Cxcl1

RNA was isolated using an RNeasy isolation kit (Qiagen) and cDNA synthesis was performed using SuperScriptII reverse transcriptase (Invitrogen). Quantitative PCR was performed using Taqman probe/primer sets to amplify the following genes: GAPDH (Mm99999915_g1), IL-6 (Mm00446190_m1), CXCL5 (Mm00436451_g1), CXCL1 (Mm04207460_m1), CCL2 (Mm00441242_m1), CCL5 (Mm01302427_m1), CCL8 (Mm01297183_m1), CCL7 (Mm00443113_m1) and CXCL14 (Mm00444699_m1) (Thermo Fisher). For experiments using isograft tissue for gene expression analysis, isografts were isolated at 11 days post injection. The tissue was snap-frozen and then pulverized using a mortar and pestle. RNA isolation and cDNA synthesis was performed as above. The samples were then pre-amplified using Taqman PreAmp Master Mix kit and Taqman primers for the following genes of interest: GAPDH (Mm99999915_g1), STAT3 (Mm01219775_m1), Nos2 (Mm00440502_m1), Arg1 (Mm00475988_m1), CXCL1 (Mm04207460_m1), M-CSF (Mm00432686_m1), GM-CSF (Mm01290062_m1), IL-6 (Mm00446190_m1), CCL2 (Mm00441242_m1), TGFβ (Mm01178820_m1), IL-1β (Mm00434228_m1), IL-1α (Mm00439620_m1) and IL-10 (Mm01288386_m1) (Thermo Fisher). All primer sequences are proprietary. For analysis, values are normalized to GAPDH.

Real-time PCR was conducted with the Bio-Rad CFX96 system employing TaqMan PCR Master Mix (Bio-Rad, Cat# 1725284) and premixed primers/probe sets (mouse: CXCL1 (Mm04207460_m1) and Hprt (Mm03024075_m1); human: CXCL1 (Hs00236937_m1), CXCL2 (Hs00601975_m1), CXCL5 (Hs01099660_g1), CXCL8 (Hs00174103_m1), HDAC5 (Hs00608351_m1), and HPRT (Hs02800695_m1)) from Thermo Fisher Scientific.

Western blot analysis was conducted using a previously described protocol.^{39 (link)} We used antibodies against ANXA1, β-catenin, caspase 3, Lrp5, Lrp6, Runx2, Sclerostin, Snail, TGFβ, NFATc1, cathepsin K (all from Cell Signaling, Danvers, MA, USA), DMP1, ANXA6, CXCL5 (all from Abcam, Cambridge, MA, USA), M-CSF, MMP9, OPN, TPM4 (all from Santa Cruz, Dallas, TX, USA), WISP1 (R&D systems, Minneapolis, MN, USA), β-actin (Sigma, Saint Louis, MO, USA), LIMA1, Trail (both from Novus, Centennial, CO, USA), p53, CXCL1 (both from Invitrogen, Carlsbad, California, USA), and DSP (ProteinTech, Rosemont, IL, USA). The expression levels of Sclerostin and Lrp5 in CM were detected by ELISA (My BioSource, San Diego, CA, USA). Proteins isolated from A5 osteocyte CM, Y4 osteocyte CM, and osteoclast control CM (RAW264.7 cells) were analyzed with an HF Hybrid Quadrupole Orbitrap mass spectrometer. Among the 549 identified proteins, 49 proteins had higher expression levels in A5 CM than in Y4 CM and control CM. Among these proteins, 11 (p53; SPARC = osteonectin; TPM1, TPM4 = tropomyosin 1 and 4; ANXA1, ANXA6 = annexin A1 and A6; FMOD = fibromodulin; OGN = osteoglycin; DSP = desmoplakin; AHNAK = desmoyokin; and LIMA1 = LIM domain actin-binding protein 1) were identified as potential tumor suppressors.

Antibodies against RanBP1 (cat. no. 8780; Cell Signaling Technology, Inc., Danvers, MA, USA), CD44 (cat. no. 3570; Cell Signaling Technology, Inc., Danvers, MA, USA), Sox2 (cat. no. 3579; Cell Signaling Technology, Inc., Danvers, MA, USA), Oct-4 (cat. no. 2750; Cell Signaling Technology, Inc., Danvers, MA, USA), Nanog (cat. no. 4893; Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ZEB1 (cat. no. sc-25388; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), SLUG (cat. no. sc-166476; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), SNAIL (cat. no. sc-10432; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Twist (cat. no. sc-15393; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALDH1A1 (cat. no. ab6192; Abcam, Cambridge, UK), ALDH1A3 (cat. no. ab129815; Abcam, Cambridge, UK), E-Cadherin (cat. no. ab15148; Abcam, Cambridge, UK), CD133 (cat. no. ab19898; Abcam, Cambridge, UK), N-Cadherin (cat. no. 610920; BD Transduction, San Diego, CA, USA), Vimentin (cat. no. MA5-14564; Invitrogen, Waltham, MA, USA), CXCL1 (cat. no. PA5-115328; Invitrogen, Waltham, MA, USA), IL-8/CXCL8 (cat. no. MAB208; R&D SYSTEMS, Minneapolis, MN, USA), and IL-18 (cat. no. AF2548; R&D SYSTEMS, Minneapolis, MN, USA) were used for Western blot analysis and Immunocytochemistry assays.

For ELISA of media, 3D cocultures were grown under 21% O₂ or 1% O₂ for 72 h. Media were collected, spun down, and assayed using the manufacturer’s protocol. ELISA assays used were IL1ɑ (#433404, BioLegend), CXCL1 (#EMCXCL1, Invitrogen), IL6 (#DY406-05, R&D Systems), and LIF (#445104, BioLegend).

Serum was tested for antibodies, cytokines and chemokines. Conventional sandwich ELISAs were used to test for anti-dsDNA Ab (Alpha Diagnostic International, San Antonio, TX), MCP-1 and CXCL1 (Invitrogen, Carlsbad, CA). Electrochemiluminescence ELISAs (Meso Scale Diagnostics, Rockville, MD) were used to test for IL-1β, IL-6, IL-17A, TNF-α. All other autoantibodies were tested for using an autoantigen microarray panel (University of Texas, Southwestern, TX). Pierce (Thermo-Fisher Scientific, Waltham, MA) LAL Chromogenic Endotoxin Quantitation Kit was used to analyze LPS levels in serum.