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Blood, Vol. 111, Issue 2, 885-893, January 15, 2008
The ubiquitin-mediated degradation of Jak1 modulates osteoclastogenesis by limiting interferon-β–induced inhibitory signaling
Blood Lee et al.
111: 885
Supplemental material for: Lee et al
Files in this Data Supplement:
- Figure S1. Jak1 expression is decreased by RANKL in RAW264.7 cells (PDF, 79.5 KB). -
RAW264.7 cells were incubated with 100 ng/ml RANKL for indicated times. Total cell lysates were subjected to Western blotting for Jak1 expression.
- Figure S2. Jak2 and Jak3 expression did not change significantly during the early osteoclast differentiation(PDF, 104 KB). -
BMMs were cultured with 30 ng/ml M-CSF and 100 ng/ml RANKL for indicated times. Total cell lysates were subjected to Western blotting for Jak2 and Jak3 expression.
- Figure S3. Jak1 mRNA expression was not affected by RANKL stimulation (PDF, 86.7 KB). -
(A) BMMs were cultured with 30 ng/ml M-CSF in the presence of indicated concentrations of RANKL. Total RNAs were isolated and subjected to reverse-transcription (RT)-PCR analysis for Jak1 mRNA expression. HPRT was also amplified to ensure the same amount of RNA between samples. (B) BMMs were cultured with 30 ng/ml M-CSF and 100 ng/ml RANKL for indicated times. RT-PCR analysis for Jak1 mRNA expression were performed. Results are representative of at least 3 independent experiments.
- Figure S4. The inhibitory effect of IFN-β on osteoclastogenesis is limited to the early stage of osteoclast differentiation (PDF, 103 KB). -
(A) BMMs were incubated with 30 ng/ml M-CSF and 100 ng/ml in the presence of 10 U/ml IFN- during the indicated periods. (B) After 3-day culture, cells were stained for TRAP activity. M and MR represents M-CSF only and M-CSF plus RANKL, respectively. Cells were treated with IFN- during different periods after RANKL stimulation, i.e. I, day1; II, day2; III, day3; IV day1~2.
- Figure S5. MG132 treatment sensitized pOCs to the IFN- β-mediated inhibition of osteoclastogenesis (PDF, 107 KB). -
(A) BMMs were first differentiated into pOCs by culturing with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 48 h. MG132 (1 µM) was added for the final 4 h of 48 h incubation. Then cells were further differentiated into osteoclasts with RANKL in the presence or absence of IFN-. (B) Osteoclast numbers were counted at the end of culture. Cells were counted in three random fields. Results are presented as mean ± S.D. and representative of at least three experiments with similar results. Asterisks represent statistical difference between the values (*, p < 0.05; **, p < 0.01). (C) BMMs were treated with MG132 up to 10 µM for 4 h and the viability of cells was tested by Cell Counting Kit-8 cytotoxicity assay (Dojindo Laboratories, Japan), which is based on the tetrazolium salt. The absorbance at 450 nm is directly proportional to the number of viable cells. (D) BMMs were treated with or without MG132 (1 µM) for 4 h and further cultured for 24 h in the presence of indicated concentrations of IFN- . The viability of cells was tested as above.
- Figure S6. STAT3 signaling pathway is involved in the IFN-β -mediated inhibition of osteoclastogenesis (PDF, 116 KB). -
(A) BMMs were first differentiated into pOCs by culturing with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 48 h. Then cells were stimulated with IFN- (100 U/ml) in combination with RANKL (500 ng/ml) for indicated times. Total cell lysates were subjected to Western blotting using phospho-specific antibodies. The expression of each protein was examined by stripping and reprobing the same membrane used to detect the phospho-form of the protein with respective antibody. (B) Control or STAT3siRNA virus-infected cells were incubated with 10 U/ml IFN- for 24 h and further incubated with 30 ng/ml M-CSF and 100 ng/ml RANKL for 3 days. At the end of culture, cells were stained for TRAP activity. MR represents M-CSF plus RANKL. (A and B) Data are representative of at least three independent experiments.
- Figure S7. STAT3 activation upon IFN- β stimulation of BMM is independent of STAT1 (PDF, 86.7 KB). -
(A) BMMs were first infected with viruses harboring empty vector or STAT1 siRNA construct. After 48 h, total cell lysates were examined for STAT1 expression by Western blotting. (B) Vector or STAT1siRNA-infected BMMs were stimulated with 100 U/ml IFN- for 30 min. Nuclear extracts were prepared and assayed for the DNA binding activities of STAT3 as described in the Materials and Methods. Excess unlabelled oligo DNA was added to confirm the specificity of assay.
- Figure S8. RANKL-induced SOCS1 and SOCS3 expression is decreased in Jak1 knocked-down BMMs (PDF, 113 KB). -
BMMs were first infected with viruses harboring empty vector or Jak1 siRNA construct. After 48 h, cells were stimulated with RANKL (100 ng/ml) for indicated times. Total cell lysates were subjected to Western blotting using anti-SOCS1 and -SOCS3 antibodies.
- Figure S9. Tyk2 may compensate for the absence of Jak1 to phosphorylate STAT1 (PDF, 63. KB). -
BMMs were first infected with viruses harboring empty vector or wild type (WT) human Tyk2 construct. After 48 h, cells were stimulated with IFN- (100 U/ml) for indicated times. Total cell lysates were subjected to Western blotting using phospho-specific anti-STAT1 antibody. The expression of Tyk2 and actin protein was examined by stripping and reprobing the same membrane.
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