dusp6 inhibitor  (TargetMol)

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: A Human normal and osteoporosis samples were collected ( n = 10). The bone density of vertebral body was analyzed. The T value of human normal samples were ranged from (0.2 to 2.1, average1.01). The T value of human osteoporosis samples were ranged from (−2.6 to −5.2, average −3.3). We isolated the RNA from the vertebral body for PCR analysis to explore the expression of DUSP6. B We fed the mice until 30 months old. The tibia was collected for uCT to explore the bone loss. And the expression of DUSP6 was analyzed in the tibia from the 30-month-old and 4-month-old mice ( n = 10). C Experimental osteoporosis murine model was established ( n = 5). The tibia was collected from the two groups. Immunohistochemistry assay was carried out to explore the expression of DUSP6. Original scale bars: 500 μm. D The tibia was collected from the SHAM and OVX groups. Immunofluorescence assay was carried out to detect the expression and location of ACP5 (green) and DUSP6 (red). The ACP5-positive cells were regarded as an osteoclast marker. Original scale bars: 500 μm. E RANKL-induced osteoclast differentiation was carried out. We explored the expression of DUSP1 to DUSP6 during this process. Original scale bars: 200 μm. F Nucleic acid gel electrophoresis and G western blot analysis was employed to confirm the mRNA and protein level during the osteoclast differentiation ( n = 3). Data in all bar graphs are expressed as mean ± SD. * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Isolation, Expressing, Immunohistochemistry, Immunofluorescence, Marker, Nucleic Acid Electrophoresis, Western Blot

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: A RANKL-induced osteoclast differentiation was established. We silenced DUSP6 using small interfering RNA (RNAi, #1) or DUSP6 inhibitor (E/Z)-BCI (1 μM). TRAP staining was carried out to explore the differentiation of osteoclast. Original scale bars: 200 μm. B TRAP-positive cells number in per well and relative osteoclast cells were quantitative analyzed. C Bone resorption analysis was used to explore the effect of RNAi or (E/Z)-BCI on the function of osteoclasts. Original scale bars: 200 μm. D F-actin ring formation was carried out between the siControl(siCtrl), siDUSP6(si#1), DMSO and (E/Z)-BCI groups. The actin rings were detected using phalloidin with fluorescence microscopy. The fluorescence intensity was calculated using Imagej. Original scale bars: 200 μm. Quantitative analysis of bone resorption analysis ( E ) and F-actin ring formation ( F ). G The osteoclast-related gene expression of NFATc1, C-FOS, ACP5, and DC-STAMP was analyzed (day 5) using real-time PCR in the control and (E/Z)-BCI-treated group. Data in all bar graphs are expressed as mean ± SD ( n = 3). * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Small Interfering RNA, Staining, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: Experimental osteoporosis model was established. Mice were treated with (E/Z)-BCI for analysis the function of DUSP6 in vivo. A uCT was analyzed to confirm the bone loss. B The tibia was collected from the indicated groups and TRAP staining was analyzed to detect the osteoclasts. Original scale bars: 500 μm. C Quantitative analysis was used to determine the average TRAP-positive cell numbers from five different versions and the TRAP-positive cell bone surface/bone surface. D Serum cross-linked C-telopeptide 1 levels were determined by ELISA (mouse CTx-I ELISA kit; Cusabio, Wuhan, China). E The bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were measured to evaluate the microstructure. F The tibia was collected from the indicated groups. Immunofluorescence assay was carried out to explore the expression of DUSP6 in the indicated groups. Original scale bars: 500 μm.The tibia was collected from the indicated groups. Immunohistochemistry and quantitative analysis were carried out to explore the expression of ACP5 and CTSK, which were identified as osteoclast markers ( G , H ). Original scale bars: 500 μm. Data in all bar graphs are expressed as mean ± SD ( n = 5). * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: A Bioinformatics approach using existed databases was used to find the regulator of DUSP6. Two databases (TargetScan and miRDB) were used to explore the potential miRNAs regulating the DUSP6. And the GSE64433 form the NCBI was used to shown the differential miRNAs between osteoporosis and normal group. The overlapping miRNAs were identified as potential regulator of DUSP6 during the osteoporosis condition. B Real-time PCR was carried out to confirm the nine miRNAs in human osteoporotic and normal bone tissues. n = 10. C Human samples were collected and classified as young (range from 22 to 58 years old) and old (range from 62 to 87 years old) person. And they were then divided as normal and osteoporosis groups. The average T value was shown in the indicated group. And the hsa-miR-181a (has-miR-181a-5p) expression was evaluated using real-time PCR. Ovariectomy-induced osteoporosis model ( D ) and age-related osteoporosis model ( E ) was established to explore the expression of miR-181a (mmu-miR-181a-5p). F mRNA was isolated in the tibia in mice from the indicated group. The expression of osteoclast-related gene was analyzed using real-time PCR. G Normal and mutated luciferase reporter containing putative miR-181a target sites of DUSP6 was established. The luciferase reporter vectors were cotransfected into HEK293 cells with miR‐181a mimics/inhibitor. The luciferase activity was quantified. Data in all bar graphs are expressed as mean ± SD. * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: A TRAP staining was carried out to explore the function of miR-181a during the osteoclastogenesis. B Quantitative analysis was established. C BMMs were transfected with NC inhibitor and miR-181a inhibitor. DUSP6 expression was silenced using RNAi. TRAP staining were carried out to evaluate the osteoclast differentiation in the indicated groups. Original scale bars: 200 μm. D Quantitative analysis were carried out to confirm the TRAP staining analysis. E The effect of miR-181a on osteoclast apoptosis was calculated using Annexin V-FITC/PI kit by a flow cytometer. F The expression of DUSP6 in the indicated groups was analyzed using real-time PCR in RNAKL-induced osteoclastogenesis. Data in all bar graphs are expressed as mean ± SD ( n = 3). * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Staining, Transfection, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: RANKL-induced osteoclast differentiation was established. A BMMs were transfected with Ad-Control (AdCtrl) and Ad-DUSP6. And phosphorylation of ERK1/2, P38, and JNK signaling pathway was analyzed for the indicated time point. B BMMs were transfected with SiControl(SiCtrl) and SiDUSP6. And the phosphorylation of ERK1/2 was examined. C The genotyping analysis was carried out using PCR to confirm the ERK2 deletion according to the following standard: ERK2 wt: only 275 bp; ERK2f/+: 275 bp + 350 bp; ERK2f/f: only 350 bp. D BMMs were isolated from the ERK2f/f mice. And then they were transfected with Adcontrol(AdCtrl) and AdCre to delete the EKR2 expression. Western blot analysis was carried out to confirm this result. E BMMs isolated from the ERK2f/f mice were transfected to SiCtrl, SiDUSP6, AdCtrl, and AdCre in the indicated group. TRAP staining was carried out to explore the osteoclast differentiation. F Quantitative analysis was carried out. Original scale bars: 200 μm. G F-actin ring formation was carried out to explore the function of mature osteoclast. Original scale bars: 200 μm. Data in all bar graphs are expressed as mean ± SD ( n = 3). * P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Transfection, Control, Isolation, Expressing, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

doi: 10.1038/s41419-021-04110-y

Figure Lengend Snippet: RANKL-induced osteoclast differentiation was established. A BMMs were transfected with SiControl (SiCtrl) and SiDUSP6. And the phosphorylation of SMAD2 was examined for the indicated time point. B Immunofluorescence assay was carried out to further explore the expression of P-SMAD2.F-actin ring formation was carried out to define the mature osteoclast. C Immunoprecipitation assay was carried out to clarify the connection between DUSP6 and P-SMAD2. D BMMs were transfected with SiCtrl and SiDUSP6. Immunofluorescence assay was carried out to detect the NFATC1 expression in the nucleus. E BMMs were transfected with SiControl (SiCtrl) and SiDUSP6. And they were then treated with the P-SMAD2 inhibitor SB31542 to further confirm the effect of P-SMAD2 in DUSP6-mediated osteoclastogenesis. TRAP staining ( E ) and quantitative analysis were carried out ( F ) to determine the osteoclast differentiation. Data in all bar graphs are expressed as mean ± SD ( n = 3). Original scale bars: 200 μm.* P < 0.05, # P < 0.01.

Article Snippet: DUSP6 inhibitor (E/Z)-BCI was purchased from Medchem Express (USA), and it was dissolved in DMSO and stored in −20 °C.

Techniques: Transfection, Immunofluorescence, Expressing, Immunoprecipitation, Staining

Journal: Biomedicines

Article Title: Inhibition of DUSP6 Activates Autophagy and Rescues the Retinal Pigment Epithelium in Sodium Iodate-Induced Retinal Degeneration Models In Vivo and In Vitro

doi: 10.3390/biomedicines10010159

Figure Lengend Snippet: Upregulation of DUSP6 and MAPK after NaIO 3 treatment. ( a ) Representative blots of DUSP6/p-ERK axis expression in ARPE-19 cells after NaIO 3 treatment and the quantitative analysis of the DUSP6 expression level. ( b ) Quantitative analysis of the p-ERK level. Representative data from three independent experiments are shown. * p < 0.05 and ** p < 0.01.

Article Snippet: Cells were treated with the autophagy inhibitor Bafilomycin A1 (Sigma, Saint Louis, MO, USA) at 75 nM or DUSP6 inhibitor BCI (Sigma, Saint Louis, MO, USA) at 1.25 µM or 2.5 µM for 6 h at 37 °C.

Techniques: Expressing

Journal: Biomedicines

Article Title: Inhibition of DUSP6 Activates Autophagy and Rescues the Retinal Pigment Epithelium in Sodium Iodate-Induced Retinal Degeneration Models In Vivo and In Vitro

doi: 10.3390/biomedicines10010159

Figure Lengend Snippet: DUSP6/p-ERK axis was upregulated in NaIO 3 -induced retinal degeneration in vivo. ( a ) Schematic diagram illustrating the drug treatment and tissue preparation. ( b ) Real-time funds and retinal structures at different times post NaIO 3 injection were observed by fundoscopy and OCT system. The red arrows indicate the irregulated structure on the RPE layer. Representative data from three independent experiments are shown. ( c ) H&E staining presenting the mice retinal structures after treatment with NaIO 3 . The abnormal deposits on the RPE layer is represented by the red arrows. Scale bar = 50 µm. ( d ) Representative blots showed the DUSP6/p-ERK axis expression level after NaIO 3 injection in C57BL/6 mice. ( e , f ) A quantitative analysis is shown in ( d ). * p < 0.05.

Article Snippet: Cells were treated with the autophagy inhibitor Bafilomycin A1 (Sigma, Saint Louis, MO, USA) at 75 nM or DUSP6 inhibitor BCI (Sigma, Saint Louis, MO, USA) at 1.25 µM or 2.5 µM for 6 h at 37 °C.

Techniques: In Vivo, Injection, Staining, Expressing

Journal: Biomedicines

Article Title: Inhibition of DUSP6 Activates Autophagy and Rescues the Retinal Pigment Epithelium in Sodium Iodate-Induced Retinal Degeneration Models In Vivo and In Vitro

doi: 10.3390/biomedicines10010159

Figure Lengend Snippet: In conclusion, our data demonstrate that the inhibition of DUSP6 could protect RPE and the retina against NaIO 3 -induced oxidative stress-mediated autophagy dysfunction involving the ERK signaling pathway.

Article Snippet: Cells were treated with the autophagy inhibitor Bafilomycin A1 (Sigma, Saint Louis, MO, USA) at 75 nM or DUSP6 inhibitor BCI (Sigma, Saint Louis, MO, USA) at 1.25 µM or 2.5 µM for 6 h at 37 °C.

Techniques: Inhibition

Journal: Veterinary Research

Article Title: Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis

doi: 10.1186/s13567-020-00866-x

Figure Lengend Snippet: Pharmacological inhibition of DUSP6 promotes ERK1/2 signaling, reduces apoptosis, and facilitates IBV replication. Vero ( A ), H1299 ( B ), and DF-1 ( C ) cells were pre-incubated with BCI (10 μM) or DMSO for 1 h, followed by IBV infection. 10 μM BCI or DMSO was present throughout the experiment. Mock infection was included as another control group. Cells were harvested at 20 and 24 hpi and subjected to cell lysis or RNA extraction. The level of p-ERK1/2, ERK1/2, PARP, Bcl-2, Mcl-1, IBV N, and β-actin were checked by Western blot analysis (upper panels). The expression of DUSP6 was analyzed by quantitative real time RT-PCR (low panels). The experiments were repeated in triplicate and the representative data are shown. The signal of protein bands was determined by Image J software. The intensities of p-ERK1/2 or p-ERK2 were normalized to total ERK1/2 or total ERK2, the intensities of Bcl-2, Mcl-1, IBV N were normalized to β-actin, and the intensities of PARP-C were normalized to PARP-FL. The ratio of PARP-C and IBV N in BCI treated cells to DMSO treated cells are shown as PARP-C (BCI:DMSO) and IBV N (BCI:DMSO). The bar graphs in the low panels show means ± SD of three independent determination of relative expression of DUSP6 mRNA. P values were calculated by Student test. *** P < 0.001, **** P < 0.0001 (highly significant).

Article Snippet: The MEK1//2 inhibitor U0126 (9903S) was purchased from Cell Signaling Technology (USA), DUSP6 inhibitor BCI (B4313) were purchased from Sigma-Aldrich (USA).

Techniques: Inhibition, Incubation, Infection, Lysis, RNA Extraction, Western Blot, Expressing, Quantitative RT-PCR, Software

Journal: Veterinary Research

Article Title: Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis

doi: 10.1186/s13567-020-00866-x

Figure Lengend Snippet: Specific knock down of DUSP6 increases the phosphorylation of ERK1/2, reduces apoptosis, and promotes IBV replication. Vero ( A ), H1299 ( B ), DF-1 ( C ) cells were transfected with non-target siRNA (sic), siDUSP6-1 or siDUSP6-2 for 36 h, followed with IBV infection. Cells were harvested at 20 and 24 hpi and subjected to cell lysis or RNA extraction. The knock down effect of DUSP6 was analyzed by quantitative real time RT-PCR (low panel). The level of p-ERK1/2, ERK1/2, PARP, Bcl-2, Mcl-1, IBV N, and β-actin were checked with Western blot analysis. The experiments were repeated in triplicate and the representative data are shown. The signal of protein bands was determined by Image J software. The intensities of p-ERK1/2 or p-ERK2 were normalized to total ERK1/2 or total ERK2, the intensities of Bcl-2, Mcl-1, IBV N were normalized to β-actin, and the intensities of PARP-C were normalized to PARP-FL. The ratio of p-ERK1/2, p-ERK2, Bcl-2, Mcl-1, PARP-C, IBV N in siDUSP6 transfected cells to sic transfected cells were shown as (siDUSP6:sic). The bar graphs in the low panels show means ± SD of three independent determinations of relative expression of DUSP6 mRNA. P values were calculated by Student test. **** P < 0.0001 (highly significant).

Article Snippet: The MEK1//2 inhibitor U0126 (9903S) was purchased from Cell Signaling Technology (USA), DUSP6 inhibitor BCI (B4313) were purchased from Sigma-Aldrich (USA).

Techniques: Transfection, Infection, Lysis, RNA Extraction, Quantitative RT-PCR, Western Blot, Software, Expressing

Journal: Veterinary Research

Article Title: Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis

doi: 10.1186/s13567-020-00866-x

Figure Lengend Snippet: The phosphatase activity of DUSP6 is sufficient for suppressing ERK1/2 signaling, promoting apoptosis, and impairing IBV replication. Vero ( A ), H1299 ( B ), and DF-1 ( C ) cells were transfected with constructs encoding HA tagged DUSP6-WT and DUSP6-DN, or vector pCMV-HA, respectively. At 24 h post-transfection, cells were infected with IBV (MOI = 1) for 20 and 24 h. The expression of HA-DUSP6-WT and HA-DUSP6-DN, the levels of p-ERK1/2, ERK1/2, PARP, Bcl-2, Mcl-1, IBV N, and β-actin were checked by Western blot analysis. The experiments were repeated in triplicate and the representative data were shown. The signal of protein bands was determined by Image J software. The intensities of p-ERK1/2 or p-ERK2 were normalized to total ERK1/2 or total ERK2, the intensities of Bcl-2, Mcl-1, IBV N were normalized to β-actin, and the intensities of PARP-C were normalized to PARP-FL. The ratio of p-ERK1/2, p-ERK2, Bcl-2, Mcl-1, and IBV N in DUSP6 or DUSP6-DN transfected cells to vector transfected cells are shown as (DUSP6:pCMV). The ratio of PARP-C in DUSP6 transfected cells to DUSP6-DN transfected cells are shown as PARP-C (DUSP6:DUSP6-DN).

Article Snippet: The MEK1//2 inhibitor U0126 (9903S) was purchased from Cell Signaling Technology (USA), DUSP6 inhibitor BCI (B4313) were purchased from Sigma-Aldrich (USA).

Techniques: Activity Assay, Transfection, Construct, Plasmid Preparation, Infection, Expressing, Western Blot, Software

Journal: Veterinary Research

Article Title: Upregulation of DUSP6 impairs infectious bronchitis virus replication by negatively regulating ERK pathway and promoting apoptosis

doi: 10.1186/s13567-020-00866-x

Figure Lengend Snippet: The proposed working model. IBV infection activates MEK1/2-ERK1/2 signaling, which protects cells from death by reducing the level of Mcl-1 and Bcl-2, thereby supporting efficient IBV replication. The activation of ERK1/2 pathway induces the phosphatase DUSP6 expression. DUSP6 forms a negative regulation loop by dephosphorylating ERK1/2, to restrict this prosurvival signal. The upregulation of DUSP6 promotes cell death and impairs IBV replication, representing one of the host antiviral responses. U0126 inhibits the activation of ERK1/2 and promotes cell death, thereby suppressing IBV replication, while BCI inhibits DUSP6 and promotes ERK1/2 signaling/cell survival, thereby promoting IBV replication.

Article Snippet: The MEK1//2 inhibitor U0126 (9903S) was purchased from Cell Signaling Technology (USA), DUSP6 inhibitor BCI (B4313) were purchased from Sigma-Aldrich (USA).

Techniques: Infection, Activation Assay, Expressing