Additional Supporting Information may be found in the online version of this article.

jbmr2026-sm-0001-sm-SupplFig-S1.tif1359KFigure S1. RAW264.7 cells transduced with MVNP retrovirus express the MVNP mRNA. Empty retrovirus (EV) and MVNP-retrovirus transduced RAW264.7 cells were analyzed by qPCR for MVNP mRNA and 18S expression and the products run on a 2% agarose gel and detected with ethidium bromide. Only the MVNP transduced RAW264.7 cells were positive for MVNP mRNA expression. The gene-specific primers for MVNP were 5′-AGTGCAGGATCATACCCTCTG-3′ (sense) and 5′-ATGCTGGATCAAAGTAAGATCG-3′ (antisense).
jbmr2026-sm-0002-sm-SupplFig-S2.tif1738KFigure S2. Tbk1 mRNA levels were not affected by MVNP. RNAs isolated from EV-NIH3T3 and MVNP-NIH3T3 cells were analyzed for Tbk1 mRNA expression by qPCR.
jbmr2026-sm-0003-sm-SupplFig-S3.tif8987KFigure S3. TBK1 expression and phosphorylation is increased in MVNP-NIH3T3 cells. EV- and MVNP-NIH3T3 cells were treated with TNF-α (10 ng/mL) for the indicated time periods. Cell lysates were harvested for Western blot analysis of total TBK1 and phosphorylated-Ser172-TBK1. The ratios of phospho-TBK1 to β-actin, of total TBK1 to β-actin, and of phospho-TBK1 to total TBK1 were quantitated by Image J software.
jbmr2026-sm-0004-sm-SupplFig-S4.tif5958KFigure S4. TBK1 over-expression stimulated IL6 promoter-driven reporter activity. (A) HEK293, MC4 and mouse primary BMM cells were infected with FUGW-GFP (EV) and FUGW-TBK1-GFP (TBK1) expression lentiviruses for 72 hrs, and cell lysates were immunoblotted for TBK1, GFP and β-actin. (B) HEK293 cells were cotransfected with IL6-promoter luciferase reporter plasmid and either pCDNA3.1 (EV1), pCNDA3.1-T7-TBK1, FUGW-GFP (EV2) or FUGW-TBK1-GFP as indicated. Regulation of the cotransfected IL6-promoter driven luciferase reporter by T7-TBK1 and TBK1-GFP was assessed by dividing the luciferase assay results from each TBK1 plasmid by the luciferase units from the respective EV plasmids to give Fold RLU. The capacity of T7-TBK1 and TBK1-GFP to induce IL6-promoter activity was similar, demonstrating that TBK1-GFP is functional.
jbmr2026-sm-0005-sm-SupplFig-S5.tif13395KFigure S5. Lentiviral transduction efficiencies of SCR and shTBK1 lentiviruses were assessed by qPCR for expression of the lentiviral puromycin resistance gene. (A) TRAP staining of RANKL-treated BMM. Original magnification 200×. (B) RNAs isolated from the cells after 4 days RANKL treatment were also analyzed by qPCR for the puromycin resistance gene (Puro) mRNA expressed by the lentiviruses. The gene-specific primers for Puro were 5′- TGCAAGAACTCTTCCTCACG-3′ (sense) and 5′- CGATCTCGGCGA ACACC-3′ (antisense). The data indicate that there were similar lentiviral infection efficiencies among all the groups.
jbmr2026-sm-0006-sm-SupplFig-S6.tif15373KFigure S6. Knockdown of Tbk1 expression impaired MVNP-induced increased response of OCL progenitors to TNF-α. BMM from WT and TRAP-MVNP mice were prepared and infected with GFP and shTBK1 lentiviruses using the methods described in Figure 5. After M-CSF (50 ng/mL) and TNF-α (20 ng/mL) treatment for 6 days, (A) Tbk1 mRNA expression levels were detected by qPCR. (B) TRAP+ multinuclear cells with 3 or more nuclei (OCL) were counted/well and the mean ± SD graphed. (C-E) RNAs isolated from the cells after 6 days TNF-α treatment were also analyzed by qPCR for (C) IL6 mRNA, (D) Tnfrsf11a (RANK), and (E) Ctsk mRNAs.

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