRP | Dako/Agilent | Cat# K4002 |
| Deposited Data | ||
| Original images were deposited to Mendeley data | This paper | https://doi:10.17632/hmg3v5s7ty.1 |
| Experimental Models: Cell Lines | ||
| Human: A375 cells | ATCC | Cat# CRL-1619; RRID: CVCL_0132 |
| Human: SK-MEL-5 cells | ATCC | Cat# HTB-70; RRID: CVCL_0527 |
| Human: PMWK cells | ATCC | Cat# CRL-2624; RRID: CVCL_A665 |
| Human: WM-266-4 cells | ATCC | Cat# CRL-1676; RRID: CVCL_2765 |
| Human: CHL-1 cells | ATCC | Cat# CRL-9446; RRID: CVCL_1122 |
| Human: A2058 cells | ATCC | Cat# CRL-11147; RRID: CVCL_1059 |
| Human: Mel-ST cells | (Kang et al., 2015) | Cat# N/A; RRID: N/A |
| Human: HEK293T cells | ATCC | Cat# CRL-3216; RRID: CVCL_0063 |
| Human: HMCB cells | ATCC | Cat# CRL-9607; RRID: CVCL_3317 |
| Human: SK-MEL-2 cells | ATCC | Cat# HTB-68; RRID: CVCL_0069 |
| Human: SK-MEL-24 cells | ATCC | Cat# HTB-71; RRID: CVCL_0599 |
| Human: WiDr cells | ATCC | Cat# CCL-218 RRID: CVCL_2760 |
| Human: HT-29 cells | ATCC | Cat# HTB-38 RRID:CVCL_0320 |
| Mouse: NIH/3T3 cells | ATCC | Cat# CRL-1658; RRID: CVCL_0594 |
| Human: EL 1 | ATCC | Cat# CRL-9854; RRID: CVCL_3680 |
| Human: KG-1α | ATCC | Cat# CRL-246.1; RRID: CVCL_3602 |
| Human: K-562 | ATCC | Cat# CCL-243; RRID: CVCL_0004 |
| Human: Molm14 | (Fan et al., 2016) | Cat# N/A; RRID: N/A |
| Human: NB4 | CLS | Cat# 300299/p638_NB-4; RRID: CVCL_0005 |
| Human: THP-1 | ATCC | Cat# TIB-202; RRID: CVCL_0006 |
| SKM-1 | DSMZ | Cat# ACC-547; RRID: CVCL_0098 |
| Human: NOMO-1 | DSMZ | Cat# ACC-542; RRID: CVCL_1609 |
| Human: BEAS-2B | ATCC | Cat# CRL-9609; RRID: CVCL_0168 |
| Human: H1299 | ATCC | Cat# CRL-5803; RRID: CVCL_0060 |
| Human: A549 | ATCC | Cat# CCL-185; RRID: CVCL_0023 |
| Human: H157 | ATCC | Cat# CRL-5802; RRID: CVCL_0463 |
| Human: NCI-H596 | ATCC | Cat# HTB-178; RRID: CVCL_1571 |
| Human: EKVX | (Fan et al., 2016) | N/A |
| Human: HCC827 | ATCC | Cat# CRL-2868; RRID: CVCL_2063 |
| Human: NCI-H358 | ATCC | Cat# CRL-5807; RRID: CVCL_1559 |
| Human: H1975 | ATCC | Cat# CRL-5908; RRID: CVCL_1511 |
| Human: HOK | (Fan et al., 2016) | N/A |
| Human: Tu212 | (Kang et al., 2010) | N/A |
| Human: 212LN | (Kang et al., 2010) | N/A |
| Human: Tu686 | (Kang et al., 2010) | N/A |
| Human: Tu167 | provided by Dr. Sumin Kang, EMORY University, Atlanta, GA | N/A |
| Human: FaDu | ATCC | Cat# HTB-43; RRID: CVCL_1218 |
| Human: UMSCC47 | (Fan et al., 2016) | N/A |
| Human: UMSCC22B | provided by Dr. Georgia Chen, EMORY University, Atlanta, GA | N/A |
| Experimental Models: Organisms/Strains | ||
| Mouse: Heterozygous Hsd:Athymic Nude-Foxn1nu/Foxn1+ | Envigo/Harlan | Cat# 069(nu)/070(nu/+) |
| Mouse: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ | The Jackson Laboratory | Stock# 005557 RRID: IMSR_JAX:005557 |
| PDX tumor carrying mouse: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ | The Jackson Laboratory | N/A |
| Oligonucleotides | ||
| shRNA targeting CSGlcA-T sequence #1: 5′-CGCTCATTTGAACTGGCCAAA-3′ | BROAD Institute/Open Biosystems | N/A |
| shRNA targeting CSGlcA-T sequence #2:5′-CGGCTAGACCAAAGTGATGAA-3′ | BROAD Institute/Open Biosystems | N/A |
| RT-PCR primers for CHPF2, Forward: 5′-GTCACGGAGTCTCCTGCTTC-3′ | This paper | N/A |
| RT-PCR primers for CHPF2, Reverse: 5′-GGTCCCTATTTTTGGCCAGT-3′ | This paper | N/A |
| RT-PCR primers for GAPDH, Forward: 5′-CTGGGCTACACTGAGCACC-3′ | This paper | N/A |
| RT-PCR primers for GAPDH, Reverse: 5′-AAGTGGTCGTTGAGGGCAATG-3′ | This paper | N/A |
| Primers: CSGlcA-T D184A mutation, Forward: 5′-CTTCATGCAGGATGCAACATATGTGCAGGC-3′ | This paper | N/A |
| Primers: CSGlcA-T D184A mutation, Reverse: 5′-GCCTGCACATATGTTGCATCCTGCATGATGAAG-3′ | This paper | N/A |
| Recombinant DNA | ||
| hPTEN CRISPR/Cas9 KO plasmid | Santa Cruz Biotechnology | sc-422475 |
| hPTEN CRISPR/Cas9 HDR plasmid | Santa Cruz Biotechnology | sc-422475-HDR |
| pBabe puro HA PIK3CA H1047R | pBabe puro HA PIK3CA H1047R(Zhao et al., 2005) | Addgene plasmid # 12522 |
| pcDNA3 HA PKB AAA | pcDNA3 HA PKB AAA was a gift from Morris Birnbaum | Addgene plasmid # 16001 |
| HA PKB T308D S473D pcDNA3 | HA PKB T308D S473D pcDNA3 (Scheid et al., 2002) | Addgene plasmid # 14751 |
| pcDNA3 GFP PTEN | 809 pcDNA3 GFP PTEN was a gift from William Sellers (Vazquez et al., 2001) | Addgene plasmid # 10759 |
| pLHCX-FLAG-CSGlcA-T | This paper | N/A |
| pLHCX-FLAG-CSGlcA-T D184A | This paper | N/A |
| pMSCV-FLAG-BRAF | (Kang et al., 2015) | N/A |
| pMSCV-FLAG-BRAF V600E | (Kang et al., 2015) | N/A |
| Software and Algorithms | ||
| Patient-derived xenografts (PDX) | The Jackson Laboratory | http://www.informatics.jax.org/ |
| Human pLKO.1 the RNAi consortium (TRC) Library | BROAD Institute/ Open Biosystems | https://www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library |
| CSGlcA-T mRNA expression | ONCOMINE™ | https://www.oncomine.org/resource |
| GraphPad Prism 7 software | GraphPad Software | https://www.graphpad.com/ |