Journal: Molecular Cancer Research

Article Title: AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis

doi: 10.1158/1541-7786.mcr-19-0530

Figure Lengend Snippet: Figure 2. LKB1 is required for EOC spheroid cell via- bility. A, Images of FTE and EOC cell lines cultured in suspension using ultra-low attachment (ULA) vessels. Images were cap- tured at the following time points (postseed- ing): FT190, FT190-STK11KO, day 7; OVCAR8, OVCAR8-STK11KO, day 5; HeyA8, HeyA8- STK11KO, iOvCa147, iOvCa147-STK11KO, day 3. Scale bars represent 1 mm (top) and 250 mm (bottom). B, Cell viability for FTE and EOC cell lines cultured in suspension using ULA vessels. Viable and nonviable cell numbers were determined by Trypan blue exclusion cell counting in a minimum of three independent experiments. Data are pre- sented as the number viable cells per well of a 24-well plate in each independent experiment and the overall mean. Parental and STK11KO groups were compared using the two-tailed Student t test (, P < 0.05; , P < 0.01; , P < 0.001). C, LKB1 is required for long-term growth and viability of EOC spheroids. EOC cells were cultured in 96-well round-bottom ULA plates to form single spheroids for the indicated time periods up to 21 days and phase contrast images were captured (top, “suspension”). At each indi- cated time point, individual spheroids were transferred to single wells of a 48-well adherent culture plate for 1 day (HeyA8, HeyA8-STK11KO) or 2 days (OVCAR8, OVCAR8-STK11KO) for sufficient reattach- ment. Following transfer of spheroids into adherent culture, cells were fixed and stained (bottom, “reattached”). Scale bars, 200 mm.

Article Snippet: Generation of DUSP4-overexpressing cell lines FT190, OVCAR8,HeyA8, and iOvCa147 cells were transfected with a plasmid to drive expression of a human DUSP4-Myc-DDK fusion protein (Origene; catalog no. RC200640) or empty vector.

Techniques: Cell Culture, Suspension, Cell Counting, Two Tailed Test, Staining

Journal: Molecular Cancer Research

Article Title: AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis

doi: 10.1158/1541-7786.mcr-19-0530

Figure Lengend Snippet: Figure 3. EOC cells lacking LKB1 have decreased metastatic potential. A, Survival analysis for parental and STK11KO EOC cell lines. EOC cells were injected intraperitoneally into female NOD/SCID mice. Mice were euthanized when humane endpoint criteria were reached. Censored subjects due to thymic lymphoma in the iOvCa147/iOvCa147- STK11KO study are indicated by tick marks on the survival curve. OVCAR8, OVCAR8-STK11KO, HeyA8, HeyA8-STK11KO, n ¼ 6 per group; iOvCa147, iOvCa147-STK11KO, n ¼ 12 per group. Parental and STK11KO survival curves were compared using the log-rank test. B, Representative images of mice at time of necropsy. Tumors are enclosed within a dotted black rectangle, and magnified images of enclosed regions are depicted underneath to illustrate tumors and surrounding tissue with greater detail (a–j). C, Quantification of total tumor burden. Mice were euthanized at a single time point postinjection (OVCAR8, OVCAR8-STK11KO, 51 days; HeyA8, HeyA8- STK11KO, 33 days) and all macroscopically visible tumors were excised and weighed together. Data are presented as the total tumor wet weight per mouse and the overall mean. Parental and STK11KO groups were compared using the two-tailed Student t test (, P < 0.05). D, Immunoblot analysis on excised tumors to confirm absence of LKB1 in OVCAR8- and HeyA8-STK11KO tumors. Tubulin was used as a loading control. Each lane represents a tumor from a different mouse.

Article Snippet: Generation of DUSP4-overexpressing cell lines FT190, OVCAR8,HeyA8, and iOvCa147 cells were transfected with a plasmid to drive expression of a human DUSP4-Myc-DDK fusion protein (Origene; catalog no. RC200640) or empty vector.

Techniques: Injection, Two Tailed Test, Western Blot, Control

Journal: Molecular Cancer Research

Article Title: AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis

doi: 10.1158/1541-7786.mcr-19-0530

Figure Lengend Snippet: Figure 5. AMPKa phosphorylation is maintained in FTE and EOC STK11KO cells. A, Immunoblot analysis to assess p-AMPKa (T172) in parental and STK11KO FTE and EOC cell lines cultured as adherent monolayers (adh) or spheroids in suspension (sph). LKB1 loss was confirmed, and tubulin was used as a loading control. B, Immunoblot analysis to assess p-AMPKa (T172) in FTE, OVCAR8, and iOvCa147 cells cultured under adherent (adh) or suspension (sph) conditions in the presence of 10 mmol/L STO-609 or the equivalent concentration (v/v) of DMSO (veh). C, Immunoblot analysis to determine relative p-AMPKa (T172) levels in xenograft tumors. LKB1 loss was confirmed and vinculin was used as a loading control. Each lane represents a tumor from a different mouse. D, Densitometric analysis of immunoblots in A. Data are presented as the pixel intensity volume for p-AMPKa (T172) normalized to AMPKa. Groups were compared using the two-tailed Student t test with no significant difference observed.

Article Snippet: Generation of DUSP4-overexpressing cell lines FT190, OVCAR8,HeyA8, and iOvCa147 cells were transfected with a plasmid to drive expression of a human DUSP4-Myc-DDK fusion protein (Origene; catalog no. RC200640) or empty vector.

Techniques: Phospho-proteomics, Western Blot, Cell Culture, Suspension, Control, Concentration Assay, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: Phosphatases differentially expressed in clinical ER − ERBB2 + versus triple-negative (TN) BC patients in this series (Agilent platform) and in GSE20194 (Affymetrix platform) (both FDR q-value <0.05).

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques:

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: Phosphatases differentially expressed between the molecular ER − ERBB2 + and the basal-like enriched BC in this series (FDR q-value <0.05) and in the NKI series (FDR q-value ≤0.01).

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques:

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: Phosphatases differentially expressed between ER + and ER − BC in common among GSE7390, GSE20194 and GSE2034 (FDR q-value ≤0.05).

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques:

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: Co-expression network analysis from the GeneMania server using DUSP4, DUSP6 and DUSP10 as query genes.

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: DUSP4, DUSP6 and phospo-ERK1/2 immuohistochemical percentage scores within the triple-negative breast carcinomas and the ERBB2-positive, ER and PR-negative breast carcinoma groups.

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques: Expressing, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers

doi: 10.3892/ijo.2014.2648

Figure Lengend Snippet: Multiphosphatase signature comprising 58 probes (48 genes) trained in GSE2034 training set and validated in GSE7390 (both Affymetrix HGU133A platform).

Article Snippet: The antibodies used were the rabbit polyclonal antibodies specific against the dual phosphorylated form of ERK1/2 (Thr202/Tyr204) (#4370, Cell Signaling, Beverly, MA, USA) at a dilution of 1:200, the polyclonal DUSP4 (MKP-2) antibody (NBP1-19592, Novus Biologicals, Littleton, CO, USA) at a dilution of 1:100, and a goat polyclonal anti-DUSP6 antibody (MKP-3) (sc-8599, Santa Cruz Biotechnology Lab Inc., Santa Cruz, CA, USA) at a dilution of 1:100, in the last case the anti-goat IG (HRP) (NB710-H, Novus Biologicals) was used as a secondary antibody at a dilution of 1:400.

Techniques:

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: Increased cellular oxidative stress after hypoxic exposure (0-3 h) leads to a time-dependent degradation of DUSP4 in RAECs. (A) Upper panel is the immunoblot against DUSP4 and lower panel is the immunoblot against actin as the loading control. 2 and 3 hr hypoxic exposure of cells leads to significant DUSP4 degradation (0.25 ± 0.07 and 0.29 ± 0.07 fold change of control level). * and ** versus control, P < 0.001. Degradation of DUSP4 is correlated to the uncontrolled activation of p38 after H/R insult. (B) Immunoblots of RAECs exposed to either control (no H/R) or 2 h H/R. Actin was used a loading control for DUSP4 and DUSP1, whereas the ratio of p-p38/p38 was used to determine the extent of phosphorylation of p38. (C) Following 2 h H/R, only 0.25 ± 0.07 of DUSP4/actin remains, yet there is no significant change in DUSP1/actin level (0.85 ± 0.17). Phosphorylation of p38 increases more than two fold (2.1 ± 0.36). There is no significant difference in ERK1/2 phosphorylation. No JNK phosphorylation is seen (data not shown). * P < 0.001, and # P < 0.05. Data is expressed as mean ± SEM, n ≥ 3.

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

Techniques: Western Blot, Control, Activation Assay, Phospho-proteomics

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: Endothelial cells with DUSP4 gene silencing are more susceptible to H/R-induced cell death and apoptosis. (A) 20X bright-field images of either control or DUSP4 siRNA-transfected RAECs. (B) H/R increases cell death (H/R 59.85% ± 4.89% compared to control, P < 0.005), pre-treatment with SB203580 prevents death (SB H/R 21.45% ± 3.98%, *P<0.0005). DUSP4 gene silencing significantly enhances H/R-induced death (93.24% ± 1.09%) compared to the H/R treatment group (# P < 0.005). This increased susceptibility is reversed by treatment with SB203580 (23.04% ± 6.93%, **P < 0.005 compared to si H/R). Data is expressed as mean ± SEM, n ≥ 3. (C) DUSP4 gene silencing in RAECs enhances H/R-induced TUNEL positive cells (45.81% ± 5.23% compared to H/R alone 29.46 ± 3.75%, #P < 0.05). However, SB203580 treatment of transfected cells significantly lowers their sensitivity to H/R-induced apoptosis (17.47% ± 1.45%, ** P < 0.005 compared to the si H/R group). Similarly the inhibition of H/R-treated cells with SB203580 also significantly diminishes H/R-induced apoptosis (12.99% ± 1.89%), * P < 0.001. Data is expressed as mean ± SEM, n ≥ 3. There is no significant effect on the scrambled siRNA control group compared to control (data not shown).

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

Techniques: Control, Transfection, TUNEL Assay, Inhibition

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: H/R-induced endothelial cell death is modulated by DUSP4 and p38 activity. (A) Immunoblotting of RAECs after H/R. DUSP4 is degraded after 2 h hypoxic treatment. The degradation of DUSP4 is correlated with the over-activation of p38. Phosphorylation of p38 subsequently activates its downstream target, MK2, which can trigger caspase-3 activation and apoptosis. Inhibition of p38 with 20 µM SB203580 prevents H/R-induced apoptosis by blocking the phosphorylation of MK2 at the T334 site. (B) DUSP4 gene silencing leads to more pronounced p38 activation and MK2 phosphorylation at the T334 site after H/R. Similarly the inhibition of p38 kinase activity with 20 µM SB203580 diminishes MK2 activation and can thus prevent H/R-induced apoptosis. * Increased p38 MAPK activity. A negative scrambled siRNA is always performed. No effect from this negative control is seen (data not shown). (C) DUSP4 gene silencing up-regulates superoxide generation from cells (3.05 ± 0.22-fold increase compared to control, * P < 0.001). This increase in superoxide generation is inhibited by the treatment of 100 µM apocynin (0.57 ± 0.06-fold change compared to control, # P < 0.05). There is no significant effect from the negative control of scrambled siRNA. Data is expressed as mean ± SEM, n ≥ 3.

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

Techniques: Activity Assay, Western Blot, Activation Assay, Phospho-proteomics, Inhibition, Blocking Assay, Negative Control, Control

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: DUSP4−/− mice are more prone to I/R-induced myocardial damage. (A) TTC-stained Langendorff-perfused heart slices from DUSP4−/− versus WT hearts subjected to 30 min global ischemia and 60 min reperfusion demonstrated a significantly greater infarct size (46.75% ± 4.19%) compared to WT (30.31% ± 3.33%). P < 0.05. n = 6 (B-G) Post-ischemic myocardial recovery following 30 min global ischemia and 30 min reperfusion for WT and DUSP4−/− hearts. (B) Rate pressure product (RPP), (C) Left ventricular developed pressure (LVDP), (D) Heart rate (HR), and (E) Coronary flow (CF) are expressed as a percentage of pre-ischemic (PI) value (100 %). (F) dP/dtmax is expressed in mmHg/s. The pre-ischemic (PI) value of dP/dtmax of WT is 4078.4 ± 183.5 mmHg/s, and PI value of dP/dtmax of DUSP4 KO is 5236.8 ± 270.4 mmHg/s. (G) Left ventricular end diastolic pressure (LVEDP) is expressed in mmHg. * P ≤ 0.05, and ** P ≤ 0.01, n > 10.

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

Techniques: Staining

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: DUSP4 gene deletion up-regulates Nox4 expression. (A) Immunoblotting against Nox4 antibody reveals that DUSP4 gene deletion promotes Nox4 protein expression (11.3 ± 3.64-fold change versus control, * P < 0.05) under basal conditions. (B) Quantitative PCR analysis shows that DUSP4 gene deletion increases Nox4 mRNA expression (4.9 ± 1.17-fold change versus control, * P < 0.01) under basal conditions. Data is expressed as mean ± SEM, n ≥ 3.

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

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

Journal: Free radical biology & medicine

Article Title: Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

doi: 10.1016/j.freeradbiomed.2015.07.013

Figure Lengend Snippet: Molecular alterations in DUSP4−/− hearts after I/R injury. (A) Increased p38 phosphorylation is evident in hearts subjected to I/R. This effect is more pronounced in DUSP4−/− mice. The ratio of p-p38/p38 of WT I/R hearts is set at 1. * versus WT I/R hearts, P < 0.001 n = 5. (B) The increase in p38 activity leads to MK2 phosphorylation at T334 and cleaved caspase-3 activation. Immunoblotting of cleaved caspase-3 is an indicator of apoptosis. WT and DUSP4−/− hearts both show an increase in caspase-3 and cleaved caspase-3 expression. * Increased p38 MAPK activity. (C) Direct inhibition of p38 activity by infusing with 10 µM SB203580 improves cardiac function at the end of 30 min reperfusion. RPP (%), LVDP (%), and CF (%) are dramatically improved for both WT and DUSP4−/− hearts. For (dP/dt)max, only DUSP4−/− hearts show significant recovery after the treatment of p38 inhibitor. There is no significant difference for heart rate and LVEDP at the end of 30 min reperfusion for both WT and DUSP4−/− hearts. * P < 0.05, and ** P < 0.01. n ≥ 7.

Article Snippet: A rat DUSP4 siRNA kit (OriGene, Rockville, MD) was utilized at a dose of 10 nM, which was previously determined sufficient for silencing of at least 80% of the constitutively expressed DUSP4 72 h post transfection [ 28 ].

Techniques: Phospho-proteomics, Activity Assay, Activation Assay, Western Blot, Expressing, Inhibition

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A The relative mRNA levels of MKP2 and MKP7 in NC and OV cells were determined with qPCR. B and C The relative MKP2 and MKP7 protein levels in both OV and NC cells were determined with Western Blotting. D The protein levels of MKP7 and NR4A1 in response to 100 μM H 2 O 2 in NC cells. E – H The protein levels of MKP7 in response to 100 μM H 2 O 2 and 0.5 μM TG at various time points in both OV and NC cells. The data represented the means of three independent experiments, * P < 0.05, ** P < 0.01 vs. ns. Error bars were shown as SD values.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: For real-time quantitative PCR.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: PSC intervention downregulates DUSP4 and upregulates the MAPK signaling pathway. (A) mRNA expression levels of DUSP2 and DUSP4 following different doses of PSC intervention. (B) Protein expression levels of DUSP4 after different doses of PSC intervention. (C) Quantification of DUSP4 protein levels shown in (B) . (D) Visualization of F-actin ring formation and DUSP4 expression in osteoclasts after different doses of PSC intervention. Green: F-actin; red: DUSP4; blue: DAPI. Magnification ×40. (E) Quantification of the F-actin ring area and the absolute and relative fluorescence intensity of DUSP4 shown in (D) . Data represent three independent experiments. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Expressing, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: Overexpression of DUSP4 affects osteoclast formation. (A) mRNA expression levels of DUSP4 in control, lentiviral-transfected null, and DUSP4-overexpression groups. (B) Protein levels of DUSP4 in the same groups. (C) Quantification of (B) . (D) Cell viability assay in the same groups. (E) TRAP staining of osteoclasts in control and DUSP4-overexpression groups after PSC intervention. Magnification ×40. (F) Quantification of the area occupied by TRAP-positive cells and the number of TRAP-positive cells shown in (E) . (G) F-actin ring staining in control and DUSP4-overexpression groups after PSC intervention green: F-actin; blue: DAPI. Magnification ×40. (H) Quantification of the F-actin ring area and fluorescence intensity shown in (G) . Data represent three independent experiments, and significance was determined using one-way ANOVA. ns, no significance; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Over Expression, Expressing, Control, Transfection, Viability Assay, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: Effects of DUSP4 on the MAPK pathway and osteoclast-related genes. (A) Western blot analysis showing changes in MAPK signaling pathway components (p-JNK, p-ERK, and p-p38) in osteoclasts after PSC intervention in control and DUSP4-overexpression groups. (B) Quantification of MAPK pathway phosphorylation levels in osteoclasts from control and DUSP4-overexpression groups, as shown in (A) . (C) mRNA expression levels of osteoclast-related proteins (DUSP4, MMP9, NFATc1, TRAP, and CTSK) after PSC intervention in control and DUSP4-overexpression groups. (D) Western blot analysis of osteoclast-related proteins (DUSP4, MMP9, NFATc1, CTSK, and c-fos) after PSC intervention in control and overexpression groups. (E) Quantification of osteoclast-related proteins expression levels in (D) . Data represent three independent experiments, and significance was determined using one-way ANOVA. ns, no significance; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Western Blot, Control, Over Expression, Phospho-proteomics, Expressing

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet: DUSP4 inhibits osteoclasts and attenuates bone destruction in osseous echinococcosis. (A) X-ray examination of long bones from mice treated with empty vector, lentiviral DUSP4, or left untreated in the affection of PSC after 6 months, with red rectangle indicating cyst. (B) Micro-CT images of long bones from each treatment group, with red arrows indicating bone defects. (C) HE staining of bone tissue sections from each treatment group. Magnification ×20. (D) Western blot analysis of osteoclast-related proteins (DUSP4 and CTSK) after PSC intervention in control and DUSP4-overexpression groups. (E) Quantification of osteoclast-related protein expression levels in osteoclasts from control and DUSP4-overexpression groups, as shown in (D) . Data are presented as mean ± SD from five mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques: Plasmid Preparation, Micro-CT, Staining, Western Blot, Control, Over Expression, Expressing

Journal: Frontiers in Microbiology

Article Title: Echinococcus granulosus sensu lato promotes osteoclast differentiation through DUSP4-MAPK signaling in osseous echinococcosis

doi: 10.3389/fmicb.2025.1558603

Figure Lengend Snippet:

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) blocking solution (BIOTEK, China) for 2 h, and then incubated overnight at 4°C with primary antibodies: DUSP4 (1:1000; Boster, #OT17C11); GAPDH (1:2000; Boster, #BG-7) ERK (1:2,000; #4695), p-ERK (1:1,000; #4370), JNK (1:2,000; #9252), p-JNK (1:1,000; #4668), p38 (1:2,000; #8690), p-p38 (1:1,000; #4511) (Cell Signaling Technology, Inc.); MMP-9 (1:1000; #ab76003); c-Fos (1:2,000; #ab190289), TRAP (1:1,000; #ab191406), NFATc1 (4 μg/mL; #ab2796), Cathepsin K (1:2,000; #ab19027) (Abcam, Inc).

Techniques:

Journal: Physiological Reports

Article Title: Multigenerational effects of fetal-neonatal iron deficiency on hippocampal BDNF signaling

doi: 10.1002/phy2.96

Figure Lengend Snippet: List of Real-Time PCR Taqman™ Probes

Article Snippet: Dusp4 , NM_022199 , Rn00573501_m1 , Phosphatase.

Techniques: Real-time Polymerase Chain Reaction, Membrane, Control

Journal: Physiological Reports

Article Title: Multigenerational effects of fetal-neonatal iron deficiency on hippocampal BDNF signaling

doi: 10.1002/phy2.96

Figure Lengend Snippet: Quantitative PCR (qPCR) analysis demonstrates increased expression of BDNF and downstream targets in the P15 F2 IS rats. Transcript levels were measured from the hippocampus of F1 iron sufficient (IS), F1 iron deficient (ID), and F2 iron-sufficient (F2 IS) rats for (A) BDNF-III and BDNF-IV, (B) trkB, and (C) Egr1, Hif1a, Cxcl12, and Dusp4. Values are means ± SEM; n = 4–6/group; * P < 0.05, ** P < 0.01, and *** P < 0.001, Unpaired t -test.

Article Snippet: Dusp4 , NM_022199 , Rn00573501_m1 , Phosphatase.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Physiological Reports

Article Title: Multigenerational effects of fetal-neonatal iron deficiency on hippocampal BDNF signaling

doi: 10.1002/phy2.96

Figure Lengend Snippet: Real-Time PCR Analysis of P65 Male Rat Hippocampus

Article Snippet: Dusp4 , NM_022199 , Rn00573501_m1 , Phosphatase.

Techniques: Real-time Polymerase Chain Reaction