Structured Review

Cell Signaling Technology Inc anti nfatc1
A schematic model of osteoclastogenesis regulated by the Blimp1-Bcl6-osteoclastic gene axis. RANKL–RANK interaction results in Blimp1 induction, leading to Bcl6 down-regulation and dissociation of Bcl6 from osteoclastic gene promoters, an event critical for osteoclastogenesis. <t>NFATc1</t> activation is induced by various factors, such as ITAM, TRAF6, c-Fos, and Ca 2+ signaling, which are also activated by RANKL.
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1) Product Images from "The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis"

Article Title: The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20091957

A schematic model of osteoclastogenesis regulated by the Blimp1-Bcl6-osteoclastic gene axis. RANKL–RANK interaction results in Blimp1 induction, leading to Bcl6 down-regulation and dissociation of Bcl6 from osteoclastic gene promoters, an event critical for osteoclastogenesis. NFATc1 activation is induced by various factors, such as ITAM, TRAF6, c-Fos, and Ca 2+ signaling, which are also activated by RANKL.
Figure Legend Snippet: A schematic model of osteoclastogenesis regulated by the Blimp1-Bcl6-osteoclastic gene axis. RANKL–RANK interaction results in Blimp1 induction, leading to Bcl6 down-regulation and dissociation of Bcl6 from osteoclastic gene promoters, an event critical for osteoclastogenesis. NFATc1 activation is induced by various factors, such as ITAM, TRAF6, c-Fos, and Ca 2+ signaling, which are also activated by RANKL.

Techniques Used: Activation Assay

Bcl6 suppresses osteoclast differentiation. (A) Total RNA was prepared from control (white bars) or Blimp1 cKO (shaded bars) cells treated with (+) or without (−) RANKL, and the expression of the osteoclastic genes NFATc1 , DC-STAMP , and Ctsk relative to β-actin was analyzed by a quantitative real-time PCR. Data are means ± SD of osteoclastic genes / β-actin . (**, P
Figure Legend Snippet: Bcl6 suppresses osteoclast differentiation. (A) Total RNA was prepared from control (white bars) or Blimp1 cKO (shaded bars) cells treated with (+) or without (−) RANKL, and the expression of the osteoclastic genes NFATc1 , DC-STAMP , and Ctsk relative to β-actin was analyzed by a quantitative real-time PCR. Data are means ± SD of osteoclastic genes / β-actin . (**, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Bcl6 is suppressed during osteoclastogenesis and inhibits osteoclast formation. (A) Bcl6 expression was examined by comparative microarray analysis between osteoclast precursors (M-CSF) and osteoclasts (M-CSF + RANKL) cultured for 6 d. (B) BMMs were cultured with or without RANKL for 8 d and subjected to immunofluorescence staining (left) and immunoblot (right) for Bcl6. Nuclei were visualized by DAPI. Bar, 25 µm. (C) Recruitment of NFATc1 and Bcl6 to the NFATc1 P1 distal promoter was detected by ChIP assay. RAW264.7 cells were stimulated with or without RANKL for 48 h and subjected to ChIP analysis. (D) RAW264.7 cells transduced with Bcl6-overexpressing (Bcl6) or mock (control) retrovirus were cultured in the presence (RANKL) or absence (control) of RANKL for 5 d and stained with TRAP. Left, TRAP staining. (right) Numbers are means ± SD of multinuclear TRAP-positive cells in control or Bcl6-overexpressing RAW264.7 cells cultured with RANKL (**, P
Figure Legend Snippet: Bcl6 is suppressed during osteoclastogenesis and inhibits osteoclast formation. (A) Bcl6 expression was examined by comparative microarray analysis between osteoclast precursors (M-CSF) and osteoclasts (M-CSF + RANKL) cultured for 6 d. (B) BMMs were cultured with or without RANKL for 8 d and subjected to immunofluorescence staining (left) and immunoblot (right) for Bcl6. Nuclei were visualized by DAPI. Bar, 25 µm. (C) Recruitment of NFATc1 and Bcl6 to the NFATc1 P1 distal promoter was detected by ChIP assay. RAW264.7 cells were stimulated with or without RANKL for 48 h and subjected to ChIP analysis. (D) RAW264.7 cells transduced with Bcl6-overexpressing (Bcl6) or mock (control) retrovirus were cultured in the presence (RANKL) or absence (control) of RANKL for 5 d and stained with TRAP. Left, TRAP staining. (right) Numbers are means ± SD of multinuclear TRAP-positive cells in control or Bcl6-overexpressing RAW264.7 cells cultured with RANKL (**, P

Techniques Used: Expressing, Microarray, Cell Culture, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Transduction

2) Product Images from "Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway"

Article Title: Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01075-15

Ifrd1 deficiency represses osteoclastogenesis by modulating the NF-κB/NFATc1 pathway by reducing HDAC-dependent deacetylation of p65 in osteoclasts at residues K122 and K123. Repression of interaction between HDAC1 and p65 in Ifrd1 -deficient BMMs
Figure Legend Snippet: Ifrd1 deficiency represses osteoclastogenesis by modulating the NF-κB/NFATc1 pathway by reducing HDAC-dependent deacetylation of p65 in osteoclasts at residues K122 and K123. Repression of interaction between HDAC1 and p65 in Ifrd1 -deficient BMMs

Techniques Used:

3) Product Images from "Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression"

Article Title: Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression

Journal: Molecules

doi: 10.3390/molecules23123139

Effect of 2N1HIA on the expression of osteoclast-related genes. Bone marrow-derived macrophages were cultured in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) and the macrophage colony-stimulating factor together, with or without 1 μM of 2N1HIA. After cultivation for the indicated time periods, the expression of ( A ) TRAF6, ( B ) NFATc1, ( C ) RANK, ( D ) OC-STAMP, ( E ) DC-STAMP, ( F ) CatK, and ( G ) CD47 mRNA was analyzed by real-time PCR. The expression levels were normalized to GAPDH and expressed relative to day zero. Statistical analysis was performed between the control (dimethyl sulfoxide) and the 2N1HIA-treated values at the indicated time points; * p
Figure Legend Snippet: Effect of 2N1HIA on the expression of osteoclast-related genes. Bone marrow-derived macrophages were cultured in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) and the macrophage colony-stimulating factor together, with or without 1 μM of 2N1HIA. After cultivation for the indicated time periods, the expression of ( A ) TRAF6, ( B ) NFATc1, ( C ) RANK, ( D ) OC-STAMP, ( E ) DC-STAMP, ( F ) CatK, and ( G ) CD47 mRNA was analyzed by real-time PCR. The expression levels were normalized to GAPDH and expressed relative to day zero. Statistical analysis was performed between the control (dimethyl sulfoxide) and the 2N1HIA-treated values at the indicated time points; * p

Techniques Used: Expressing, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction

4) Product Images from "Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6"

Article Title: Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20205196

PPOA- N -Ac-2-Cl inhibits RANKL-induced osteoclast-specific gene expression in vitro. Relative mRNA expression of osteoclast-specific genes, TRAF6 , c-fos , NFATc1 , ATP6v0d2 , DC-STAMP , MMP9 , CtsK , and Acp5 (TRAP) , was analyzed by qRT-PCR in the presence or absence of treatment with 6 µM PPOA- N -Ac-2-Cl for 24 h (day 1), 48 h (day 2), or 96 h (day 4). Black bars indicate control, without PPOA- N -Ac-2-Cl treatment, and white bars indicate PPOA- N -Ac-2-Cl-treated group. Transcript levels were normalized to the expression levels of the control at day 0. The primers used for this experiment are listed in Table S1 . * p
Figure Legend Snippet: PPOA- N -Ac-2-Cl inhibits RANKL-induced osteoclast-specific gene expression in vitro. Relative mRNA expression of osteoclast-specific genes, TRAF6 , c-fos , NFATc1 , ATP6v0d2 , DC-STAMP , MMP9 , CtsK , and Acp5 (TRAP) , was analyzed by qRT-PCR in the presence or absence of treatment with 6 µM PPOA- N -Ac-2-Cl for 24 h (day 1), 48 h (day 2), or 96 h (day 4). Black bars indicate control, without PPOA- N -Ac-2-Cl treatment, and white bars indicate PPOA- N -Ac-2-Cl-treated group. Transcript levels were normalized to the expression levels of the control at day 0. The primers used for this experiment are listed in Table S1 . * p

Techniques Used: Expressing, In Vitro, Quantitative RT-PCR

5) Product Images from "Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy"

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy

Journal: Scientific Reports

doi: 10.1038/s41598-018-37091-8

The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p
Figure Legend Snippet: The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p

Techniques Used: Expressing, Incubation, Western Blot

6) Product Images from "CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway"

Article Title: CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13254

Western blot and real‐time quantitative PCR results showed that CD11b blockage or deficiency down‐regulated NFATc1 via the Syk signalling pathway. Mouse OCPs were treated with sRANKL (100 ng/ml) and M‐CSF (10 ng/ml), together with anti‐CD11b antibody (10 μg/ml) for 7 days. Periostea were from Itgam ‐KO group and WT group. ( A ) Cell lysates and periosteum lysates were analysed by Western blotting. ( B ) Relative expression levels of proteins were presented as mean ± S.D., n = 3. ( C ) Total RNA was obtained for RT‐PCR and quantitative real‐time PCR, n = 3. # P
Figure Legend Snippet: Western blot and real‐time quantitative PCR results showed that CD11b blockage or deficiency down‐regulated NFATc1 via the Syk signalling pathway. Mouse OCPs were treated with sRANKL (100 ng/ml) and M‐CSF (10 ng/ml), together with anti‐CD11b antibody (10 μg/ml) for 7 days. Periostea were from Itgam ‐KO group and WT group. ( A ) Cell lysates and periosteum lysates were analysed by Western blotting. ( B ) Relative expression levels of proteins were presented as mean ± S.D., n = 3. ( C ) Total RNA was obtained for RT‐PCR and quantitative real‐time PCR, n = 3. # P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction

7) Product Images from "Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy"

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy

Journal: Scientific Reports

doi: 10.1038/s41598-018-37091-8

The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p
Figure Legend Snippet: The expression of typical proteins involved in the osteoclastogenesis by RAW264.7 after incubation for 7 days in Mg alloy extracts. Total proteins are extracted from RAW264.7 cells and analyzed by Western blotting. Expression of β-actin is used as an internal control. The semi-quantitative analysis and corresponding histogram indicate that ZA coating Mg-Sr alloy could decrease the NFATC1, CTSK, IKK-α and p-p65 protein expression (p

Techniques Used: Expressing, Incubation, Western Blot

8) Product Images from "AAV-Anti-miR-214 Prevents Collapse of the Femoral Head in Osteonecrosis by Regulating Osteoblast and Osteoclast Activities"

Article Title: AAV-Anti-miR-214 Prevents Collapse of the Femoral Head in Osteonecrosis by Regulating Osteoblast and Osteoclast Activities

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2019.09.030

Inhibitory Role of AAV-Anti-miR-214 on Osteoclastic Activity and the Promotion of AAV-Anti-miR-214 on Osteogenic Activity (a) RT-PCR and quantitative real-time PCR analyses of ALP , Bglap , and Col1α1 mRNA levels in the femoral heads of rats among the various groups at 8 weeks postsurgery. For each group, n = 6. (B) RT-PCR and quantitative real-time PCR analyses of Acp5 , Ctsk , Mmp9 , and Clcn7 mRNA levels in the femoral heads of rats from control, model, model + NC, and model + AAV-anti-miR-214 groups at 8 weeks postsurgery. For each group, n = 6. (C) ELISA analyses of ALP and OCN levels in rat serum from control, model, model + AAV-NC, and model + AAV-anti-miR-214 groups at 8 weeks postsurgery. For each group, n = 6. (D) ELISA analyses of CTX-1 and NTX-1 levels in rat serum from control, model, model + AAV-NC, and model + AAV-anti-miR-214 groups at 8 weeks post-surgery. For each group, n = 6. (E) Western blot analyses of Nfatc1, p-Akt, PTEN, and ATF4 protein levels in the femoral heads of rats in the various groups at 8 weeks postsurgery. GAPDH was used as the internal control. (F) Western blot analyses of NFATC1, p-AKT, PTEN, and ATF4 protein levels in the femoral heads of patients with osteonecrosis. GAPDH was used as the internal control. All data are presented as mean ± SEM from three independent experiments.
Figure Legend Snippet: Inhibitory Role of AAV-Anti-miR-214 on Osteoclastic Activity and the Promotion of AAV-Anti-miR-214 on Osteogenic Activity (a) RT-PCR and quantitative real-time PCR analyses of ALP , Bglap , and Col1α1 mRNA levels in the femoral heads of rats among the various groups at 8 weeks postsurgery. For each group, n = 6. (B) RT-PCR and quantitative real-time PCR analyses of Acp5 , Ctsk , Mmp9 , and Clcn7 mRNA levels in the femoral heads of rats from control, model, model + NC, and model + AAV-anti-miR-214 groups at 8 weeks postsurgery. For each group, n = 6. (C) ELISA analyses of ALP and OCN levels in rat serum from control, model, model + AAV-NC, and model + AAV-anti-miR-214 groups at 8 weeks postsurgery. For each group, n = 6. (D) ELISA analyses of CTX-1 and NTX-1 levels in rat serum from control, model, model + AAV-NC, and model + AAV-anti-miR-214 groups at 8 weeks post-surgery. For each group, n = 6. (E) Western blot analyses of Nfatc1, p-Akt, PTEN, and ATF4 protein levels in the femoral heads of rats in the various groups at 8 weeks postsurgery. GAPDH was used as the internal control. (F) Western blot analyses of NFATC1, p-AKT, PTEN, and ATF4 protein levels in the femoral heads of patients with osteonecrosis. GAPDH was used as the internal control. All data are presented as mean ± SEM from three independent experiments.

Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, ALP Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Adeno-Associated Virus-Anti-miR-214 Increased Osteogenic Activity and Decreased Osteoclastic Activity In Vitro (A and B) Representative images of GFP in mouse primary osteoblasts (A) and osteoclasts (B) incubated with AAV-anti-miR-214 or AAV-normalized control (NC). (C) RT-PCR and quantitative real-time PCR analyses of miR-214 levels in mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (D) RT-PCR and quantitative real-time PCR analyses of ALP , Bglap , and Col1α1 mRNA levels in mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (E) Representative images of ALP staining of mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. Scale bar, 7.8 mm. (F) Western blot analyses of ATF4 levels in primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (G) RT-PCR and quantitative real-time PCR analyses of miR-214 levels in receptor activator of nuclear factor κB ligand (RANKL)-induced bone marrow-derived macrophages (BMMs) following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (H) RT-PCR and quantitative real-time PCR analyses of Nfatc1 , Acp5 , Ctsk , and Clcn7 mRNA levels in RANKL-induced BMMs following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (I) Western blot analyses of PTEN protein levels in RANKL-induced BMMs following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (J) Representative images of tartrate-resistant acid phosphatase (TRAP) staining of RANKL-induced BMMs following incubation with AAV-anti-miRNA-214 or AAV-NC for 4 days. All data are presented as mean ± SEM from three independent experiments. **p
Figure Legend Snippet: Adeno-Associated Virus-Anti-miR-214 Increased Osteogenic Activity and Decreased Osteoclastic Activity In Vitro (A and B) Representative images of GFP in mouse primary osteoblasts (A) and osteoclasts (B) incubated with AAV-anti-miR-214 or AAV-normalized control (NC). (C) RT-PCR and quantitative real-time PCR analyses of miR-214 levels in mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (D) RT-PCR and quantitative real-time PCR analyses of ALP , Bglap , and Col1α1 mRNA levels in mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (E) Representative images of ALP staining of mouse primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. Scale bar, 7.8 mm. (F) Western blot analyses of ATF4 levels in primary osteoblasts following incubation with anti-miR-214, NC, AAV-anti-miR-214, or AAV-NC for 2 days. (G) RT-PCR and quantitative real-time PCR analyses of miR-214 levels in receptor activator of nuclear factor κB ligand (RANKL)-induced bone marrow-derived macrophages (BMMs) following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (H) RT-PCR and quantitative real-time PCR analyses of Nfatc1 , Acp5 , Ctsk , and Clcn7 mRNA levels in RANKL-induced BMMs following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (I) Western blot analyses of PTEN protein levels in RANKL-induced BMMs following incubation with AAV-anti-miR-214 or AAV-NC for 4 days. (J) Representative images of tartrate-resistant acid phosphatase (TRAP) staining of RANKL-induced BMMs following incubation with AAV-anti-miRNA-214 or AAV-NC for 4 days. All data are presented as mean ± SEM from three independent experiments. **p

Techniques Used: Activity Assay, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, ALP Assay, Staining, Western Blot, Derivative Assay

9) Product Images from "The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs"

Article Title: The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-020-0441-x

Hypoxia activated the calcineurin/NFAT signaling pathway through suppression of RCAN1.4. a Relative gene expression of RCAN1.1 and RCAN1.4 in human NP cells under hypoxia or b DMOG treatment compared with that in the normoxia group. c Relative protein level of RCAN1.4 in human NP cells under hypoxia or d DMOG treatment compared with that in the normoxia group. e The RCAN1.4 protein level was visibly decreased in a time-dependent manner under hypoxia or f DMOG treatment. g The RCAN1.4 protein level in human NP cells was reduced with HIF-1α overexpression. h Several microRNAs targeting the RCAN1 were selected. Under hypoxia or i DMOG treatment, rno-miR-124-3p was slightly increased in rat NP cells. j Overexpression of HIF-1α in rat NP cells upregulated rno-miR-124-3p. k An rno-miR-124-3p mimic could suppress RCAN1.4 expression in rat NP cells. l The activity of calcineurin in rat NP cells was upregulated under hypoxia or m RCAN1.4 siRNA treatment. n Immunofluorescence and o western blotting demonstrated the nuclear translocation of NFATc1 was obviously evaluated in rat NP cells under hypoxia. p The quantified results of the assessment of nuclear translocation of NFATc1 are shown. All experiments were performed at least three times. Scale bar: 100 μm. * P
Figure Legend Snippet: Hypoxia activated the calcineurin/NFAT signaling pathway through suppression of RCAN1.4. a Relative gene expression of RCAN1.1 and RCAN1.4 in human NP cells under hypoxia or b DMOG treatment compared with that in the normoxia group. c Relative protein level of RCAN1.4 in human NP cells under hypoxia or d DMOG treatment compared with that in the normoxia group. e The RCAN1.4 protein level was visibly decreased in a time-dependent manner under hypoxia or f DMOG treatment. g The RCAN1.4 protein level in human NP cells was reduced with HIF-1α overexpression. h Several microRNAs targeting the RCAN1 were selected. Under hypoxia or i DMOG treatment, rno-miR-124-3p was slightly increased in rat NP cells. j Overexpression of HIF-1α in rat NP cells upregulated rno-miR-124-3p. k An rno-miR-124-3p mimic could suppress RCAN1.4 expression in rat NP cells. l The activity of calcineurin in rat NP cells was upregulated under hypoxia or m RCAN1.4 siRNA treatment. n Immunofluorescence and o western blotting demonstrated the nuclear translocation of NFATc1 was obviously evaluated in rat NP cells under hypoxia. p The quantified results of the assessment of nuclear translocation of NFATc1 are shown. All experiments were performed at least three times. Scale bar: 100 μm. * P

Techniques Used: Expressing, Over Expression, Activity Assay, Immunofluorescence, Western Blot, Translocation Assay

NFATc1 directly bound the SOX9/MMP13 promoter region. a Overexpression of NFATc1 upregulated SOX9 and MMP13 expression in rat NP cells. b Five constructs derived from 2000 bp upstream of the rat SOX9 promoter. c NFATc1-drived luciferase activity of the P1, P2, P3, P4, and P5 fragments in rat NP cells. d NFATc1 increasingly bound the SOX9 promotor site (bp −470 to −323) by ChIP assay. e Mutations in the putative NFATc1-binding site of the P4 fragment. f The P4 mutant decreased NFATc1-drived luciferase activity in rat NP cells. g NFATc1 could also bind sites in the MMP13 promotor (bp −1463 to −1361 and −321–216). h Similarly, NFATc1 promoted the transcriptional level by binding the MMP13 promotor region. i Hypoxia caused accumulation of the HIF-1α and NFATc1 protein complex. All experiments were performed at least three times. * P
Figure Legend Snippet: NFATc1 directly bound the SOX9/MMP13 promoter region. a Overexpression of NFATc1 upregulated SOX9 and MMP13 expression in rat NP cells. b Five constructs derived from 2000 bp upstream of the rat SOX9 promoter. c NFATc1-drived luciferase activity of the P1, P2, P3, P4, and P5 fragments in rat NP cells. d NFATc1 increasingly bound the SOX9 promotor site (bp −470 to −323) by ChIP assay. e Mutations in the putative NFATc1-binding site of the P4 fragment. f The P4 mutant decreased NFATc1-drived luciferase activity in rat NP cells. g NFATc1 could also bind sites in the MMP13 promotor (bp −1463 to −1361 and −321–216). h Similarly, NFATc1 promoted the transcriptional level by binding the MMP13 promotor region. i Hypoxia caused accumulation of the HIF-1α and NFATc1 protein complex. All experiments were performed at least three times. * P

Techniques Used: Over Expression, Expressing, Construct, Derivative Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Mutagenesis

RCAN1.4 the ECM synthesis and remodeling by regulating the calcineurin/NFAT signaling pathway. a Relative SOX9 and MMP13 gene expression in rat NP cells treated with or without FK506 (a calcineurin inhibitor) and stimulated with hypoxia or DMOG treatment. b Western blot analysis of rat NP cells treated with or without FK506 and stimulated with hypoxia or DMOG treatment. The quantified results are shown below. c Immunofluorescence analysis of SOX9 and d type II collagen in rat NP cells treated with or without FK506 and stimulated with hypoxia or DMOG treatment for 48 h. Results of the quantification of SOX9 and type II collagen expression are shown on the right. e Western blot analysis of rat NP cells treated with vector or RCAN1.4 plasmid and stimulated with hypoxia or DMOG treatment. f Immunofluorescence demonstrated that RCAN1.4 overexpression suppressed the nuclear translocation of NFATc1 in rat NP cells under hypoxia. g Relative expression of SOX9 and MMP13 in rat NP cells treated with or without FK506 and stimulated with negative control siRNA or 100 nM RCAN1.4 siRNA. All experiments were performed at least three times. Scale bar: 100 μm. * P
Figure Legend Snippet: RCAN1.4 the ECM synthesis and remodeling by regulating the calcineurin/NFAT signaling pathway. a Relative SOX9 and MMP13 gene expression in rat NP cells treated with or without FK506 (a calcineurin inhibitor) and stimulated with hypoxia or DMOG treatment. b Western blot analysis of rat NP cells treated with or without FK506 and stimulated with hypoxia or DMOG treatment. The quantified results are shown below. c Immunofluorescence analysis of SOX9 and d type II collagen in rat NP cells treated with or without FK506 and stimulated with hypoxia or DMOG treatment for 48 h. Results of the quantification of SOX9 and type II collagen expression are shown on the right. e Western blot analysis of rat NP cells treated with vector or RCAN1.4 plasmid and stimulated with hypoxia or DMOG treatment. f Immunofluorescence demonstrated that RCAN1.4 overexpression suppressed the nuclear translocation of NFATc1 in rat NP cells under hypoxia. g Relative expression of SOX9 and MMP13 in rat NP cells treated with or without FK506 and stimulated with negative control siRNA or 100 nM RCAN1.4 siRNA. All experiments were performed at least three times. Scale bar: 100 μm. * P

Techniques Used: Expressing, Western Blot, Immunofluorescence, Plasmid Preparation, Over Expression, Translocation Assay, Negative Control

The role of RCAN1.4-calcineurin/NFAT in an ex vivo rat disc model. a Safranin O/fast green and Alcian blue staining of sections of discs treated with or without DMOG (1 μm), or FK506 (1 μm) for 14 days was carried out. b Histological scores for discs in different groups. c Relative SOX9, type II collagen, MMP13, and NFATc1 gene expression in NP tissues from different groups. d IHC analysis of RCAN1 and HIF-1α expression in NP disc sections. Results of quantitative analysis of the percentage of positive cells among NP cells are shown on the right. e Immunofluorescence analysis of MMP13 and f type II collagen in NP disc sections. Results of quantitative analysis of the percentage of positive cells among NP cells are shown on the right. g Schematic representation of major molecular pathway was presented. n = 6. Scale bar: 100 μm. * P
Figure Legend Snippet: The role of RCAN1.4-calcineurin/NFAT in an ex vivo rat disc model. a Safranin O/fast green and Alcian blue staining of sections of discs treated with or without DMOG (1 μm), or FK506 (1 μm) for 14 days was carried out. b Histological scores for discs in different groups. c Relative SOX9, type II collagen, MMP13, and NFATc1 gene expression in NP tissues from different groups. d IHC analysis of RCAN1 and HIF-1α expression in NP disc sections. Results of quantitative analysis of the percentage of positive cells among NP cells are shown on the right. e Immunofluorescence analysis of MMP13 and f type II collagen in NP disc sections. Results of quantitative analysis of the percentage of positive cells among NP cells are shown on the right. g Schematic representation of major molecular pathway was presented. n = 6. Scale bar: 100 μm. * P

Techniques Used: Ex Vivo, Staining, Expressing, Immunohistochemistry, Immunofluorescence

10) Product Images from "RasGRP1 Is an Essential Signaling Molecule For Development of B1a Cells With Autoantigen Receptors"

Article Title: RasGRP1 Is an Essential Signaling Molecule For Development of B1a Cells With Autoantigen Receptors

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1502132

RasGRP1 is dispensable for BCR signaling outcomes beyond p-ERK in B1 cells (A) RasGRP1 is dispensable for BCR-stimulated p-JNK. B1a cells were sorted from wild type (WT) and RasGRP1-deficient (KO) mice and were stimulated with anti-Ig (15 μg/ml) (αIg) for 5 and 10 min. p-JNK was evaluated by immunoblot. Membranes were stripped and reprobed with JNK-specific antibody as a loading control. (B) RasGRP1 is dispensable for BCR-stimulated p-p38. Peritoneal lymphocytes from wild type and RasGRP1-deficient mice were unstimulated (US) (WT: red line; KO: blue line) or stimulated (WT: green line; KO: orange line) with anti-Ig (αIg) (15 μg/ml) for 1 min. p-p38 in B1 cells (CD19+CD43+) was evaluated by intracellular immunofluorence staining and flow cytometry. (C) Constitutive expression of NFATc1 is intact in RasGRP1-deficient B1 cells. NFATc1 protein expression was evaluated in WT B1 cells, WT B2 cells, KO B1 cells, and KO B2 cells by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. (D) BCR-stimulated NFATc1 protein expression is intact in RasGRP1-deficient B1 cells. WT B1 cells (left upper panels), WT B2 cells (left lower panels), KO B1 cells (right upper panels), and KO B2 cells (right lower panels) were stimulated with anti-Ig (15 μg/ml) (αIg) for 0, 1, or 2 days. NFATc1 protein expression was evaluated by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. Results represent one of three comparable experiments.
Figure Legend Snippet: RasGRP1 is dispensable for BCR signaling outcomes beyond p-ERK in B1 cells (A) RasGRP1 is dispensable for BCR-stimulated p-JNK. B1a cells were sorted from wild type (WT) and RasGRP1-deficient (KO) mice and were stimulated with anti-Ig (15 μg/ml) (αIg) for 5 and 10 min. p-JNK was evaluated by immunoblot. Membranes were stripped and reprobed with JNK-specific antibody as a loading control. (B) RasGRP1 is dispensable for BCR-stimulated p-p38. Peritoneal lymphocytes from wild type and RasGRP1-deficient mice were unstimulated (US) (WT: red line; KO: blue line) or stimulated (WT: green line; KO: orange line) with anti-Ig (αIg) (15 μg/ml) for 1 min. p-p38 in B1 cells (CD19+CD43+) was evaluated by intracellular immunofluorence staining and flow cytometry. (C) Constitutive expression of NFATc1 is intact in RasGRP1-deficient B1 cells. NFATc1 protein expression was evaluated in WT B1 cells, WT B2 cells, KO B1 cells, and KO B2 cells by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. (D) BCR-stimulated NFATc1 protein expression is intact in RasGRP1-deficient B1 cells. WT B1 cells (left upper panels), WT B2 cells (left lower panels), KO B1 cells (right upper panels), and KO B2 cells (right lower panels) were stimulated with anti-Ig (15 μg/ml) (αIg) for 0, 1, or 2 days. NFATc1 protein expression was evaluated by immunoblot. Membranes were stripped and reprobed with actin-specific antibody as a loading control. Results represent one of three comparable experiments.

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing

11) Product Images from "Effect of echinalkamide identified from Echinacea purpurea (L.) Moench on the inhibition of osteoclastogenesis and bone resorption"

Article Title: Effect of echinalkamide identified from Echinacea purpurea (L.) Moench on the inhibition of osteoclastogenesis and bone resorption

Journal: Scientific Reports

doi: 10.1038/s41598-020-67890-x

Echinalkamide inhibits the RANKL-mediated expression of c-Fos/NFATc1. BMMs were stimulated with 100 ng/mL RANKL with 30 μM echinalkamide for the indicated periods (0, 24, 48, 72 or 96 h). ( A ) The effect of echinalkamide on the protein expression levels of RANKL-induced transcription factors was evaluated using western blot analysis. Actin was used as the internal control. Full blots are provided in Supplementary Fig. S01 . ( B , C ) Total RNA was then isolated using easy blue kit, and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. All the data were confirmed by technical replicated (n = 3). The results are presented as the mean ± SD. Values with different letters (a, b, c, d, e) are significantly different one from another (one-way ANOVA followed by Newman-Keuls multiple range test, p
Figure Legend Snippet: Echinalkamide inhibits the RANKL-mediated expression of c-Fos/NFATc1. BMMs were stimulated with 100 ng/mL RANKL with 30 μM echinalkamide for the indicated periods (0, 24, 48, 72 or 96 h). ( A ) The effect of echinalkamide on the protein expression levels of RANKL-induced transcription factors was evaluated using western blot analysis. Actin was used as the internal control. Full blots are provided in Supplementary Fig. S01 . ( B , C ) Total RNA was then isolated using easy blue kit, and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. All the data were confirmed by technical replicated (n = 3). The results are presented as the mean ± SD. Values with different letters (a, b, c, d, e) are significantly different one from another (one-way ANOVA followed by Newman-Keuls multiple range test, p

Techniques Used: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction

12) Product Images from "Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression"

Article Title: Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression

Journal: Molecules

doi: 10.3390/molecules23123139

Effect of 2N1HIA on the expression of osteoclast-related genes. Bone marrow-derived macrophages were cultured in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) and the macrophage colony-stimulating factor together, with or without 1 μM of 2N1HIA. After cultivation for the indicated time periods, the expression of ( A ) TRAF6, ( B ) NFATc1, ( C ) RANK, ( D ) OC-STAMP, ( E ) DC-STAMP, ( F ) CatK, and ( G ) CD47 mRNA was analyzed by real-time PCR. The expression levels were normalized to GAPDH and expressed relative to day zero. Statistical analysis was performed between the control (dimethyl sulfoxide) and the 2N1HIA-treated values at the indicated time points; * p
Figure Legend Snippet: Effect of 2N1HIA on the expression of osteoclast-related genes. Bone marrow-derived macrophages were cultured in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) and the macrophage colony-stimulating factor together, with or without 1 μM of 2N1HIA. After cultivation for the indicated time periods, the expression of ( A ) TRAF6, ( B ) NFATc1, ( C ) RANK, ( D ) OC-STAMP, ( E ) DC-STAMP, ( F ) CatK, and ( G ) CD47 mRNA was analyzed by real-time PCR. The expression levels were normalized to GAPDH and expressed relative to day zero. Statistical analysis was performed between the control (dimethyl sulfoxide) and the 2N1HIA-treated values at the indicated time points; * p

Techniques Used: Expressing, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction

Related Articles

Blocking Assay:

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy
Article Snippet: .. Non-specific binding were removed by blocking buffer (1% BSA) for 1 h. The corresponding bands positively reacted with specific anti-NFATC1 (1:1000, CST), -CTSK (1:500, abcam), -IKK-α(1:10000, abcam), -phospho-NF-κB p65 (1:1000, CST) and -β-actin (1:2000, CST) at 4 °C overnight, and then with secondary antibodies (1:10000, ThermoFisher Scientific) labelled with horseradish peroxidase (HRP) at 37 °C for 1 h. The chemiluminescence detection method (Millipore) and ChemiDoc MP imaging system (Bio-Rad Laboratories) were then used to visualize and scan the signals of immunoblots, respectively. .. Co-culture of pre-osteoblasts with pre-osteoclasts and cytokine production analysis Transwell chamber culture system (Corning life sciences) was used to evaluate the intercellular contact of pre-osteoblasts with pre-osteoclasts.

Immunoprecipitation:

Article Title: The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis
Article Snippet: .. Immunoprecipitation was performed using anti-NFATc1 (7A6), anti-Bcl6 (N-3), and anti-Blimp1 (C14A4; Cell Signaling Technology). .. DNA was purified by QIAquick PCR purification kit (QIAGEN) and analyzed using primers corresponding to the following promoters: NFATc1 -P1 promoter, 5′-CCGGGACGCC­CATGCAATCTG­TTAGTAATT-3′ (sense) and 5′-GCGGGTGCCC­TGAGAAAGCTA­CTCTCCCTT-3′ (antisense); the DC-STAMP promoter, 5′-GGGGGTCCTCATTTCTACAACTCAT-3′ (sense) and 5′-GCCACATCACCCTGAATCAATCTT-3′ (antisense); the Ctsk promoter, 5′-CCTTAAACTGGCTCCTGTCAAAGA-3′ (sense) and 5′-CCCTTCTTCAGAAGCCCTGTAAT-3′ (antisense); NFATc2 promoter, 5′-TTATCAGGGA­GCACTGCCCAT­CTCCGCTTT-3′ (sense) and 5′-CGGTCTGGCC­TGAGCGACAGG­CCCAGACAA-3′ (antisense); and Bcl6 promoter, 5′-CAGCCACCCTGAGTTTACAA-3′ (sense) and 5′-CGTTCCAGCACTGTTTTGAA-3′ (antisense).

Incubation:

Article Title: Inhibitory Effects of 2N1HIA (2-(3-(2-Fluoro-4-Methoxyphenyl)-6-Oxo-1(6H)-Pyridazinyl)-N-1H-Indol-5-Ylacetamide) on Osteoclast Differentiation via Suppressing Cathepsin K Expression
Article Snippet: .. The membranes were then incubated with anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), anti-cathepsin K (Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD47 (Abcam, Cambridge, MA, USA), anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-NFATc1 (Cell Signaling Technology, Boston, MA, USA), anti-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), anti-phospho-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), anti-p38 (Cell Signaling Technology, Boston, MA, USA), anti-phospho-p38 (Cell Signaling Technology, Boston, MA, USA), anti-JNK (Cell Signaling Technology, Boston, MA, USA), anti-phospho-JNK (Cell Signaling Technology, Boston, MA, USA), anti-p65 (Cell Signaling Technology, Boston, MA, USA), or anti-phospho-p65 (Cell Signaling Technology, Boston, MA, USA). .. This was followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology Boston, MA, USA) which was detected using an ECL system (iNtRON, Seoul, Korea).

Article Title: CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway
Article Snippet: .. Total protein was transferred to nitrocellulose membranes (Bio‐Rad) and incubated overnight with primary antibody: rabbit polyclonal anti‐Syk, anti‐P‐Syk (phospho‐Y352), anti‐c‐Fos, anti‐Erk1/2, anti‐P‐Erk, anti‐NFATc1, anti‐CD11b and anti‐β‐actin (Cell Signaling Technology, Boston, USA). .. Blots were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (donkey anti‐rabbit IgG, Jackson Immunoresearch, Baltimore, USA) in blocking buffer.

Imaging:

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy
Article Snippet: .. Non-specific binding were removed by blocking buffer (1% BSA) for 1 h. The corresponding bands positively reacted with specific anti-NFATC1 (1:1000, CST), -CTSK (1:500, abcam), -IKK-α(1:10000, abcam), -phospho-NF-κB p65 (1:1000, CST) and -β-actin (1:2000, CST) at 4 °C overnight, and then with secondary antibodies (1:10000, ThermoFisher Scientific) labelled with horseradish peroxidase (HRP) at 37 °C for 1 h. The chemiluminescence detection method (Millipore) and ChemiDoc MP imaging system (Bio-Rad Laboratories) were then used to visualize and scan the signals of immunoblots, respectively. .. Co-culture of pre-osteoblasts with pre-osteoclasts and cytokine production analysis Transwell chamber culture system (Corning life sciences) was used to evaluate the intercellular contact of pre-osteoblasts with pre-osteoclasts.

Western Blot:

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy
Article Snippet: .. Non-specific binding were removed by blocking buffer (1% BSA) for 1 h. The corresponding bands positively reacted with specific anti-NFATC1 (1:1000, CST), -CTSK (1:500, abcam), -IKK-α(1:10000, abcam), -phospho-NF-κB p65 (1:1000, CST) and -β-actin (1:2000, CST) at 4 °C overnight, and then with secondary antibodies (1:10000, ThermoFisher Scientific) labelled with horseradish peroxidase (HRP) at 37 °C for 1 h. The chemiluminescence detection method (Millipore) and ChemiDoc MP imaging system (Bio-Rad Laboratories) were then used to visualize and scan the signals of immunoblots, respectively. .. Co-culture of pre-osteoblasts with pre-osteoclasts and cytokine production analysis Transwell chamber culture system (Corning life sciences) was used to evaluate the intercellular contact of pre-osteoblasts with pre-osteoclasts.

Binding Assay:

Article Title: Regulation of osteogenesis and osteoclastogenesis by zoledronic acid loaded on biodegradable magnesium-strontium alloy
Article Snippet: .. Non-specific binding were removed by blocking buffer (1% BSA) for 1 h. The corresponding bands positively reacted with specific anti-NFATC1 (1:1000, CST), -CTSK (1:500, abcam), -IKK-α(1:10000, abcam), -phospho-NF-κB p65 (1:1000, CST) and -β-actin (1:2000, CST) at 4 °C overnight, and then with secondary antibodies (1:10000, ThermoFisher Scientific) labelled with horseradish peroxidase (HRP) at 37 °C for 1 h. The chemiluminescence detection method (Millipore) and ChemiDoc MP imaging system (Bio-Rad Laboratories) were then used to visualize and scan the signals of immunoblots, respectively. .. Co-culture of pre-osteoblasts with pre-osteoclasts and cytokine production analysis Transwell chamber culture system (Corning life sciences) was used to evaluate the intercellular contact of pre-osteoblasts with pre-osteoclasts.

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    Cell Signaling Technology Inc primary antibodies against nfatc1
    (A) Tetrandrine influenced the intracellular calcium oscillation caused by RANKL in bone marrow monocytes (BMMs). (B) Fluorescence intensity was detected by flow cytometry. The mean fluorescence intensity was analyzed by FlowJo. (C) RAW264.7 cells were harvested after induced by RANKL (50 ng/ml) and tetrandrine (0, 0.125, 0.25, 0.5, and 1 μM) for 5 days. The proteins were used to detect osteoclastogenic proteins levels of <t>NFATc1,</t> CTR9, CTSK, TRAP, and MMP9. (D) Tetrandrine down‐regulates osteoclastogenic gene expression of NFATc1, TRAP, CTSK , and c-fos . RAW264.7 cells were stimulated with RANKL and cultured with different concentrations of tetrandrine. RNA was extracted after 5 days. A qPCR analysis was carried to detect osteoclastogenic gene expression. Data were analyzed using the 2−ΔΔCT method. The specific primers used are listed in Table 1 . *P
    Primary Antibodies Against Nfatc1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc dnm2
    Adult dynamin 2-depleted Schwann cells are replaced by dynamin 2-positive Schwann cells in remyelinated nerves. ( A ) Immunostainings for <t>DNM2,</t> SOX10 (SCs) and YFP (reporter-recombined SCs) on SN cross-sections of control* (Mpz CreERT2 :Rosa26-stop loxP/loxP -YFP) and P0ERT2-Dnm2 KO * (Mpz CreERT2 :Dnm2 loxP/loxP : Rosa26-stop loxP/loxP -YFP) mice at 4 wpT and 14 wpT. White arrows: YFP-recombined SCs that express DNM2 (in controls at 4 wpt and 14 wpt); black arrowheads: YFP-recombined SCs lacking DNM2 (in mutants at 4 wpt); white arrowheads: Non-recombined SCs expressing DNM2 (in controls and mutants at 4 wpt and 14 wpt). Scale bar = 25 μm for entire panel. ( B ) Quantification related to ( A ). Percentage of YFP+ cells among SOX10+ SCs/SN cross-sections at 4 wpT, 6 wpT, and 14 wpT. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( C ) Western blot analysis of cleaved caspase 3 (cC3) in SN lysates of control* and P0ERT2-Dnm2 KO * mice at 4 wpT, 6 wpT and 14 wpT. Full-length blots in Supplementary file 1F . ( D ) Quantification referring to ( C ). Control average was set to 1. N = 3 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. Results in graphs represent means ±s.e.m.; ***p
    Dnm2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit antibody against nfatc1
    Iguratimod blocks PPAR-γ/c-Fos signaling. Proteins were extracted, and the protein expression levels of PPAR-γ, c-Fos and <t>NFATc1</t> were detected (A) and quantified (B). The experiments were repeated 3 times independently. Data are presented as means ± SD. *P
    Rabbit Antibody Against Nfatc1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Tetrandrine influenced the intracellular calcium oscillation caused by RANKL in bone marrow monocytes (BMMs). (B) Fluorescence intensity was detected by flow cytometry. The mean fluorescence intensity was analyzed by FlowJo. (C) RAW264.7 cells were harvested after induced by RANKL (50 ng/ml) and tetrandrine (0, 0.125, 0.25, 0.5, and 1 μM) for 5 days. The proteins were used to detect osteoclastogenic proteins levels of NFATc1, CTR9, CTSK, TRAP, and MMP9. (D) Tetrandrine down‐regulates osteoclastogenic gene expression of NFATc1, TRAP, CTSK , and c-fos . RAW264.7 cells were stimulated with RANKL and cultured with different concentrations of tetrandrine. RNA was extracted after 5 days. A qPCR analysis was carried to detect osteoclastogenic gene expression. Data were analyzed using the 2−ΔΔCT method. The specific primers used are listed in Table 1 . *P

    Journal: Frontiers in Pharmacology

    Article Title: Tetrandrine Prevents Bone Loss in Ovariectomized Mice by Inhibiting RANKL-Induced Osteoclastogenesis

    doi: 10.3389/fphar.2019.01530

    Figure Lengend Snippet: (A) Tetrandrine influenced the intracellular calcium oscillation caused by RANKL in bone marrow monocytes (BMMs). (B) Fluorescence intensity was detected by flow cytometry. The mean fluorescence intensity was analyzed by FlowJo. (C) RAW264.7 cells were harvested after induced by RANKL (50 ng/ml) and tetrandrine (0, 0.125, 0.25, 0.5, and 1 μM) for 5 days. The proteins were used to detect osteoclastogenic proteins levels of NFATc1, CTR9, CTSK, TRAP, and MMP9. (D) Tetrandrine down‐regulates osteoclastogenic gene expression of NFATc1, TRAP, CTSK , and c-fos . RAW264.7 cells were stimulated with RANKL and cultured with different concentrations of tetrandrine. RNA was extracted after 5 days. A qPCR analysis was carried to detect osteoclastogenic gene expression. Data were analyzed using the 2−ΔΔCT method. The specific primers used are listed in Table 1 . *P

    Article Snippet: Primary antibodies against NFATc1, P-PI3K, AKT, P-AKT, P50, P-P50, P65, P-P65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK, P38, and P-P38 were obtained from Cell Signaling Technologies (Beverly, MA, USA).

    Techniques: Fluorescence, Flow Cytometry, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Diagram of the proposed mechanism of AA-induced antitumoral activity. This mechanism includes the NFATc1-dependent down-regulation of GLI1 and its target genes ( BCL2 , BFL1/A1 , and 4-1BB ). ROS , reactive oxygen species.

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid *

    doi: 10.1074/jbc.M115.691972

    Figure Lengend Snippet: Diagram of the proposed mechanism of AA-induced antitumoral activity. This mechanism includes the NFATc1-dependent down-regulation of GLI1 and its target genes ( BCL2 , BFL1/A1 , and 4-1BB ). ROS , reactive oxygen species.

    Article Snippet: GLI1 and NFATc1 antibodies were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Activity Assay

    NFATc1 mediates AA silencing of GLI1 expression. A , PANC1 cells were transfected with an expression construct encoding NFATc1-GFP for 48 h and then treated with AA (60 μg/ml) or vehicle for 15 or 30 min. Cells were fixed, stained with DAPI, and

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid *

    doi: 10.1074/jbc.M115.691972

    Figure Lengend Snippet: NFATc1 mediates AA silencing of GLI1 expression. A , PANC1 cells were transfected with an expression construct encoding NFATc1-GFP for 48 h and then treated with AA (60 μg/ml) or vehicle for 15 or 30 min. Cells were fixed, stained with DAPI, and

    Article Snippet: GLI1 and NFATc1 antibodies were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Transfection, Construct, Staining

    Adult dynamin 2-depleted Schwann cells are replaced by dynamin 2-positive Schwann cells in remyelinated nerves. ( A ) Immunostainings for DNM2, SOX10 (SCs) and YFP (reporter-recombined SCs) on SN cross-sections of control* (Mpz CreERT2 :Rosa26-stop loxP/loxP -YFP) and P0ERT2-Dnm2 KO * (Mpz CreERT2 :Dnm2 loxP/loxP : Rosa26-stop loxP/loxP -YFP) mice at 4 wpT and 14 wpT. White arrows: YFP-recombined SCs that express DNM2 (in controls at 4 wpt and 14 wpt); black arrowheads: YFP-recombined SCs lacking DNM2 (in mutants at 4 wpt); white arrowheads: Non-recombined SCs expressing DNM2 (in controls and mutants at 4 wpt and 14 wpt). Scale bar = 25 μm for entire panel. ( B ) Quantification related to ( A ). Percentage of YFP+ cells among SOX10+ SCs/SN cross-sections at 4 wpT, 6 wpT, and 14 wpT. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( C ) Western blot analysis of cleaved caspase 3 (cC3) in SN lysates of control* and P0ERT2-Dnm2 KO * mice at 4 wpT, 6 wpT and 14 wpT. Full-length blots in Supplementary file 1F . ( D ) Quantification referring to ( C ). Control average was set to 1. N = 3 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. Results in graphs represent means ±s.e.m.; ***p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Adult dynamin 2-depleted Schwann cells are replaced by dynamin 2-positive Schwann cells in remyelinated nerves. ( A ) Immunostainings for DNM2, SOX10 (SCs) and YFP (reporter-recombined SCs) on SN cross-sections of control* (Mpz CreERT2 :Rosa26-stop loxP/loxP -YFP) and P0ERT2-Dnm2 KO * (Mpz CreERT2 :Dnm2 loxP/loxP : Rosa26-stop loxP/loxP -YFP) mice at 4 wpT and 14 wpT. White arrows: YFP-recombined SCs that express DNM2 (in controls at 4 wpt and 14 wpt); black arrowheads: YFP-recombined SCs lacking DNM2 (in mutants at 4 wpt); white arrowheads: Non-recombined SCs expressing DNM2 (in controls and mutants at 4 wpt and 14 wpt). Scale bar = 25 μm for entire panel. ( B ) Quantification related to ( A ). Percentage of YFP+ cells among SOX10+ SCs/SN cross-sections at 4 wpT, 6 wpT, and 14 wpT. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( C ) Western blot analysis of cleaved caspase 3 (cC3) in SN lysates of control* and P0ERT2-Dnm2 KO * mice at 4 wpT, 6 wpT and 14 wpT. Full-length blots in Supplementary file 1F . ( D ) Quantification referring to ( C ). Control average was set to 1. N = 3 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. Results in graphs represent means ±s.e.m.; ***p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Mouse Assay, Expressing, Western Blot

    Lack of dynamin 2 in Schwann cells of developing nerves causes Schwann cell loss and invasion of perineurial cells. ( A ) Quantification of total nuclei on full cross-sections of control and P0-Dnm2 KO SNs at P5 and P14 (P5-representative picture in Figure 3E ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( B ) Quantification of total SOX10+ SCs on cross-sections of control and P0ERT2-Dnm2 KO SNs at P5 and P14 (P5-representative picture in Figure 3E ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( C ) Percentage of SOX10+ SCs among total DAPI+ cells in controls and P0ERT2-Dnm2 KO SNs at P5 and P14. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( D ) Immunostainings of GLUT1 on cross-sections of control and P0ERT2-Dnm2 KO SNs at P5 and P14. Scale bar = 100 μm for entire panel. ( E ) Quantification of ( D ). Relative GLUT1-immunopositive area/total area of SN cross-sections of control and P0ERT2-Dnm2 KO mice. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( F ) Quantification of ( D ). SN endoneurial area (mm 2 ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( G ) Exemplary EM images showing ultrastructural organization of control and P0-Dnm2 KO SNs at P14. Perineurial cells are false colored in red. N = 3 mice/genotype. Scale bar = 10 μm for entire panel. Results in graphs represent means ±s.e.m.; ***p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Lack of dynamin 2 in Schwann cells of developing nerves causes Schwann cell loss and invasion of perineurial cells. ( A ) Quantification of total nuclei on full cross-sections of control and P0-Dnm2 KO SNs at P5 and P14 (P5-representative picture in Figure 3E ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( B ) Quantification of total SOX10+ SCs on cross-sections of control and P0ERT2-Dnm2 KO SNs at P5 and P14 (P5-representative picture in Figure 3E ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( C ) Percentage of SOX10+ SCs among total DAPI+ cells in controls and P0ERT2-Dnm2 KO SNs at P5 and P14. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( D ) Immunostainings of GLUT1 on cross-sections of control and P0ERT2-Dnm2 KO SNs at P5 and P14. Scale bar = 100 μm for entire panel. ( E ) Quantification of ( D ). Relative GLUT1-immunopositive area/total area of SN cross-sections of control and P0ERT2-Dnm2 KO mice. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( F ) Quantification of ( D ). SN endoneurial area (mm 2 ). N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( G ) Exemplary EM images showing ultrastructural organization of control and P0-Dnm2 KO SNs at P14. Perineurial cells are false colored in red. N = 3 mice/genotype. Scale bar = 10 μm for entire panel. Results in graphs represent means ±s.e.m.; ***p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Mouse Assay

    Dynamin 2 ablation in Schwann cells inhibits their differentiation and myelination. ( A ) Schematic representation of Dnm2 ablation in SCs. Exon 2 of Dnm2 is flanked by LoxP sites and excised upon expression of Cre recombinase under the control of the SC-specific Mpz promoter. ( B ) Quantification referring to Figure 1A and Supplementary file 1A (full-length blots). Relative amounts of DNM2 protein in SN extracts derived from Dnm2 loxP/loxP (control) and Mpz Cre :Dnm2 loxP/loxP (P0-Dnm2 KO ) mice at P1. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) Representative immunoblot of the myelin proteins MAG, P0, and MBP in SN lysates from P1 control and P0-Dnm2 KO mice. N = 4 mice/genotype. Full-length blots in Supplementary file 1D . ( D ) Relative amounts of myelin proteins in P1 SNs of control and P0-Dnm2 KO mice. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( E ) Representative immunoblot analysis of the differentiation markers KROX20 and cJUN in SN lysates from P1 control and P0-Dnm2 KO mice. N = 4 mice/genotype, full-length blots in Supplementary file 1D . ( F ) Relative amounts of KROX20 and cJUN in P1 SNs of control and P0-Dnm2 KO mice. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. Results in graphs represent means ±s.e.m.; ***p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Dynamin 2 ablation in Schwann cells inhibits their differentiation and myelination. ( A ) Schematic representation of Dnm2 ablation in SCs. Exon 2 of Dnm2 is flanked by LoxP sites and excised upon expression of Cre recombinase under the control of the SC-specific Mpz promoter. ( B ) Quantification referring to Figure 1A and Supplementary file 1A (full-length blots). Relative amounts of DNM2 protein in SN extracts derived from Dnm2 loxP/loxP (control) and Mpz Cre :Dnm2 loxP/loxP (P0-Dnm2 KO ) mice at P1. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) Representative immunoblot of the myelin proteins MAG, P0, and MBP in SN lysates from P1 control and P0-Dnm2 KO mice. N = 4 mice/genotype. Full-length blots in Supplementary file 1D . ( D ) Relative amounts of myelin proteins in P1 SNs of control and P0-Dnm2 KO mice. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( E ) Representative immunoblot analysis of the differentiation markers KROX20 and cJUN in SN lysates from P1 control and P0-Dnm2 KO mice. N = 4 mice/genotype, full-length blots in Supplementary file 1D . ( F ) Relative amounts of KROX20 and cJUN in P1 SNs of control and P0-Dnm2 KO mice. Control average is set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. Results in graphs represent means ±s.e.m.; ***p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Expressing, Derivative Assay, Mouse Assay, Two Tailed Test

    Dynamin 2 ablation in adult Schwann cells induces transient cellular dedifferentiation. ( A ) Schematic representation of inducible Dnm2 ablation and YFP expression in adult SCs. The inducible Cre-ERT2 fusion recombinase is expressed under the SC-specific Myelin Protein Zero ( Mpz ) promoter. Upon tamoxifen injection, the recombinase shuttles into the nucleus leading to excision of floxed exon 2. The Cre-dependent RosaYFP-reporter allele was used in some experiments to track recombination in SCs. In this case, the transcription stop signal flanked by loxP sites is excised upon tamoxifen administration leading to YFP expression. ( B ) Quantification referring to Figure 2A , full-length blots in Supplementary file 1B . Relative amounts of DNM2 protein in sciatic nerve (SN) extracts derived from Dnm2 loxP/loxP (control) and Mpz CreERT2 :Dnm2 loxP/loxP (P0ERT2-Dnm2 KO ) mice at 4 wpT. Control average was set to 1. N = 3 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) Average g-ratios of control and P0ERT2-Dnm2 KO SNs indicate a slight hypomyelination of mutant mice at 41 wpT. At least 90 axons/sample were analyzed (random EM fields). N = 3 mice/genotype, two-tailed unpaired Student´s t-test. ( D ) G-ratios as function of axon diameters. ( E ) (left) Exemplary picture of internodes of teased osmicated fibers from SNs of control and P0ERT2-Dnm2 KO mice at 14 wpT. (right) Quantification of internodal lengths. 100 internodes/animal were measured, n = 3 mice/genotype, two-tailed unpaired Student´s t-test. Black arrowheads: Nodes of Ranvier. Scale bar = 100 μm (for both panels). ( F ) (left) Immunostaining of Mpz CreERT2 :Rosa26-stop loxP/loxP -YFP (control*) and Mpz CreERT2 :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (P0ERT2-Dnm2 KO *) SN cross-sections for p75 NTR at different time points. There was a pronounced, but transient increase of p75 NTR staining at 6 wpT in P0ERT2-Dnm2 KO* mice. (right) Quantification of relative signal intensity of p75 NTR immunostaining/SN cross-section. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. Scale bar = 200 μm. ( G ) qRT-PCR of P75 NTR , cyclinD1 and Krox24 from control and P0ERT2-Dnm2 KO SNs at the indicated time points. Transcript levels were normalized to GAPDH. Control values were set to one for each individual transcript at each time point. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( H ) Exemplary picture of immunoblot analysis of cJUN in SN extracts from control and P0ERT-Dnm2 KO mice at the indicated time points. N = 3 mice/genotype. Full-length blots in Supplementary file 1E . ( I ) Quantification referring to Figure 2H , Supplementary file 1E . Control average was set to one for each time point individually. N = 3 mice/genotype, two-tailed unpaired Student’s t-test. Results in graphs represent means ±s.e.m.; *p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Dynamin 2 ablation in adult Schwann cells induces transient cellular dedifferentiation. ( A ) Schematic representation of inducible Dnm2 ablation and YFP expression in adult SCs. The inducible Cre-ERT2 fusion recombinase is expressed under the SC-specific Myelin Protein Zero ( Mpz ) promoter. Upon tamoxifen injection, the recombinase shuttles into the nucleus leading to excision of floxed exon 2. The Cre-dependent RosaYFP-reporter allele was used in some experiments to track recombination in SCs. In this case, the transcription stop signal flanked by loxP sites is excised upon tamoxifen administration leading to YFP expression. ( B ) Quantification referring to Figure 2A , full-length blots in Supplementary file 1B . Relative amounts of DNM2 protein in sciatic nerve (SN) extracts derived from Dnm2 loxP/loxP (control) and Mpz CreERT2 :Dnm2 loxP/loxP (P0ERT2-Dnm2 KO ) mice at 4 wpT. Control average was set to 1. N = 3 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) Average g-ratios of control and P0ERT2-Dnm2 KO SNs indicate a slight hypomyelination of mutant mice at 41 wpT. At least 90 axons/sample were analyzed (random EM fields). N = 3 mice/genotype, two-tailed unpaired Student´s t-test. ( D ) G-ratios as function of axon diameters. ( E ) (left) Exemplary picture of internodes of teased osmicated fibers from SNs of control and P0ERT2-Dnm2 KO mice at 14 wpT. (right) Quantification of internodal lengths. 100 internodes/animal were measured, n = 3 mice/genotype, two-tailed unpaired Student´s t-test. Black arrowheads: Nodes of Ranvier. Scale bar = 100 μm (for both panels). ( F ) (left) Immunostaining of Mpz CreERT2 :Rosa26-stop loxP/loxP -YFP (control*) and Mpz CreERT2 :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (P0ERT2-Dnm2 KO *) SN cross-sections for p75 NTR at different time points. There was a pronounced, but transient increase of p75 NTR staining at 6 wpT in P0ERT2-Dnm2 KO* mice. (right) Quantification of relative signal intensity of p75 NTR immunostaining/SN cross-section. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. Scale bar = 200 μm. ( G ) qRT-PCR of P75 NTR , cyclinD1 and Krox24 from control and P0ERT2-Dnm2 KO SNs at the indicated time points. Transcript levels were normalized to GAPDH. Control values were set to one for each individual transcript at each time point. N = 3 mice/genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( H ) Exemplary picture of immunoblot analysis of cJUN in SN extracts from control and P0ERT-Dnm2 KO mice at the indicated time points. N = 3 mice/genotype. Full-length blots in Supplementary file 1E . ( I ) Quantification referring to Figure 2H , Supplementary file 1E . Control average was set to one for each time point individually. N = 3 mice/genotype, two-tailed unpaired Student’s t-test. Results in graphs represent means ±s.e.m.; *p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Expressing, Injection, Derivative Assay, Mouse Assay, Two Tailed Test, Mutagenesis, Immunostaining, Staining, Quantitative RT-PCR

    Schwann cells lacking dynamin 2 show impaired cell cycle progression, reduced mitosis rate, and cytokinesis defects. ( A ) Schematic representation of the cell cycle phases marked by Ki-67 and EdU. ( B ) EdU-labeling, combined with immunostainings for Ki-67 and SOX10 on control and P0-Dnm2 KO SN cross-sections at P5. Arrows: EdU+ Ki-67+ SOX10+ SCs, arrowheads: Ki-67+ SOX10+ SCs. Scale bar = 25 μm for entire panel. ( C ) Quantification of ( B ). Percentage of EdU+ SCs/SN cross-sections. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( D ) Quantification of ( B ). Percentage of Ki-67+ SCs/SN cross-sections at P5. N = 5 mice/genotype, two-tailed unpaired Student´s t-test. ( E ) Quantification of ( B ). Percentage of EdU+ among Ki-67+ SCs/SN cross-section at P5. N = 5 mice/genotype, two-tailed unpaired Student´s t-test. ( F ) Quantification of mitotic events in cultured mouse SCs isolated from Mpz Cre :Rosa26-stop loxP/loxP -YFP (control*) and Mpz Cre :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (P0-Dnm2 KO *) SNs at P1, monitored by time-lapse microscopy for 24 hr. Each data point represents one individual animal (at least 42 cells/animal analyzed). N = 5 controls and seven mutant mice, two-tailed unpaired Student´s t-test. ( G ) Quantification of mitosis duration (minutes) of SCs of control* and P0-Dnm2 KO * mice, monitored by time-lapse microscopy for 24 hr. Each data point represents one cell derived from a total of 5 control and seven mutant mice. Cells derived from each animal were isolated and analyzed separately, but pooled in one graph; two-tailed unpaired Student´s t-test. ( H ) Exemplary picture of a multinucleated Dnm2 KO SCs after 48 hr in culture. Scale bar = 25 μm. ( I ) Quantification of multinucleated cells in control and Dnm2 KO SCs after 24 hr, 48 hr and 76 hr in culture. N = 6 mice/genotype for 48 hr; N = 6 control* and n = 5 P0-Dnm2 KO * mice for 24 hr and 72 hr, two-Way ANOVA with Sidak’s multiple comparisons test. ( J ) Exemplary time-lapse images of the mitosis of control and Dnm2 KO SCs (YFP+ cells). Arrowheads: cell body of dividing SCs, black arrows: cytokinesis site. ( K ) Representative time-lapse images of Dnm2 KO SC (YFP+) failing cytokinesis. The SC is undergoing mitosis, but fails to divide (arrowheads point to the cell body). Results in graphs represent means ±s.e.m.; *p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Schwann cells lacking dynamin 2 show impaired cell cycle progression, reduced mitosis rate, and cytokinesis defects. ( A ) Schematic representation of the cell cycle phases marked by Ki-67 and EdU. ( B ) EdU-labeling, combined with immunostainings for Ki-67 and SOX10 on control and P0-Dnm2 KO SN cross-sections at P5. Arrows: EdU+ Ki-67+ SOX10+ SCs, arrowheads: Ki-67+ SOX10+ SCs. Scale bar = 25 μm for entire panel. ( C ) Quantification of ( B ). Percentage of EdU+ SCs/SN cross-sections. N = 5 mice/time point and genotype, Two-Way ANOVA with Sidak’s multiple comparisons test. ( D ) Quantification of ( B ). Percentage of Ki-67+ SCs/SN cross-sections at P5. N = 5 mice/genotype, two-tailed unpaired Student´s t-test. ( E ) Quantification of ( B ). Percentage of EdU+ among Ki-67+ SCs/SN cross-section at P5. N = 5 mice/genotype, two-tailed unpaired Student´s t-test. ( F ) Quantification of mitotic events in cultured mouse SCs isolated from Mpz Cre :Rosa26-stop loxP/loxP -YFP (control*) and Mpz Cre :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (P0-Dnm2 KO *) SNs at P1, monitored by time-lapse microscopy for 24 hr. Each data point represents one individual animal (at least 42 cells/animal analyzed). N = 5 controls and seven mutant mice, two-tailed unpaired Student´s t-test. ( G ) Quantification of mitosis duration (minutes) of SCs of control* and P0-Dnm2 KO * mice, monitored by time-lapse microscopy for 24 hr. Each data point represents one cell derived from a total of 5 control and seven mutant mice. Cells derived from each animal were isolated and analyzed separately, but pooled in one graph; two-tailed unpaired Student´s t-test. ( H ) Exemplary picture of a multinucleated Dnm2 KO SCs after 48 hr in culture. Scale bar = 25 μm. ( I ) Quantification of multinucleated cells in control and Dnm2 KO SCs after 24 hr, 48 hr and 76 hr in culture. N = 6 mice/genotype for 48 hr; N = 6 control* and n = 5 P0-Dnm2 KO * mice for 24 hr and 72 hr, two-Way ANOVA with Sidak’s multiple comparisons test. ( J ) Exemplary time-lapse images of the mitosis of control and Dnm2 KO SCs (YFP+ cells). Arrowheads: cell body of dividing SCs, black arrows: cytokinesis site. ( K ) Representative time-lapse images of Dnm2 KO SC (YFP+) failing cytokinesis. The SC is undergoing mitosis, but fails to divide (arrowheads point to the cell body). Results in graphs represent means ±s.e.m.; *p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Labeling, Mouse Assay, Two Tailed Test, Cell Culture, Isolation, Time-lapse Microscopy, Mutagenesis, Derivative Assay

    Various immune cells invade sciatic nerves after dynamin 2 depletion in adult Schwann cells. ( A ) (Left) Immunostainings for the macrophage marker CD68 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD68+ cells (random fields). N = 3 mice/genotype for 4, 6, 41 wpT; N = 4 mice/genotype for 14 wpT. ( B ) (Left) Immunostainings for the T-cell marker CD3 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD3+ cells (random fields). N = 3 mice/genotype for 6, 14, and 41 wpT. At 4 wpT, N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. ( C ) (Left) Immunostainings for the neutrophil marker GR1 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of GR1+ cells (random fields). N = 3 mice/genotype for 6, 14, and 41 wpT. At 4 wpT, N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. ( D ) (Left) Immunostainings for the mast cell marker CD117 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD117+ cells (random fields). N = 3 mice/time point and genotype. Two-Way ANOVA with Sidak´s multiple comparisons test for all statistical analyses. Scale bars = 50 μm for all panels. Results in graphs represent means ±s.e.m.; *p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Various immune cells invade sciatic nerves after dynamin 2 depletion in adult Schwann cells. ( A ) (Left) Immunostainings for the macrophage marker CD68 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD68+ cells (random fields). N = 3 mice/genotype for 4, 6, 41 wpT; N = 4 mice/genotype for 14 wpT. ( B ) (Left) Immunostainings for the T-cell marker CD3 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD3+ cells (random fields). N = 3 mice/genotype for 6, 14, and 41 wpT. At 4 wpT, N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. ( C ) (Left) Immunostainings for the neutrophil marker GR1 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of GR1+ cells (random fields). N = 3 mice/genotype for 6, 14, and 41 wpT. At 4 wpT, N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. ( D ) (Left) Immunostainings for the mast cell marker CD117 on cross-sections of control and P0ERT2-Dnm2 KO SNs at 4 wpT, 6 wpT, 14 wpT, and 41 wpT. (Right) Quantification of CD117+ cells (random fields). N = 3 mice/time point and genotype. Two-Way ANOVA with Sidak´s multiple comparisons test for all statistical analyses. Scale bars = 50 μm for all panels. Results in graphs represent means ±s.e.m.; *p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Marker, Mouse Assay

    Dynamin 2-depleted oligodendrocytes do not show detectable defects in differentiation and myelination. ( A ) Schematic representation of Dnm2 ablation in the OL lineage. Exon 2 of Dnm2 is flanked by LoxP sites and excised upon expression of Cre recombinase under the control of CNPase gene regulatory elements. ( B ) Quantification referring to Figure 5A , full-length blot Supplementary file 1C . Relative amount of DNM2 in spinal cord extracts from Dnm2 loxP/loxP (control) and Cnp Cre :Dnm2 loxP/loxP (CNP-Dnm2 KO ) mice (P14, P60). Control average was set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) (left panel) Exemplary immunoblot of DNM2 in extracts of primary mouse OLs derived from Cnp Cre :Rosa26-stop loxP/loxP -YFP (control*) and Cnp Cre :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (CNP-Dnm2 KO* ) from P7 brain (after 14 day in culture). (right panel) Relative amounts of DNM2, full-length blot in Supplementary file 1G . N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( D ) Detailed quantification of Figure 5D (spinal cord hemisections). Total OL lineage cells (OLIG2+), OPC (PDGFRα+OLIG2+) and mature OL (CC1+ OLIG2+), in spinal cord dorsal white matter (dWM) and grey matter (GM) of control and CNP-Dnm2 KO mice. N = 5 mice/genotype, two-tailed unpaired Student’s t-test. ( E ) Average g-ratios of control and CNP-Dnm2 KO myelinated axons in spinal cord ventral WM (SpC; P14, P60) and CC (P60). At least 108 axons/sample were analyzed (random EM fields). N = 4 or 5 mice/time point and genotype, two-tailed unpaired Student´s t-test. ( F ) G-ratios as function of axon diameter. Results in graphs represent means ±s.e.m.; ***p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Dynamin 2-depleted oligodendrocytes do not show detectable defects in differentiation and myelination. ( A ) Schematic representation of Dnm2 ablation in the OL lineage. Exon 2 of Dnm2 is flanked by LoxP sites and excised upon expression of Cre recombinase under the control of CNPase gene regulatory elements. ( B ) Quantification referring to Figure 5A , full-length blot Supplementary file 1C . Relative amount of DNM2 in spinal cord extracts from Dnm2 loxP/loxP (control) and Cnp Cre :Dnm2 loxP/loxP (CNP-Dnm2 KO ) mice (P14, P60). Control average was set to 1. N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( C ) (left panel) Exemplary immunoblot of DNM2 in extracts of primary mouse OLs derived from Cnp Cre :Rosa26-stop loxP/loxP -YFP (control*) and Cnp Cre :Dnm2 loxP/loxP :Rosa26-stop loxP/loxP -YFP (CNP-Dnm2 KO* ) from P7 brain (after 14 day in culture). (right panel) Relative amounts of DNM2, full-length blot in Supplementary file 1G . N = 4 mice/genotype, two-tailed unpaired Student’s t-test. ( D ) Detailed quantification of Figure 5D (spinal cord hemisections). Total OL lineage cells (OLIG2+), OPC (PDGFRα+OLIG2+) and mature OL (CC1+ OLIG2+), in spinal cord dorsal white matter (dWM) and grey matter (GM) of control and CNP-Dnm2 KO mice. N = 5 mice/genotype, two-tailed unpaired Student’s t-test. ( E ) Average g-ratios of control and CNP-Dnm2 KO myelinated axons in spinal cord ventral WM (SpC; P14, P60) and CC (P60). At least 108 axons/sample were analyzed (random EM fields). N = 4 or 5 mice/time point and genotype, two-tailed unpaired Student´s t-test. ( F ) G-ratios as function of axon diameter. Results in graphs represent means ±s.e.m.; ***p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Expressing, Mouse Assay, Two Tailed Test, Derivative Assay

    Macrophages are beneficial for recovery after demyelination due to loss of dynamin 2 in adult Schwann cells. ( A ) Schematic drawing of the experimental timeline. 10 weeks-old control ( Dnm2 loxP/loxP ) and P0ERT2-Dnm2 KO ( Mpz CreERT2 :Dnm2 loxP/loxP ) mice were injected intraperitoneally with tamoxifen on five consecutive days. At 4 wpT, the mice were injected intravenously with clodronate liposomes (CL) or saline (Sal) three times (4 wpT, 4 wpT +3 d, 4 wpT +6 d). ( B ) Spleen weights in controls and P0ERT2-Dnm2 KO at 6 wpT are consistent with effective macrophage deprivation after CL treatment in both genotypes. N = 3 mice/genotype and treatment, One-way ANOVA with Tukey´s multiple comparisons test. ( C ) Immunostainings for the macrophage marker CD68 on SN cross-sections derived from control and P0ERT2-Dnm2 KO mice, treated with CL or Sal, at 6 wpT. N = 3 mice/genotype and treatment. Scale bar = 50 μm for entire panel. ( D ) Quantification of ( C ) (random fields). N = 3 mice/condition (color code as in B ), One-Way ANOVA with Tukey´s multiple comparisons test. ( E ) CL- and Sal-treated control and P0ERT2-Dnm2 KO mice were phenotypically assessed over a period of 5 weeks. All P0ERT2-Dnm2 KO developed a transient moderate paraparesis around 6 wpT. CL-treated P0ERT2-Dnm2 KO recovered more slowly compared to Saline-treated P0ERT2-Dnm2 KO mice. N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. Clinical score: 0 = normal, 1 = less lively, 2 = impaired righting, 3 = absent righting, 4 = ataxic gait, 5 = mild paraparesis, 6 = moderate paraparesis, 7 = severe paraparesis. Results in graphs represent means ±s.e.m.; *p

    Journal: eLife

    Article Title: Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function

    doi: 10.7554/eLife.42404

    Figure Lengend Snippet: Macrophages are beneficial for recovery after demyelination due to loss of dynamin 2 in adult Schwann cells. ( A ) Schematic drawing of the experimental timeline. 10 weeks-old control ( Dnm2 loxP/loxP ) and P0ERT2-Dnm2 KO ( Mpz CreERT2 :Dnm2 loxP/loxP ) mice were injected intraperitoneally with tamoxifen on five consecutive days. At 4 wpT, the mice were injected intravenously with clodronate liposomes (CL) or saline (Sal) three times (4 wpT, 4 wpT +3 d, 4 wpT +6 d). ( B ) Spleen weights in controls and P0ERT2-Dnm2 KO at 6 wpT are consistent with effective macrophage deprivation after CL treatment in both genotypes. N = 3 mice/genotype and treatment, One-way ANOVA with Tukey´s multiple comparisons test. ( C ) Immunostainings for the macrophage marker CD68 on SN cross-sections derived from control and P0ERT2-Dnm2 KO mice, treated with CL or Sal, at 6 wpT. N = 3 mice/genotype and treatment. Scale bar = 50 μm for entire panel. ( D ) Quantification of ( C ) (random fields). N = 3 mice/condition (color code as in B ), One-Way ANOVA with Tukey´s multiple comparisons test. ( E ) CL- and Sal-treated control and P0ERT2-Dnm2 KO mice were phenotypically assessed over a period of 5 weeks. All P0ERT2-Dnm2 KO developed a transient moderate paraparesis around 6 wpT. CL-treated P0ERT2-Dnm2 KO recovered more slowly compared to Saline-treated P0ERT2-Dnm2 KO mice. N = 3 controls and N = 4 P0ERT2-Dnm2 KO mice. Clinical score: 0 = normal, 1 = less lively, 2 = impaired righting, 3 = absent righting, 4 = ataxic gait, 5 = mild paraparesis, 6 = moderate paraparesis, 7 = severe paraparesis. Results in graphs represent means ±s.e.m.; *p

    Article Snippet: Antibodies and chemicals The following primary antibodies were used: DNM2 (Pineda, 1:100; or GeneTex, #GRX109652, RRID: AB_1950134 , 1:200), cJUN (CST, #9165, RRID: AB_2130165 , 1:1000), MAG (Invitrogen, # 34–6200, RRID: AB_2533179 , 1:1000), P0 (Millipore, #ab9352, RRID: AB_571090 , 1:1000), MBP (Serotec, MCA409S, RRID: AB_325004 , 1:200), cleaved caspase 3 (CST, #9664, RRID: AB_2070042 , 1:1000) α-tubulin (Sigma-Aldrich, T5168, RRID: AB_477579 , 1:1000), GAPDH (Hytest, 5G4 6C5, RRID: AB_1616722 , 1:10000), SOX10 (R and D Systems, AF2864, RRID: AB_442208 , 1:200), GFP/YFP (Aves Lab, #GFP-1020, RRID: AB_10000240 , 1:1000), CD45 (BD, #550539, RRID: AB_2174426 , 1:200), Ki-67 (abcam, #ab15580, RRID: AB_443209 , 1:200), CC1 (Calbiochem, OP80, RRID: AB_2057371 , 1:500), OLIG2 (Millipore, #MABN50, RRID: AB_10807410 , 1:1000 or Millipore, #AB9610, RRID: AB_570666 , 1:500), PDGFRα (CST, #3174, RRID: AB_2162345 , 1:600), p75NTR (Millipore, #ab1554, RRID: AB_11211656 , 1:200), CD68 (Serotec, #MCA1957, RRID: AB_322219 , 1:100), CD3 (Dako, #A0452, RRID: AB_2335677 , 1:200), GR1 (BD, #553127, RRID: AB_394643 , 1:200), CD117 (BD, #562417, RRID: AB_11154233 , 1:200), GLUT-1 (Millipore, #07–1401, RRID: AB_1587074 , 1:200).

    Techniques: Mouse Assay, Injection, Marker, Derivative Assay

    Iguratimod blocks PPAR-γ/c-Fos signaling. Proteins were extracted, and the protein expression levels of PPAR-γ, c-Fos and NFATc1 were detected (A) and quantified (B). The experiments were repeated 3 times independently. Data are presented as means ± SD. *P

    Journal: Molecular Medicine Reports

    Article Title: Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-γ

    doi: 10.3892/mmr.2017.7648

    Figure Lengend Snippet: Iguratimod blocks PPAR-γ/c-Fos signaling. Proteins were extracted, and the protein expression levels of PPAR-γ, c-Fos and NFATc1 were detected (A) and quantified (B). The experiments were repeated 3 times independently. Data are presented as means ± SD. *P

    Article Snippet: Rabbit antibody against NFATc1 (no. 8032, dilution 1:1,000) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing

    Iguratimod inhibits the expression of c-Fos, NFATc1 and osteoclast marker genes. (A) The mRNA levels of c-Fos, NFATc1 and osteoclast marker genes were detected using RT-qPCR. Data are presented as means ± SD. (B and C) Proteins were extracted at indicated times and protein expression levels of c-Fos and NFATc1 were detected by western blotting (B) and quantified (C). The experiments were repeated 3 times independently. Data are presented as means ± SD. **P

    Journal: Molecular Medicine Reports

    Article Title: Iguratimod prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator-activated receptor-γ

    doi: 10.3892/mmr.2017.7648

    Figure Lengend Snippet: Iguratimod inhibits the expression of c-Fos, NFATc1 and osteoclast marker genes. (A) The mRNA levels of c-Fos, NFATc1 and osteoclast marker genes were detected using RT-qPCR. Data are presented as means ± SD. (B and C) Proteins were extracted at indicated times and protein expression levels of c-Fos and NFATc1 were detected by western blotting (B) and quantified (C). The experiments were repeated 3 times independently. Data are presented as means ± SD. **P

    Article Snippet: Rabbit antibody against NFATc1 (no. 8032, dilution 1:1,000) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing, Marker, Quantitative RT-PCR, Western Blot