Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: DUSP2 dephoshorylates the MAP kinases ERK1/2, p38 MAPK, and JNK but not STAT3 in T cells (A)Western blot of Jurkat cells either left untreated or pretreated with CHX (10 μg/mL for 30 min) prior to activation with 5 μg/mL aCD3 and 10 nM PMA. Cell extracts were collected at indicated time points and western blots were probed with the antibodies detecting phosphorylated MAP kinases. (B) Relative mRNA expression levels of the inducible DUSPs (DUSP1, 2, 4, 5) in Jurkat cells unstimulated or stimulated for 2 h or 4 h with 250 ng/mL aCD3/and 250 ng/mL PMA ( n = 3). (C) RT-qPCR analysis was conducted to verify the induction of DUSP2 mRNA expression in Jurkat WT cells within 6 h after stimulation with 5 μg/mL aCD3 and 10 nM PMA or (D) within 4 h after stimulation with aCD3/aCD28 coated beads. Data are presented as DUSP2 mRNA expression normalized to geometric mean of GAPDH and TBP as control ( n = 3). (E) Western blot analysis of stimulated (5 μg/mL aCD3/10 nM PMA) Jurkat cells showed an inverse correlation of DUSP2 protein levels with MAPK phosphorylation levels after initial induction. (F) DUSP2 KO led to sustained MAPK phosphorylation after stimulation. (G) The rescue of Jurkat DUSP2 KO cells with wild-type DUSP2 (DUSP2 resWT ) restored the DUSP2 phosphatase function completely. This effect was abrogated in Jurkat cells (H) rescued with DUSP2 resKIM or (I) with DUSP2 resCS mutants. (J) Stimulation (5 μg/mL aCD3/10 nM PMA) of doxycycline pretreated (2 μg/mL, 3h) DUSP2 resWT (Tet) cells showed a DUSP2 dependent dephosphorlyation of ERK1/2 and p38, (K) while STAT3 was not dephosphorylated in a DUSP2 dependent manner in DUSP2 resWT (Tet) cells after doxycycline pretreatment (2 μg/mL, 3 h) and INFα (100 ng/mL) stimulation. For all experiments cells were starved overnight, and majority of experiments were performed in at least three replicates. Expression data are presented as mean ± SEM. Unpaired student’s t test was calculated compared to unstimulated cells, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; CHX = cycloheximide, PMA = phorbol-12-myristate-13-acetate; INFα = interferon alpha, TPM = transcripts per million, ° unspecific band.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Western Blot, Activation Assay, Expressing, Quantitative RT-PCR, Control, Phospho-proteomics

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: Mutations outside DUSP2 key residues can impair its phosphatase activity (A) Schematic representation of the amino acid sequence of wild-type DUSP2 compared to mutated allele leading to a C-terminal extended DUSP2 protein (41 kDa) of 380aa. (B–D) Western blot analysis of (B) Jurkat cells, (C) single-allele knock-out Jurkat cells expressing DUSP2 32kDA and (D) single-allele knock-out Jurkat cells expressing DUSP2 41kDA after stimulation with 5 μg/mL aCD3/10 nM PMA with the indicated antibodies. (E) Western blot analysis of extracts derived from stimulated (aCD3/PMA) Jurkat DUSP2 KO cells rescued with a lentiviral expression vector for the 41 kDa allele of DUSP2 and probed with the indicated antibodies. For all western blot experiments cells were starved overnight. (F) Microscopic analysis showed U2OS cells transfected with myc-tagged expression vectors encoding either DUSP2 WT or DUSP2 41kDa and stained with an anti-myc antibody to detect DUSP2. The scale bars indicated a length of 25 μm. (G–I) In vitro phosphatase assays were performed using 300 ng of (G) wild-type human DUSP2 WT protein, (H) catalytically inactive human DUSP2 protein (DUSP2 CS ), or (I) human DUSP2 41kDa protein in combination with the indicated MAPKs (1 μg). Data are presented as mean ± SD of relative increased fluorescence signal (RFU 60 min/RFU 0 min), n = 3. Unpaired student’s t test, ∗∗∗ p < 0.001. DSP, dual specificity phosphatase; PMA, phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Activity Assay, Sequencing, Western Blot, Knock-Out, Expressing, Derivative Assay, Plasmid Preparation, Transfection, Staining, In Vitro, Fluorescence

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: Loss of DUSP2 is compensated by DUSP5 (A) RT-qPCR analysis of DUSP5 mRNA expression in Jurkat and DUSP2 KO cells stimulated with aCD3/aCD28 coated beads (bead/cell ration 1:1) for 0–4 h. (B) RT-qPCR analysis of DUSP5 mRNA expression in Jurkat and DUSP2 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA for 0–6 h. (C–E) Western blots analysis of DUSP2, DUSP5, and EGR1 protein levels and phosphorylation of ERK1/2, and p38 in (C) Jurkat, (D) DUSP2 KO , (E) and DUSP2/DUSP5 KO cells after stimulation with 5 μg/mL aCD3/10 nM PMA. The faint DUSP5 band occurred only in this replicate of the DUSP2/DUSP5 KO blot, but was absent in all other replicates (Document S2). (F) RT-qPCR confirmation of EGR1 mRNA expression in Jurkat and DUSP2 KO cells stimulated with aCD3/aCD28 coated beads (bead/cell ration 1:1) for 0–4 h. (G) RT-qPCR confirmation of EGR1 mRNA expression in Jurkat and DUSP2 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA for 0–6 h. (H) Comparison of DUSP2 and DUSP5 mRNA expression levels in Jurkat and EGR1 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA. (I) Western blot analysis of extracts from Jurkat and EGR1 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA and probed with indicated antibodies. (J) Western blot analysis of Jurkat cells either untreated or pretreated with ERK inhibitor (SCH772984, 1 μM, 30 min) prior to stimulation with 5 μg/mL aCD3/10 nM PMA, probed with indicated antibodies. Cells for all experiments were starved overnight prior to stimulation with at least in three replicates. Gene expression data are presented as target gene mRNA expression normalized to geometric mean of GAPDH and TBP as control ( n = 3). Data are presented as mean ± SD, unpaired student’s t test, ∗ p < 0.05, ∗∗∗ p < 0.001. PMA = phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Phospho-proteomics, Comparison, Gene Expression, Control

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: DUSP2 and DUSP5 act in concert to regulate IL2 production in T cells (A–C) RT-qPCR analysis of Jurkat, DUSP2 KO , DUSP2 resWT and DUSP/DUSP5 KO cells stimulated with 250 ng/mL aCD3/250 ng/mL PMA. The analysis determined mRNA expression of (A) the EGR1 (B) DUSP5 and (C) IL2 . Gene expression data are presented as target gene mRNA expression normalized to geometric mean of GAPDH and TBP as control and for statistics compared to 0 h of respective cell line. (D) ELISA analysis of IL2 secretion of Jurkat, DUSP2 KO , DUSP2 resWT , and DUSP/DUSP5 KO cells 24 h after stimulation with 250 ng/mL aCD3/250 ng/mL PMA. (E) ELISA analysis of IL2 secretion of Jurkat, DUSP2 KO , DUSP2 resWT , and DUSP/DUSP5 KO stimulated for 24 h with 250 ng/mL aCD3/250 ng/mL PMA. Cells were either left unstimulated, stimulated, treated with ERK inhibitor (SCH772984, 1 μM) 30 min before stimulation or treated with ERK inhibitor 2 h or 3 h after stimulation. All experiments were performed at least in three replicates ( n ≥ 3), data are presented as mean ± SD, unpaired student’s t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. PMA = phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Control, Enzyme-linked Immunosorbent Assay