Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Western Blot, Cell Culture, Staining, Fluorescence, Transduction, shRNA, Control, Transfection

Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Activity Assay, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Expressing, Fluorescence, Generated, Isolation, Injection, Transduction, shRNA, Western Blot, Plasmid Preparation, Recombinant, In Vitro, Immunostaining, Flow Cytometry, Cell Culture

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: (A) Volcano plot analysis showing the differentially expressed genes; genes with log 2 foldchange > 1 and P -value < 0.05 are indicated in red; genes with the log 2 foldchange < −1 and P -value < 0.05 are indicated in blue; genes with the |log 2 foldchange| < 1 or P -value > 0.05 are indicated in grey. (B) ERK protein phosphorylation levels in JAR cells with DOCK1 repression or overexpression. (C) ERK phosphorylation levels in HTR-8 cells treated with 10 ng/ml EGF for 0, 30, and 60 min. (D) Knockdown efficacy of DUSP4 siRNA and DOCK1 expression after DUSP4 knockdown detected in HTR-8 cells by Western blotting. (E) 293T cells were transfected with ectopic Flag-DOCK1, Myc-DUSP4, and HA-UB for 48 h, and then treated with 20 μM MG132 for 6 h. The cell lysates were immunoprecipitated with Myc antibody and ubiquitination assays showed the DUSP4 protein ubiquitination levels. (F) mRNA expression of DUSP4 after HUWE1 knockdown in HTR-8 cells. (G) Knockdown efficiency of HUWE1 siRNA and DUSP4 protein abundance after HUWE1 knockdown detected in HTR-8 cells by Western blotting.

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques: Phospho-proteomics, Over Expression, Knockdown, Expressing, Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Quantitative Proteomics

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: (A) DUSP4 mRNA expression in DOCK1-knockout HTR-8 cells. (B) DUSP4 protein levels in DOCK1-depleted HTR-8 cells (left). The protein levels were quantified (right). (C) Wound healing ability after DUSP4 inhibition by siRNA in DOCK1-knockout HTR-8 cells (left). Rate of wound closure was quantified (right). Scale bar: 200 μm. (D) Migration and invasion ability after DUSP4 inhibition in DOCK1 knockout HTR-8 cells (top). The number of migrating and invading cells was quantified (bottom). Scale bar: 100 μm. (E) ERK phosphorylation and metastasis-related protein levels after DUSP4 siRNA treatment in DOCK1-knockout HTR-8 cells. In (A, C, D), each group, n = 3 biological replicates (* P < 0.05; ** P < 0.01; unpaired two-tailed t test). In (B), each group, n = 4 biological replicates (*** P < 0.001; unpaired two-tailed t test).

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques: Expressing, Knock-Out, Inhibition, Migration, Phospho-proteomics, Two Tailed Test

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: (A) DUSP4 protein levels in DOCK1-overexpressing HTR-8 cells. (B) Interaction of DOCK1 and DUSP4 detected by CO-IP in HEK293T cells transfected with ectopic Flag-DOCK1 and Myc-DUSP4. (C) Interaction of DOCK1 and DUSP4 in the cytoplasm observed by immunofluorescence staining with anti-DOCK1 (red) and anti-DUSP4 (green) in HTR-8 cells. Scale bar: 25 μm. (D) DOCK1 knockout HTR-8 cells were treated with 50 μg/ml cycloheximide (CHX) for 0, 1.5, 3, and 6 h, and DOCK1 and DUSP4 proteins were assayed by Western blot. (E) HTR-8 cells overexpressing DOCK1 were treated with 50 μg/ml CHX for 0, 1.5, 3, and 6 h, and Western blot was used to detect DOCK1 and DUSP4 proteins. (F) HTR-8 cells were treated with 50 μg/ml CHX alone or 50 μg/ml CHX and 20 μM MG132 for 6 h. (G) HTR-8 cells were treated with 50 μg/ml CHX alone or 50 μg/ml CHX and 10 μM CQ or 50 μg/ml CHX plus 1 mM 3-MA for 6 h. (H) DOCK1-overexpression and control HTR-8 cells were treated with 20 μM MG132 or left untreated for 6 h.

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Staining, Knock-Out, Western Blot, Over Expression, Control

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: (A) DOCK1-knockout HTR-8 cells were transfected with HA-UB for 48 h and treated with 20 μM MG132 for 6 h. The cell lysates were immunoprecipitated with the DUSP4 antibody, and the ubiquitination assay showed the ubiquitination levels of DUSP4 protein. (B) HUWE1 protein levels after DOCK1 knockout or overexpression in HTR-8 cells. (C) After DOCK1 overexpression followed by HUWE1 knockdown, HTR-8 cells were treated with 50 μg/ml cycloheximide for 0, 1.5, 3, and 6 h, and Western blot was performed to detect DOCK1 and DUSP4 protein levels. (D) 293T cells were transfected with ectopic Flag-DOCK1, Myc-DUSP4, and HA-UB for 24 h, and then transfected with HUWE1 siRNA for 48 h. Finally, 20 μM MG132 was added and the protein was extracted after 6 h of incubation. The cell lysates were immunoprecipitated with Myc antibody and the ubiquitination assay showed DUSP4 protein ubiquitination levels.

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques: Knock-Out, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Over Expression, Knockdown, Western Blot, Incubation

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: (A) Schematic illustration of the protocol for inhibiting DOCK1 during murine pregnancy. Experimental mice received vehicle control or TBOPP on GDs 6, 8, 10, and 12 through intraperitoneal injections, and were euthanatized on GDs 13.5. (B) Morphology of pregnant uteri with embryo implantation sites on GD 13.5 in mouse models after vehicle control or TBOPP treatments (n = 6 mice for each group). Scale bar: 1 cm. (C) Embryo resorption rate was calculated. (D) Western blot assays were performed to detect the levels of DUSP4, phosphorylated ERK, and HUWE1 after TBOPP treatment. (E) Quantification of DUSP4, phosphorylated ERK, and HUWE1 protein levels. (F) Representative HE-stained images of placentas and CK7 immunofluorescence of trophoblast cell invasion into the decidua after TBOPP treatment (left). Based on the HE and CK7 staining, distinct layers—Dec (decidua), Jz (junctional zone), and Lab (labyrinth)—are delineated by black lines in HE images and white lines in immunofluorescence images. Boxes on the left correspond to magnified sections on the right, with the region between the yellow and white lines indicating the trophoblast cell invasion area into the decidua. Scale bar: 500 μm. (G) The area of trophoblast cell invasion into the decidua was quantified. (H) Representative images of Ki67 immunostaining in placental tissues after TBOPP treatment (left). Percentage of Ki67-positive cells was calculated (right). Scale bar: 200 μm. In (C, E, G, H), each group, n = 6 mice (* P < 0.05; ** P < 0.01; *** P < 0.001, unpaired two-tailed t test).

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques: Control, Western Blot, Staining, Immunofluorescence, Immunostaining, Two Tailed Test

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: Details of antibodies used.

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques:

Journal: Life Science Alliance

Article Title: DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway

doi: 10.26508/lsa.202302247

Figure Lengend Snippet: Primers used for real-time quantitative polymerase chain reaction.

Article Snippet: DUSP4 , IF(1:200) , Proteintech , #66349-1-Ig.

Techniques:

Journal: Journal of cellular physiology

Article Title: Mitogen-Dependent Regulation of DUSP1 Governs ERK and p38 Signaling During Early 3T3-L1 Adipocyte Differentiation

doi: 10.1002/jcp.25248

Figure Lengend Snippet: DUSP and inflammatory genes analyzed in this study

Article Snippet: For adipocytes, these stimuli include insulin and the synthetic glucocorticoid dexamethasone ( Kusari et al., 1997 ; Kassel et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Symbol Name/alias Accession ABI number C T Dual specificity phosphatases GP MKB NLS NES EJP Dusp1 MKP-1,hVH1 I ● ● NM_013642 Mm00457274_g1 24 Dusp2 PAC-1 I ● ● NM_010090 Mm00839675_g1 30 Dusp4 MKP-2, hVH2 I ● ● NM_176933 Mm00723761_m1 25 Dusp5 hVH3 I ● ● NM_001085390 Mm01266104_m1 27 Dusp6 MKP-3, rVH6 II ● ● E NM_026268 Mm00650255_g1 24 Dusp7 MKP-X II ● ● E NM_153459 Mm00463228_m1 27 Dusp9 MKP-4 II ● ● E NM_029352 Mm00512646_m1 27 Dusp8 M3/6, hVH5 III ● ● ● J/P NM_008748 Mm00456230_m1 27 Dusp10 MKP-5 III ● J/P NM_022019 Mm00517678_m1 24 Dusp16 MKP-7, MKP-M III ● ● ● J/P NM_130447 Mm00459935_m1 25 Inflammatory genes IL-6 Interleukin-6 NM_031168 Mm99999064_m1 32 TNFα Tumor necrosis factor-alpha NM_013693 Mm99999068_m1 28 Reference gene 18S 18 Ribosomal RNA X03205 4342930E 9 Open in a separate window DUSP group (GP), MAPK binding domain (MKB), nuclear localization sequence (NLS), nuclear export sequence (NES), ERK (E), JNK (J), p38 (P), threshold cycle (C T ) measured in lean adipose tissue.

Techniques:

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: