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Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

  1. Naga Suresh Adapala
  2. Gaurav Swarnkar
  3. Manoj Arra
  4. Jie Shen
  5. Gabriel Mbalaviele
  6. Ke Ke
  7. Yousef Abu-Amer  Is a corresponding author
  1. Department of Orthopaedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, United States
  2. Bone and Mineral Division, Department of Medicine, Washington University School of Medicine, United States
  3. Shriners Hospital for Children, United States
Research Article
Cite this article as: eLife 2020;9:e56095 doi: 10.7554/eLife.56095
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
NEMOK270A mutant expression in BMMs exacerbates RANKL-induced osteoclastogenesis.

(A) Domain structure of NEMO (B) Western blot showing expression of pMX-NEMOWT (NM) and pMX-NEMO mutants (NM-K270A, NEMO-D304N and NM-K319A). (C) BMMs from WT and (LysM-cre-NEMO f/f) NEMO-cKO mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMOWT (NM-WT) and NEMOK270A (NM-KA) and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml). (D) Representative TRAP staining for osteoclast (n = 8) and (D) quantification of TRAP positive OCs. qPCR analysis for OC marker genes (E) TRAP, (F) CTSK, (G) MMP9, (H) β3integrin, (I) DC-STAMP and (J) NFATC1 (p=0.057). Representative data (n = 3 independent experiments). (K) BMMs from RelA_luc reporter mice expressing NM-WT and NM-KA were cultured in the presence of MCSF (10 ng/ml) for 3 days followed by RANKL stimulation with RANKL (50 ng/ml) for 6 hr and RelA-luciferase activity measurement (n = 3). pMX-Flag-NEMOWT-RFP (NM-WT), pMX-Flag-NEMOK270A-RFP (NM-KA). (*p<0.05, **p<0.01 and ***p<0.001).

Figure 1—source data 1

Western blot showing expression ofpMX-NEMOWT(NM) andpMX-NEMOmutants (NM-K270A, NEMO-D304N and NM-K319A).

https://cdn.elifesciences.org/articles/56095/elife-56095-fig1-data1-v2.pdf
Figure 1—source data 2

qPCR analysis for OC marker genes.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig1-data2-v2.xlsx
Figure 1—source data 3

RelA-luciferase activity.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
BMMs from wild type mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMOWT (NM-WT), NEMOK270A (NM-KA), NEMO-D304N and NEMO-K319A constructs followed by culture in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days.

(A) Representative images for TRAP staining for osteoclast (n = 8). (B) Wild type BMMs were transduced with different dilution (1x, 0.5x, 0.25x, 0.1x and 0.05x) of retroviral particles expressing NEMOK270A (NM-KA). Wild type BMMs were also transduced with viral particles expressing NEMO-D304N and NEMO-K319N at 1x dilution. Western-blot using anti-flag antibody shows higher expression of NEMOK270A even at low viral dilution. pMX-Flag-NEMOWT-RFP (NM-WT), pMX-Flag-NEMOK270A-RFP (NM-KA).

Figure 2 with 1 supplement
Expression of NEMOK270A in vivo leads to inflammatory osteolysis and joint destruction.

NEMOK270A was conditionally expressed in myeloid cells (NM-KA mice) by crossing NEMOK270A f/f mice with LysozymeM cre expressing mice. (A) Whole body images of NM-KA mice compared to littermate wild type control mice (6 weeks old). The arrows point to deformed joints and swelling. (B) Photomicrograph of spleen and bone from NM-WT and NM-KA mice. MicroCT analysis of bone from NM-WT and NM-KA mice showing (C) femur trabecular bone, (D) knee joint osteolysis (arrow) and quantification of (E) Bone volume/total volume (BV/TV), (F) Connectivity density, (G) Trabecular number (Tb.N), (H) Trabecular thickness (Tb.Th) and (I) Trabecular separation (Tb.Sp) in the femur trabecular region (n = 6). Long bones from 6 weeks old NM-WT and NM-KA mice were processed for histology and stained for TRAP to visualize TRAP+ osteoclasts in (K) bone sections and (K) Articular surfaces of knee joint (arrow). Representative images (n = 6) Serum was collected from NM-WT and NM-KA to measure serum (L) TRAP and (M) CTX concentration as an indicator of increased osteoclast activity (n = 6–8). LysM-cre-NEMOWT-f/f (NM-WT), LysM-cre-NEMOK270A-f/f (NM-KA) mice. (*p<0.05, **p<0.01 and ***p<0.001).

Figure 2—figure supplement 1
Generation of NEMO transgenic mice.

Schematic diagram showing generation of conditional (A) NEMO-K270A-KI (NM-KA) and (B) NEMO-WT-Tg (NM-WT-Tg) mice (6 weeks old). (C) MicroCT images of paw and ankle joint showing osteolysis in NM-KA mice (arrow). X-ray images of NM-WT and NM-KA mice showing significant bone loss and deformities in (D) whole body (E) Knee joint (F) ankle and paw (arrow pointing towards osteolysis and deformed bones). (G) Whole body images of NM-WT and NM-WT-Tg mice (6 weeks old). (H) Photomicrograph of spleen and bone from NM-WT and NM-WT-Tg mice. MicroCT analysis showing quantification of (I) Bone volume/total volume (BV/TV), (J) Trabecular number (Tb.N), (K) Trabecular thickness (Tb.Th) and (L) Trabecular separation, between NM-WT and NM-WT-Tg mice (n = 3).

Figure 3 with 1 supplement
NEMOK270A mutation instigates systemic inflammation.

Serum was collected from NM-WT and NM-KA mice (n = 8–10) to measure concentration of inflammatory cytokines (A) Interleukin (IL) 1b, (B) IL-4, (C) IL-6, (D) IL-10, (E) IL-13, (F) IL-17, (G) Monocyte chemoattractant protein1 or CCL2, (H) Tumor necrosis factor alpha, (I) Macrophage colony stimulating factor, (J) macrophage Inflammatory protein (MCP)−1 or CCL3, (K) keratinocyte chemoattractant or neutrophil activating protein three or CXCL1 and (L) granulocyte colony stimulating factor (GCSF). (M) BMMs from NM-WT and NM-KA mice were isolated and cultured in the presence of MCSF (10 ng/ml) and RANKL (10 ng/ml). Representative TRAP staining for osteoclast (n = 8) is shown. (N–R) Representative qPCR analysis for OC marker genes TRAP, CTSK, β3integrin, DC-STAMP and NFATC1 (n = 3). (S) BMMs from NM-WT and NM-KA mice were isolated and cultured in the presence of MCSF (10 ng/ml) four days followed by serum starvation and stimulation with RANKL (50 ng/ml) for different time points (n = 8). Representative western-blot showing activation of p65 (phos-p65/p65 ratio) post RANKL stimulation in BMMs from NM-WT and NM-KA mice. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05, **p<0.01 and ***p<0.001).

Figure 3—source data 1

Serum concentration of cytokines from NM-WT and NM-KA mice measured by ELISA.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig3-data1-v2.xlsx
Figure 3—source data 2

Representative qPCR analysis for OC marker genes.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig3-data2-v2.xlsx
Figure 3—source data 3

Representative western-blot of p65 (phos-p65/p65 ratio) post RANKL stimulation in BMMs from NM-WT and NM-KA mice.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig3-data3-v2.pdf
Figure 3—figure supplement 1
BrdU was injected to NM-WT and NM-KA mice.

1 day after injection single cell suspensions from bone marrow were prepared by flushing the marrow out of femur and tibia. Following RBC lysis, cells were stained with different antibody cocktails. Flow analysis showing percentage of (A) Lin-Sca1+Kit+ (LSK) cells in haemopoietic stem cells (HSC), (B) percentage of common myeloid progenitor (CMP), granulocyte-monocyte progenitors (GMP) cells; proliferation of (C) LSK, (D) CMP and (E) GMP cells, (F) Myeloid cells and (G–J) proliferation of neutrophils populations. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice.

Figure 4 with 1 supplement
NEMOK270A mutation hampers autophagy.

PLAT-E cells were transfected with retroviral pMX-Flag-NEMO-WT-RFP (NM-WT) and pMX-flag-NEMO-K270-RFP (NM-KA) expression vector. (A) Fluorescence images showing distribution of NM-WT-RFP in cytoplasm compared to puncta (yellow arrows) (juxtaposed to nuclei- DAPI stained) formation in case of NM-KA-RFP in PLAT-E cells. (B) Western blot for LC3 using WES (protein simple). BMMs were cultured for 2 days with RANKL (preOC) followed by 6 hr of serum starvation and western blotting. Fold change of LC3 relative to actin is indicated on top. (C) Quantification of LC3+ cells per high magnification field. (D) For flow cytometry, BMMs were transduced with pMX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells, and flow analysis was done to detect GFP signal or LC3 flux. Contour plots showing LC3-GFP+ expressing cells in NM-WT and NM-KA preOC (Blue: NM-WT without serum starvation, Red: NM-KA without serum starvation, and Black: after 6 hr of serum starvation), (E) Histograms representing shift in LC3-GFP+ cells following induction of autophagy (Red histogram: background signal in uninfected cells, Blue histogram: No serum starvation or 10% FBS control, yellow: 6 hr serum starvation, and pink: chloroquine), (F) Change in Mean fluorescent intensity (MFI) showing LC3-GFP signal in NM-WT and NM-KA preOC cells post autophagy induction. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05). (*p<0.05, **p<0.01 and ***p<0.001).

Figure 4—figure supplement 1
Autophagy is negatively impacted in NEMOK270A cells.

(A) Pre-OC (RANKL-treated BMMs) from NM-WT and NM-KA mice were pelleted and processed for electron microscopic and Immunofluorescence (IF) analysis after 6 hr of serum starvation. Representative Electron microscopic images (x7500) showing nucleus, Cell membrane (CM) lysosome (L) in NM-WT preOC and cytoplasmic aggregates (yellow arrow) in NM-KA preOC. (B) Representative IF images for NEMO (red), LC3 (green) and NEMO-LC3 colocalization (yellow). Arrows indicate accumulation of NEMO in LC3 positive vacuole-like structures. (C) Representative western blot showing expression of LC3 from BMMs starved and stimulated with RANKL as shown. (D) Representative Western blot for mTOR expression in BMMs from NM-WT and NM-KA mice treated as shown. (E) Pre-osteoclasts were treated with chloroquine as indicated and number of TRAP+ multi nucleated osteoclasts (MNC) per well were counted in triplicate wells from three independent experiments (*p<0.05).

Figure 5 with 1 supplement
NEMOK270A is restricted to autophagosomes whereas NEMOWT is delivered to lysosomes.

preOC from NM-WT and NM-KA mice were pelleted and processed for Immunofluorescence (IF) and electron microscopic analyses after 6 hr of serum starvation. (A) Representative IF images showing NEMO (red) and LAMP1 (green). Arrows indicate colocalization of NEMO in LAMP1 positive vacuole-like structures in NM-WT, which is decreased in NM-KA preOC. (B) Representative electron microscopic images (x7500) lysosome (L), Autophagosome (AP) and APL (Autophagolysosome). (C) Representative IF images showing changes in cellular NEMO organization in response to autophagy induction by serum starvation in NM-WT and NM-KA preOC cells. NEMO-puncta (white) and nucleus (blue). (D) NEMO-puncta quantification. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05, **p<0.01 and ***p<0.001).

Figure 5—figure supplement 1
preOC from NM-WT and NM-KA mice were processed for Immunofluorescence (IF) analysis after 6 hr of serum starvation.

(A) NEMO-puncta determination in IgG control and (B) chloroquinone (CQ) treated pOC and quantification. (C) NEMO puncta quantification in presence of chloroquinone (CQ).

Figure 6 with 1 supplement
Intact NEMO K270 residue is essential for post-translational modification (PTM) by ISG15.

(A) Volcano plot showing changes in autophagy and PTM related proteins in immunoprecipitated lysates from NM-WT compared with NM-KA BMMs using anti-NEMO antibody. preOC from NM-WT and NM-KA mice were processed for Immunofluorescence (IF) and Immuno-electron microscopy (EM) analysis after 6 hr of serum starvation. (B) Representative IF images of NEMO (red) and ISG15 (green) co-localization in preOC. White arrows indicate foci of expression of ISG15 (enlarged inset at bottom of panel B). Yellow arrows indicating NEMO-ISG15 co-localization. (C) ISGylated proteins (upper panel) and free ISG15 in response to RANKL treatment. (D) BMMs from WT and ISG15-KO mice were isolated and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for four days. Representative TRAP staining for osteoclast (D) and quantification (E). (**p<0.01).

Figure 6—source data 1

Proteomic data from immunoprecipitated lysates from NM-WT compared with NM-KA BMMs.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig6-data1-v2.xlsx
Figure 6—source data 2

Western blots for ISGylated proteins and free ISG15 in response to RANKL treatment.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig6-data2-v2.pdf
Figure 6—source data 3

Osteoclast quantification from WT and ISG15-KO in vitro cultures.

https://cdn.elifesciences.org/articles/56095/elife-56095-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
Representative Immuno-EM images (x7,500) showing localization of NEMO (black arrows) and ISG15 (blue arrow) in NM-WT and NM-KA cells; lysosomes (L), autophagosome (AP).

Large black dot: NEMO (18 nm gold particle), small black dot: ISG15 (12 nm gold particle). BMMs from NM-WT and NM-KA mice cells treated with RANKL for different time points followed by western blot.

Figure 7 with 1 supplement
ISGylation of NEMO is essential to restrain osteoclastogenesis.

BMMs from NM-WT and NM-KA mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing ISG15 and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days. (A) Representative TRAP staining for osteoclast (n = 6) and (B) quantification of TRAP positive OCs. (C) BMMs from NM-WT and NM-KA mice were transduced with ISG15 and pMRX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells. The cells were cultured for 2 days (preOC) followed by 6 hr of serum starvation and flow analysis to detect GFP signal or LC3 flux. (C) Histograms representing shift in LC3-GFP+ cells following induction of autophagy. Blue histogram: serum starvation, yellow histogram: serum starvation + ISG15 expression (D) Change in Mean Fluorescent Intensity (MFI) showing LC3-GFP signal. (E) Wild type BMMs transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMO+/-ISG15, NEMO-K270A+/-ISG15 NEMO-WT::ISG15 (fused) and NEMO-K270A::ISG15 (fused) protein and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days.(E) Representative TRAP staining for osteoclast (n = 3) and (F) quantification of TRAP positive OCs. (G) NEMO puncta regulation by ISG15: Live images of preOC expressing RFP-NEMOWT+/-ISG15, RFP-NEMOK270A+/-ISG15, GFP-NEMOWT::ISG15 and GFP-NEMOK270A::ISG15 fusion protein. Yellow arrows indicate NEMOK270A puncta. ISG15 panel which is not tagged serves as background control. (H) Quantification of LC3 puncta+ preOC cells shown in Figure 7—figure supplement 1. (I) WB for LC3 in preOC expressing NEMOWT+/-ISG15, NEMOK270A+/-ISG15, NEMOWT::ISG15 and NEMOK270A::ISG15 fusion protein. (*p<0.05, **p<0.01 and ***p<0.001). (::) denotes fusion. (J) Representative IF images (NEMO (Red); LAMP1(green)). NEMO localization in preOC expressing NEMOWT+/-ISG15, NEMOK270A+/-ISG15, NEMOWT::ISG15 and NEMOK270A::ISG15 fusion protein. Green arrow- Lysosome and Yellow arrow-localization of NEMO in Lysosome.

Figure 7—figure supplement 1
LC3 puncta accumulation of NEMOK270A is reduced by forced fusion with ISG15.

(A) Representative IF images for LC3 puncta+ cells (arrow) in preOC expressing NEMOWT+/-ISG15, NEMOK270A+/-ISG15, NEMOWT::ISG15 and NEMOK270A::ISG15 fusion protein (quantified in Figure 7H).

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, Strain backgroud Mus musculusIkbkg (Nemo)-floxedDr. Manolis Pasparakis, Cologne, GermanyNM-f/fC57BL/6 background
Strain, Strain backgroud Mus musculusIkbkg (Nemo)-K270A-floxedMouse Genetics Core, Washington University in St.LouisNM-KA-f/fC57BL/6 background
Strain, Strain backgroud Mus musculusIkbkg (Nemo)-WT-Tg-floxedMouse Genetics Core, Washington University in St.LouisNM-WT-Tg f/fC57BL/6 background
Strain, Strain backgroud Mus musculusLyz2 (Lysozyme M)-creLysM-creC57BL/6 background
Strain, Strain backgroud Mus musculusLysM-cre-NEMO-floxThis PaperNM-cKOC57BL/6 background
Strain, Strain backgroud Mus musculusLysM-cre-NEMO-K270A-f/fThis PaperNM-KAC57BL/6 background
Strain, Strain backgroud Mus musculusLysM-cre-NEMO-WT-f/fThis PaperNM-WT-TgC57BL/6 background
Strain, Strain backgroud Mus musculusRELA (NF-ĸB)-GFP-luciferase reporterThe Jackson LaboratoryNF-ĸB reporter miceC57BL/6 background
Recombinant DNA reagentpMX- retroviral vectorCell biolabsCat# RTV-010Retroviral vector
Recombinant DNA reagentpMX-GFPThis paperGFP version of pMX retroviral vector
Recombinant DNA reagentpMX-flag-NEMO-WT-RFPThis paperNEMO WT with flag tag and RFP on pMX backbone-Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-K270A-RFPThis paperNEMO K270A mutant with flag tag and RFP on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-D304NThis paperNEMO D304N mutant on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-K319AThis paperNEMO K319A mutant with Flag tag on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-WT-GFPThis paperNEMO WT with Flag tag and GFP on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-HA-ISG15This paperISG15 with HA tag on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-WT-ISG15-GFPThis paperNEMO WT-ISG15 fusion construct with GFP tag on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentpMX-flag-NEMO-K270A-ISG15-GFPThis paperNEMO K270A-ISG15 fusion construct with GFP tag on pMX backbone -Available in Dr. Yousef Abu-Amer’s lab
Recombinant DNA reagentPMRX-GFP-LC3-RFP retrovirusAddGeneCat# 84573LC3 wth GFP and RFP on PMRX backbone
Recombinant DNA reagentXtreme gene 9RocheCat# 6365809001Transfection reagent
Cell line (Homo-sapiens)PLAT-ECell biolabsCat# RV-101For generating retroviruses
Commercial assay or kitTRAP-Leukocyte kitMillipore-SigmaCat# 387A-1KTIdentify osteoclasts
Commercial assay or kitluciferase activityGoldBioCat# I920-50NFkB activity assay
Commercial assay or kitBCA assayThermo FisherCat# 23227Quantitation of protein
OtherCell lysis bufferCell SignalingCat# 9803SWestern blot reagent
Antibodydonkey anti-rabbit and anti-mouseLI-COR BiosciencesCat# 926–32213, RRID:AB_621848WB(1:10,000)
AntibodyNEMO
(Rabbit polyclonal/Mouse monoclonal)
Santa CruzCat# SC-8330,
RRID:AB_2124846
IF(1:200), WB(1:1000)
AntibodyLAMP-1
(Mouse monoclonal)
Santa CruzCat# SC-20011,
RRID:AB_626853
IF(1:200)
AntibodyISG15
(Mouse monoclonal)
Santa CruzCat# SC-166755, RRID:AB_2126308IF(1:200), WB(1:1000)
Antibodyphos-p65
(Rabbit polyclonal)
Cell SignalingCat# 3031, RRID:AB_330559WB(1:1000)
Antibodyp65
(Rabbit polyclonal)
Cell Signaling Technology,Cat# 8242, RRID:AB_10859369WB(1:1000)
AntibodyLC3
(Rabbit polyclonal)
Cell Signaling Technology,Cat# 3868, RRID:AB_2137707IF(1:200), WB(1:1000)
AntibodyFlag
(Rabbit polyclonal)
Millipore-SigmaCat# F1804, RRID:AB_262044WB(1:1000)
Antibodyβ-actin
(Mouse monoclonal)
Millipore-SigmaCat# A2228, RRID:AB_476697WB(1:5000)
Antibodyanti-B220
(Rat monoclonal)
Thermo FisherCat# 14-0452-82, RRID:AB_467254FACS (1 µL per test)
Antibodyanti-CD3e
(Armenian hamster monoclonal)
BiolegendCat# 100301, RRID:AB_312666FACS (1 µL per test)
Antibodyanti-Gr1
(Rat monoclonal)
Thermo FisherCat# 14-5931-82, RRID:AB_467730FACS (1 µL per test)
Antibodyanti-Ter119
(Rat monoclonal)
BD BioscienceCat#550565, RRID:AB_393756FACS (1 µL per test)
Antibodyanti-Sca1 PerCP Cy5.5 (Rat monoclonal)Thermo FisherCat# 122523, RRID:AB_893621FACS (1 µL per test)
Antibodyanti-c-Kit APC eFluor 780 (Mouse monoclonal)Thermo FisherCat# 47-1171-82, RRID:AB_1272177FACS (1 µL per test)
Antibodyanti-CD34 FITC (Mouse monoclonal)Thermo FisherCat# 343503,
RRID:AB_343503
FACS (1 µL per test)
AntibodyCD16/32 eFluor450 (Rat monoclonal)Thermo FisherCat# 48-0161-82, RRID:AB_1272191FACS (1 µL per test)
Antibodycolloidal gold conjugated secondary antibodiesJackson ImmunoResearch LaboratoriesCat# 715-205-150, RRID:AB_2340822Electron microscopy
(1:25)
AntibodyAlexa Fluor 568 (goat anti-mouse IgG)Thermo FisherCat# A11031, RRID:AB_144696IF
(1:2000)
AntibodyAlexa Fluor 488 (goat-anti-rabbit IgG)Thermo FisherCat# A11034, RRID:AB_2576217IF (1:2000)
Commercial assay or kitmultiplex mouse cytokine kitsR and D SystemsCat# AYR006Inflammation markers
Commercial assay or kitmultiplex mouse cytokine kitsMillipore-SigmaCat# MCYTMAG-70K-PX32Inflammation markers
Commercial assay or kitRatLaps (CTX-1) EIAImmunodiagnostic SystemsCat# AC-06F1Serum cross‐linked telopeptide of type I collagen (CTX‐I)-bone resorption marker
Commercial assay or kitMouse TRAP (TRAcP 5b) kitsImmunodiagnostic SystemsCat# SB TR-103osteoclast marker
Commercial assay or kitPureLink RNA mini kitThermo FisherCat# 12183025RNA isolation
OtheriTaq universal SYBR green super-mixBioRadCat# 1725120Real-Time PCR reagent
Sequence-based reagentTRAP_FIDTPCR primerCGACCATTGTTAGCCACATACG
Sequence-based reagentTRAP_RIDTPCR primerCACATAGCCCACACCGTTCTC
Sequence-based reagentCTSK_FIDTPCR primerATGTGGGTGTTCAAGTTTCTGC
Sequence-based reagentCTSK_RIDTPCR primerCCACAAGATTCTGGGGACTC
Sequence-based reagentMMP9_FIDTPCR primerACTGGGCTTAGATCATTCCAGCGT
Sequence-based reagentMMP9_RIDTPCR primerACACCCACATTTGACGTCCAGAGA
Sequence-based reagentNFATC1_FIDTPCR primerCCGGGACGCCCATGCAATCTGTTAGT
Sequence-based reagentNFATC1_RIDTPCR primerGCGGGTGCCCTGAGAAAGCTACTCTC
Software, algorithmImageJImagej.nih.govIF image processing, count
Software, algorithmGraphPadGraphpad prism-8 softwareStatistical Analysis softwareGraph preparation, statistical analysis

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