KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
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| Rabbit polyclonal anti-PFKFB4 | Abcam | Cat# ab137785; RRID: AB_2722775 |
| Rabbit polyclonal anti-PFKFB4 | Abnova | Cat# PAB4031; RRID: AB_1578645 |
| Rabbit polyclonal anti-PFKFB4 | Sino Biological | Cat# 202954-T36 |
| Rabbit polyclonal anti-LYVE1 | Abcam | Cat# ab14917; RRID: AB_301509 |
| Mouse monoclonal anti-Ki-67 (clone MIB-1) | Agilent Dako | Cat# M7240; RRID: AB_2142367 |
| Rabbit monoclonal anti-Integrin β3 (clone D7X3P) | Cell Signaling Technology | Cat# 13166; RRID: AB_2798136 |
| Mouse monoclonal anti-pimonidazole (clone 4.3.11.3) | Hypoxyprobe | Cat# HP1-1000Kit; RRID: AB_2811309 |
| Mouse monoclonal anti-β-actin (clone AC-15), peroxidase conjugate | Sigma-Aldrich | Cat# A3854; RRID: AB_262011 |
| Mouse monoclonal anti-AIB-1 (clone 34/AIB-1) | BD Biosciences | Cat# 611105; RRID: AB_2151198 |
| Mouse monoclonal anti-HIF-1α (clone H1alpha67) | Novus Biologicals | Cat# NB100-105; RRID: AB_10001154 |
| Rabbit monoclonal anti-HIF-1α (clone D1S7W) | Cell Signaling Technology | Cat# 36169; RRID: AB_2799095 |
| Rabbit polyclonal anti-Lamin A/C | Cell Signaling Technology | Cat# 2032S; RRID: AB_2136278 |
| Rabbit monoclonal β-Tubulin (clone 9F3) | Cell Signaling Technology | Cat# 2128S; RRID: AB_823664 |
| Anti-Rabbit IgG (H + L), HRP conjugate | Promega | Cat# W4011; RRID: AB_430833 |
| Anti-Mouse IgG (H + L), HRP conjugate | Promega | Cat# W4021; RRID: AB_430834 |
| Goat anti-rabbit, Alexa Fluor 488 conjugate | Invitrogen | Cat# A11008; RRID: AB_143165 |
| Donkey anti-mouse, Alexa Fluor 594 conjugate | Invitrogen | Cat# A21203; RRID: AB_141633 |
| Goat anti-rabbit, Alexa Fluor 594 conjugate | Invitrogen | Cat# A11012; RRID: AB_141359 |
| Goat anti-mouse, Alexa Fluor 488 conjugate | Invitrogen | Cat# A11001; RRID: AB_2534069 |
| Goat anti-rat, Alexa Fluor 647 conjugate | Invitrogen | Cat# A21247; RRID: AB_141778 |
| Goat anti-mouse, Alexa Fluor 568 conjugate | Invitrogen | Cat# A11031; RRID: AB_144696 |
| Goat anti-mouse, Alexa Fluor 405 conjugate | Invitrogen | Cat# A31553; RRID: AB_221604 |
| Normal mouse IgG | Sigma-Aldrich | Cat# 12-371; RRID:AB_145840 |
| Rabbit monoclonal anti-phospho-SRC-3 (Ser857) (Clone 10A6) | Cell Signaling Technology (Dasgupta et al., 2018)47 | Cell culture supernatant produced from hybridoma; Gift from Cell Signaling Technology |
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| Biological samples | ||
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| Human breast cancer tissue microarrays (TMAs) | RPCCC | N/A |
|
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| Chemicals, peptides, and recombinant proteins | ||
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| Doxycycline hyclate | Sigma-Aldrich | Cat# D9891 |
| Doxycycline diet (2000mg/kg) | Envigo | Cat# TD.09633 |
| Lipofectamine 2000 | Cat# 11668500 | |
| Polybrene Infection/Transfection Reagent | Thermo Fisher Scientific Sigma | Cat# TR1003G |
| Vitronectin | Sigma-Aldrich | Cat# 5051-0.1MG |
| Fibronectin Human Protein, Plasma | Thermo Fisher Scientific | Cat# 33016015 |
| Falcon™ Cell Culture Inserts, 8μm | Sigma-Aldrich | Cat# 08-771-21 |
| Corning® BioCoat® Matrigel® Invasion Chambers with 8.0 μm PET Membrane | Corning | Cat# 354480 |
| Protein Ladder | Thermo Fisher Scientific | Cat# LC5800 |
| Pierce™ ECL Plus Western Blotting Substrate | Thermo Fisher Scientific | Cat# 32132X3 |
| RIPA buffer | Thermo Fisher Scientific | Cat# 89900 |
| NP40 lysis buffer | Thermo Fisher Scientific | Cat# FNN0021 |
| Protease Inhibitor Cocktail Set I – Calbiochem | Sigma-Aldrich | Cat# 539131-10VL |
| Halt™ Phosphatase Inhibitor Cocktail | Thermo Fisher Scientific | Cat# 78427 |
| Micro BCA Protein Assay Kit | Thermo Fisher Scientific | Cat# 23235 |
| Acetone | Sigma-Aldrich | Cat# 179973-1L |
| 10% buffered formalin | Sigma-Aldrich | Cat# HT501320 |
| Sucrose | Sigma-Aldrich | Car# 84097 |
| Tissue-Tek O.C.T. Compound | Sakura Finetek | Cat# 4583 |
| Vector® TrueVIEW® Autofluorescence Quenching Kit | Vector Labs | Cat# SP-8400 |
| Antigen Unmasking Solution, Citrate-Based | Vector Labs | Cat# H-3300 |
| Normal Goat Serum Blocking Solution, 2.5% | Vector Labs | Cat# S-1012-50 |
| VectaMount Permanent Mounting Medium, 60 mL | Vector Labs | Cat# H-5000-60 |
| Vectashield vibrance with DAPI, mounting media | Vector Labs | Cat# H-1800-10 |
| Bond Dewax Solution | Leica | Cat# AR9222 |
| Bond Epitope Retrieval 2 | Leica | Cat# AR9640 |
| Bond Polymer Refine Detection kit | Leica | Cat# DS9800 |
| Rabbit Envision | Agilent | Cat# K4003 |
| D-luciferin, potassium salt | Goldbio | Cat# LUCK-1G |
| Matrigel™ Membrane Matrix | Corning | Cat# 354234 |
| Restore™ PLUS Western Blot Stripping Buffer | Thermo Fisher Scientific | Cat# 46430 |
| Protein G magnetic beads, 5mL | Thermo Fisher Scientific | Cat# 88848 |
| RNeasy Mini Kit | Qiagen | Cat# 74106 |
| TURBO DNA-free kit | Invitrogen | Cat# AM1907 |
| SuperScript VILO cDNA synthesis kit | Invitrogen | Cat# 11754250 |
| PowerUp™ SYBR™ Green Master Mix | Thermo Fisher Scientific | Cat# A25777 |
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| Critical commercial assays | ||
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| ONE-Glo™ EX Luciferase Assay System | Promega | Cat# E8110 |
| NADP/NADPH-Glo™ Assay | Promega | Cat# G9081 |
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| Deposited data | ||
|
| ||
| RNA-seq data of WT and PFKFB4-KO cells | This paper | GEO: GSE217132 |
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| Experimental models: Cell lines | ||
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| Human: MDA-MB-468 cells | ATCC | HTB-132 |
| Human: MDA-MB-231 cells | ATCC | HTB-26 |
| Human: LM3.3 cells | Minn et al., 200564 | N/A |
| Mouse: EMT6 cells | ATCC | CRL-2755 |
| Mouse: 4T1 cells | ATCC | CRL-2539 |
| Human: HEK 293T cells | RPCCC Gene Modulation Services Shared Resource | N/A |
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| Experimental models: Organisms/strains | ||
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| Mouse: C.B-Igh-1b/IcrTac-Prkdcscid | RPCCC in-house | N/A |
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| Oligonucleotides | ||
|
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| Primers used for qPCR analysis, see Table S2 | Integrated DNA Technologies | N/A |
| Primers for Targeted Amplicon Sequencing, see Table S3 | Integrated DNA Technologies | N/A |
|
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| Recombinant DNA | ||
|
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| pLenti-U6-human PFKFB4 gRNA-Cas9-puro | CBASS Core at Baylor College of Medicine | N/A |
| pLenti-U6-mouse PFKFB4 gRNA-Cas9-puro | CBASS Core at Baylor College of Medicine | N/A |
| pLKO.1-puro-shRNA targeting human SRC-3 | Sigma-Aldrich | TRCN0000370321, TRCN0000365196 |
| pLKO.1-puro non-targeting shRNA | Sigma-Aldrich | SHC216 |
| pTRIPZ inducible shRNA targeting human PFKFB4 | Horizon Discovery | V3THS_378097, V3THS_378099 |
| SMARTvector inducible shRNA targeting mouse PFKFB4 | Horizon Discovery | V3IMMHER_13710683, V3IMMHER_14783876 |
| SMARTvector Inducible Nontargeting hEF1a-TurboGFP (control 1) | Horizon Discovery | Cat# VSC11653 |
| pLenti ORF clone of Human ITGB3 tagged with mGFP | Origene | RC221606L4 |
| pLenti ORF clone of Human PFKFB4 tagged with myc-DDK | Origene | RC201573L3 |
| pLenti-luciferase-P2A-Neo | Xu et al., 201865 | Addgene #105621 |
| pLenti HRE-Luc pGK Hygro | Briggs et al., 201648 | Addgene #118706 |
| pMD2.G | Didier Trono Lab | Addgene #12259 |
| psPAX2 | Didier Trono Lab | Addgene #12260 |
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| Software and algorithms | ||
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| ImageJ v1.52a | https://imagej.nih.gov/ij/index.html | |
| Image Lab Software v6.0.1 | Bio-Rad | https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z |
| Aperio ImageScope v12.4.3.7009 | Leica Biosystems | https://www.leicabiosystems.com/us/digital-pathology/manage/aperio-imagescope/ |
| Graphpad Prism v9.3.1 | GraphPad Software | https://www.graphpad.com/ |
| Leica Application Suite X v3.7.4.23463 | Leica Microsystems Inc. | https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/downloads/ |
| Vevo LAZR | VisualSonics Inc. | https://www.visualsonics.com/product/imaging-systems/vevo-lazr |
| Living Image Software v4.7.3 | PerkinElmer | https://www.perkinelmer.com/lab-products-and-services/resources/in-vivo-imaging-software-downloads.html |
| FCS Express v7.14.0020 | De Novo Software | https://denovosoftware.com/ |
| QuantStudio Real-Time PCR Software v1.3 | Applied Biosystems | https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-flex-software.html |