Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: (Left) Mycelium phenotypes of the indicated strains grown on M2 medium. (Right) CG test of WT and the ΔPaMpk1 and ΔPaMpk2 mutants. Slices of the indicated strains were inoculated on M2 medium (M2) and M2 medium supplemented with yeast extract (M2 + YE). Parts of the inoculated slices corresponding to the growing edge are on the right; parts corresponding to the stationary phase area are on the left. The top of the slice corresponding to the aerial mycelium is oriented toward the top of the plate. Wild-type develops CG on M2 + YE (visible on the left of the culture as the pigmented area that lacks aerial hyphae) but not on M2 as previously described (Haedens et al. 2005), while the MAPK mutants never develop CG on either medium. Note the similar mycelium phenotypes of the ΔPaMpk1 and ΔPaMpk2 mutants.

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques:

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: Phenotypes associated with inactivation of the PaMpk1, PaMpk2, and PaMpk3 MAPK modules

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques:

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: Immunoblot analysis of amounts of PaMpk1 and PaMpk2 (MAPK) and phosphorylation (phospho-MAPK) in wild type throughout the life cycle. Extracts from ΔPaMpk1 and ΔPaMpk2 mycelia were loaded as controls. Mycelium of 3-day-old WT cultures was separated into three zones corresponding to 1 day of growth (1), 2 days (2), and 3 days (3) or extracted as a whole (mycelium). Gel loading with extract corresponding to 3 days contained more proteins. PaMpk1 and PaMpk2 are present and phosphorylated in all three zones in roughly equal amounts. They are also present and phosphorylated in maturing 2-day- (perithecia 2 d) and mature 4-day-(perithecia 4 d)-old fruiting bodies. They are present in an unphosphorylated form in ascospores not induced for germination (NI) and PaMpk2 is phosphorylated upon induction of germination (I).

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques: Western Blot, Phospho-proteomics

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: Amounts and phosphorylation of PaMpk1 and PaMpk2 were measured in the indicated strains in 3-day-old mycelia. Legend as in Figure 2.

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques: Phospho-proteomics

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: (A) Ascospores carrying the PaMKK2c mutation issued from a wild-type (WT) × PaMKK2c mutant cross germinate on a noninducing medium, while wild-type ascospores (issued from a wild-type × wild-type cross) do not. Note that the structures that decorate the ascospores on the WT × WT panel are appendages and not germinating hyphae. (B) Phosphorylation of PaMpk1 and PaMpk2 was measured on noninduced ascospores in WT and in two strains carrying constitutive PaMKK2c alleles (MKK2c). Extract from 3-day-old mycelia from ΔPaMpk1 (Δ1) and ΔPaMpk2 (Δ2) mutants were loaded to identify both MAPKs. (C) Phenotypes of strains carrying PaMKK2c constitutive allele. Fruiting bodies are visible as small black dots. They are numerous in WT and present in low amounts in strains carrying a PaMKK2c constitutive allele. Arrows point toward matured fructifications. Inactivation of PaMpk1 and PaMKK2 abolished fertility in strains with or without PaMKK2c. (D) CG is slightly diminished in strains carrying PaMKK2c. The presence of PaMKK2c does not rescue the CG defect of the ΔPaMpk1 mutants. (E) Phosphorylation of PaMpk1 and PaMpk2 is not modified in 3-day-old mycelia of strains carrying PaMKK2c in association with either a wild-type PaMKK2 allele (+) or ΔPaMKK2 (Δ). Fewer proteins were loaded in the well located on the far right.

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques: Mutagenesis, Phospho-proteomics, Modification

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: Localization of PaMpk1–GFP in stationary phase hyphae of wild type, ΔPaMpk2, and PaMKK2c mutants. In WT, PaMpk1–GFP accumulates in 10 to 20% of the nuclei (arrows). In the ΔPaMpk2 and the PaMKK2c mutants, PaMpk1–GFP never accumulates in nuclei (n > 500 nuclei). In these strains, fluorescence of the hyphae carrying the PaMpk1–GFP transgene is similar to autofluorescence of hyphae with no transgene (control). Hyphae have an abnormal morphology in the ΔPaMpk2 mutant, since they look crooked and bulging. “GFP” are hyphae observed with a GFP–3035B filter from Semrock (Ex, 472 nm/30; dichroïc, 495 nm; and Em, 520 nm/35); “GFP” + DAPI are overlays of PaMpk1–GFP (in green) and DAPI-stained nuclei (in blue).

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques: Fluorescence, Control, Mutagenesis, Staining

Journal: Genetics

Article Title: A Non-Mendelian MAPK-Generated Hereditary Unit Controlled by a Second MAPK Pathway in Podospora anserina

doi: 10.1534/genetics.112.139469

Figure Lengend Snippet: Growing edge showing branching and outward growth of the wild type and MAPK mutants. Δ1, ΔPaMpk1; Δ2, ΔPaMpk2; and Δ3, ΔPaMpk3.

Article Snippet: Involvement of the PaMpk1 MAPK pathway was discovered through a traditional genetics approach, i.e. , we identified mutants unable to develop CG (IDC mutants with I mpaired D evelopment of C G) and found that two of the mutated genes encode the MAPKKK and MAPKK of the cascade ( Kicka and Silar 2004 ; Kicka et al. 2006 ).

Techniques:

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: TGF-β1 mediated activation of signaling pathways. Confluent monolayers of HK2 cells were stimulated by the addition of TGF-β1 (1 ng/ml) for up to 6 hours. At the time points indicated, total cell extracts were generated, and immunoblot analysis of lysate samples for phosphorylated-Smad/total Smad (A), phosphorylated ERK/total-ERK (B), and phosphorylated p38/total p38 (C) was performed.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Activation Assay, Protein-Protein interactions, Generated, Western Blot

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: Smad activation occurs independently of MAP kinase activation. To determine the role of ERK and p38 MAP kinase in TGF-β1-mediated Smad activation, cells were stimulated with TGF-β1 (1 ng/ml) either alone or in the presence of PD98059 (A) or SB203580 (B) for 30 minutes. Subsequently, total cell extracts were generated and immunoblot analysis of lysate samples for phosphorylated Smad/total Smad.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Activation Assay, Generated, Western Blot

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: MAP kinase activation occurs after expression of Smad dominant-negative expression vector. Inhibition of Smad3 activation was achieved by transient transfection with a c-myc-tagged dominant-negative Smad3 expression vector. A: Transfection efficiency of the dominant-negative Smad3 expression vector. Histogram of FL-1 fluorescence in EGFP-transfected versus mock-transfected cells. Transfection efficiency, based on the proportion of cells in gated region M1, is estimated at 55%. Efficacy of the expression vector was confirmed by co-transfection of 1.0 μg of the dominant-negative (DN) expression vector or empty vector (EV) together with 0.9 μg of the Smad-responsive (SBE)4-Lux reporter and 0.1 μg of the Renilla luciferase construct using 6 μl of the mixed lipofection reagent FuGene6. B: Twenty-four hours after transfection cells were stimulated with 1 ng/ml TGF-β1 for 6 hours before quantitation of luciferase content. Results are expressed as ratios of firefly/Renilla luciferase and represent mean ± SD, n = 3. In parallel experiments, cells were transiently transfected with 1 μg of the c-myc-tagged dominant-negative Smad3 expression vector using 3 μl of FuGene6. Twenty-four hours after transfection, cells were stimulated with TGF-β1 (1 ng/ml) for 30 minutes. Subsequently, total cell extracts were generated, and immunoblot analysis of lysate samples for ERK/total-ERK (C) and phosphorylated p38/total p38 (D) was performed. E: In parallel experiments, overexpression of the vector was confirmed by c-myc immunoblot.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Activation Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation, Inhibition, Transfection, Fluorescence, Cotransfection, Luciferase, Construct, Quantitation Assay, Generated, Western Blot, Over Expression

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: MAP kinase activation occurs after gene silencing of Smad3 by Smad3 siRNA. Cells were transfected with Smad3 siRNA for 48 hours before cell lysis by the addition of Trireagent. A: Smad3 mRNA was subsequently analyzed by quantitative PCR. In parallel experiments, cells transfected with Smad3 siRNA were stimulated with TGF-β1 (1 ng/ml) for 30 minutes. B: Subsequently, total cell extracts were generated and immunoblot analysis of lysate samples for ERK/total-ERK and phosphorylated p38/total p38 was performed.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Activation Assay, Transfection, Lysis, Real-time Polymerase Chain Reaction, Generated, Western Blot

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: Inhibition of ERK and Smad3, but not p38, inhibits autocrine TGF-β1 transcription. HK2 cell monolayers were stimulated with TGF-β1 (1 ng/ml) for 24 hours either alone or in the presence of either PD98059 or SB203580 at the concentrations indicated. After RNA isolation and reverse transcription, TGF-β1 mRNA was quantified by quantitative PCR. Data represent mean ± SD, n = 4. Inhibition of Smad3 was achieved by transient transfection of the Smad3 dominant-negative expression vector (A) or by gene silencing using Smad3 siRNA (B). Cells were transiently transfected with either the c-myc-tagged dominant-negative Smad3 expression vector or Smad3 siRNA. Twenty-four hours after transfection, cells were stimulated with TGF-β1 (1 ng/ml) for a further 24 hours, and TGF-β1 mRNA was quantified by quantitative PCR. Data represent mean ± SD, n = 4.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Inhibition, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: Transcription factor activation. Mobility shift experiments were performed with nuclear extracts from HK-2 cells cultured for up to 6 hours in the presence of TGF-β1 (1 ng/ml). Nuclear extract was incubated with probes for AP-1 (A–C) and NF-κB (D, E). Identification of proteins involved in AP-1 activation was determined by mobility shift analysis with antibodies to c-fos/c-jun (B) and NF-κB activation using antibodies to p50, p65, cRel, RelB, or p52 as indicated (E). The role of Smad3 in either AP-1 (C) or NF-κB (F) activation was examined by TGF-β1 (1 ng/ml) stimulation of HK2 cells transiently transfected with Smad3 dominant-negative expression vector (DN) for 24 hours before performing the mobility shift assay. In the control experiment, cells transiently transfected with the empty vector (EV) were stimulated with TGF-β1. Likewise, the role of the ERK MAP kinase and p38 MAP kinase pathways in AP-1 activation was examined by TGF-β1 (1 ng/ml) stimulation of HK2 cells for 10 minutes in the presence of either 8 μmol/L PD98059 (PD) or 0.8 μmol/L SB203580 (SB) before analysis by mobility shift assay (C), and their role in NF-κB activation was similarly examined in cells exposed to TGF-β1 for 6 hours (F).

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Activation Assay, Mobility Shift, Cell Culture, Incubation, Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation, Control

Journal:

Article Title: ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-?1 Autoinduction in Proximal Tubular Epithelial Cells

doi: 10.2353/ajpath.2006.050921

Figure Lengend Snippet: Inhibition of ERK MAP kinase and P38 MAP kinase inhibits TGF-β1-stimulated TGF-β1 de novo protein synthesis. A: HK-2 cells were stimulated with TGF-β1 (1 ng/ml) either alone or in combination with PD98059 (8 μmol/L) or SB203580 (0.8 μmol/L) for 48 hours. B: In parallel experiments, to demonstrate specificity of the kinase inhibitors used, cells were stimulated with TGF-β1 in combination with the JNK MAP kinase inhibitor recombinant L-JNKI1 (JNKi) at a concentration of 10 μmol/L. All experiments were performed in the presence of 40 μCi of 3H-radiolabeled amino acid mixture (1000 μCi/ml; Amersham). Supernatant samples were subsequently collected for TGF-β1 immunoprecipitation and radiolabeled TGF-β1 was detected by autoradiography.

Article Snippet: In contrast, inhibition of ERK MAP kinase activation attenuated TGF-β1 stimulated binding to the NF-κB consensus probe . p38 MAP Kinase Inhibits de Novo Protein Synthesis Previously, we have demonstrated the involvement of the p38 MAP kinase pathway in TGF-β1 synthesis.

Techniques: Inhibition, Recombinant, Concentration Assay, Immunoprecipitation, Autoradiography

Journal: Scientific Reports

Article Title: Esketamine attenuates bone cancer pain by suppressing MAPK signaling and glial activation in the spinal dorsal horn of rats

doi: 10.1038/s41598-026-38137-y

Figure Lengend Snippet: ESK inhibits MAPK pathway activation in the SDH of BCP rats. ( A–D ) Representative WB images and quantification of phosphorylated JNK ( B ), p38 ( C ), and ERK ( D ) levels. Data are mean ± SEM of biological replicates n = 3. *** P < 0.001 versus sham plus vehicle group, # P < 0.05, ## P < 0.01, ### P < 0.001 versus BCP plus vehicle group, ns, not significant. One-way ANOVA with repeated measures followed by post hoc Tukey test. ( E ) Schematic of experimental timeline showing repeated intrathecal administration of ESK, SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor) on POD 13 to 16. ( F ) Co-treatment with ESK enhanced the MAPK inhibitors induced increase in PWT after intrathecal administration. Notably, co-administration of MAPK inhibitors did not further enhance ESK’s analgesic effect, suggesting that its antinociceptive action mainly involves MAPK pathway inhibition. Data are mean ± SEM of biological replicates n = 7–8 rats/group. Two-way ANOVA with repeated measures followed by post hoc Tukey test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. BCP + DMSO; # P < 0.05, BCP + ESK+SB203580 vs. BCP + SB203580, BCP + ESK+SP600125 vs. BCP + SP600125 at corresponding time points. ( G ) Western blot of IL-1β, IL-6, and TNF-α expression in the SDH. ( H–J ) Quantification of cytokine levels showed further reductions in IL-1β ( H ), IL-6 ( I ), and TNF-α ( J ) with combined ESK and MAPK inhibitor treatment. Data are mean ± SEM of biological replicates n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. One-way ANOVA with repeated measures followed by post hoc Tukey test.

Article Snippet: To assess MAPK pathway involvement, the JNK inhibitor SP600125 (5 μg/10 μl; Cell Signaling Technology, USA) and the p38 inhibitor SB203580 (10 μg/10 μl; Abcam, UK) were dissolved in DMSO to a final DMSO concentration of 1%.

Techniques: Activation Assay, Inhibition, Western Blot, Expressing

Journal: BMC Cell Biology

Article Title: A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media

doi: 10.1186/1471-2121-11-76

Figure Lengend Snippet: Comparison of gene expression and GeneGo canonical pathway maps among T3/HDF, T3/CMHDF, T3/MEF and T3/CMMEF cells . A. Parameters for comparison are set at threshold of 3 with p-value of 0.05. The common genes are indicated by blue/white strips. The white area denotes similar genes in which three of four are the same. The unique genes are marked as color bands: 1) T3/HDF, orange; 2) T3/CMHDF, blue; 3) T5/MEF, red; 4) T3/CMMEF, green. B. The top 10 GeneGo canonical pathway maps.

Article Snippet: Among these common and/or similar genes, cell adhesion was also involved in three of the top 10 GeneGo canonical pathway maps and two of the top 10 GO process networks.

Techniques: Comparison, Gene Expression