Journal: Oncotarget

Article Title: Connexin 32 dysfunction promotes ethanol-related hepatocarcinogenesis via activation of Dusp1-Erk axis

doi:

Figure Lengend Snippet: Up-regulated and down-regulated genes in the liver of EtOH-treated Tg rats

Article Snippet: Thereafter a polyclonal rabbit antibody against GST-P was used with biotin-conjugated anti-rabbit IgG and Cy5-labeled streptavidin (Thermo Fischer Scientific Inc.), and finally the section was incubated with an Alexa Fluor 350 conjugated rabbit anti-DUSP1 polyclonal antibody (Bioss Inc., Boston, MA).

Techniques:

Journal: Oncotarget

Article Title: Connexin 32 dysfunction promotes ethanol-related hepatocarcinogenesis via activation of Dusp1-Erk axis

doi:

Figure Lengend Snippet: Levels of Dusp1 mRNA A. and protein B. in whole liver tissues measured by quantitative RT-PCR and western blotting, respectively. Data of quantitative RT-PCR are presented as mean ± SD, n = 4 per groups. **, ***: P < 0.01 and 0.001 respectively. Representative image of laser microdissected tissue (upper) and Dusp1 mRNA level in GST-P positive foci as determined by quantitative RT-PCR (lower) C. Immunohistochemical staining for Dusp1 in GST-P positive foci of Tg and Wt rats that received 5%EtOH (upper) and the intensity score of Dusp1 in GST-P positive foci as compared to Wt-control rats (lower) D. Data are presented as mean ± SD, n = 5 per group, 10 foci/rat. *: P < 0.05.

Article Snippet: Thereafter a polyclonal rabbit antibody against GST-P was used with biotin-conjugated anti-rabbit IgG and Cy5-labeled streptavidin (Thermo Fischer Scientific Inc.), and finally the section was incubated with an Alexa Fluor 350 conjugated rabbit anti-DUSP1 polyclonal antibody (Bioss Inc., Boston, MA).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: m 6 A mediates DUSP1 transcript stability during bacterial infection in RAW264.7 cells. (A) Tracks of m 6 A peaks on the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa . (B) Expression levels of the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa quantified by transcriptome sequencing (RNA-seq). (C) m 6 A levels on the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa examined by MeRIP-qPCR. (D) Expression levels of the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa quantified by RT-qPCR. (E) Examination of DUSP1 and ALKBH5 protein levels following ALKBH5 knockdown in RAW264.7 cells by Western blotting. (F) m 6 A levels on the DUSP1 transcript following ALKBH5 knockdown in RAW264.7 cells examined by MeRIP-qPCR. (G) Expression levels of the DUSP1 transcript following ALKBH5 knockdown examined by RT-qPCR. (H) Alterations in the half-lives (T1/2) of the DUSP1 transcript following ALKBH5 knockdown during P. aeruginosa infection examined by RT-qPCR at the indicated time points following the addition of 10 μg/mL actinomycin D. siALKBH5, siRNA targeting ALKBH5.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Infection, Expressing, Sequencing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: YTHDF2 mediates m 6 A-dependent DUSP1 transcript stability during bacterial and viral infections in RAW264.7 cells. (A) Protein levels of the m 6 A writers METTL3, METTL14, and WTAP; the erasers ALKBH5 and FTO; and the readers YTHDF1 and YTHDF2 with or without infection by C. diphtheriae , P. aeruginosa , or HSV-1 at the indicated time points examined by Western blotting. (B) Examination of DUSP1 and YTHDF2 protein levels following YTHDF2 knockdown by Western blotting. (C) Expression levels of the DUSP1 transcript following YTHDF2 knockdown examined by RT-qPCR. (D) Alterations of the half-lives of the DUSP1 transcript following YTHDF2 knockdown in RAW264.7 cells during P. aeruginosa infection examined by RT-qPCR at the indicated time points following the addition of 10 μg/mL actinomycin D. (E) Binding of YTHDF2 to the DUSP1 transcript at the indicated time points following P. aeruginosa infection examined by RIP-qPCR.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Binding Assay

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: DUSP1 regulates p38 and JNK phosphorylation and the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) DUSP1 knockdown enhanced p38 and JNK phosphorylation during infection by P. aeruginosa , C. diphtheriae , HSV-1, or the HSV-1 ICP34.5 mutant. (B) DUSP1 knockdown enhanced the expression of the IL-1β, CSF3, TGM2, and SRC genes during infection by P. aeruginosa , C. diphtheriae , HSV-1, or the HSV-1 ICP34.5 mutant. (C) DUSP1 knockdown enhanced the protein level of IL-1β during infection by P. aeruginosa , C. diphtheriae , or the HSV-1 ICP34.5 mutant.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Expressing, Infection, Mutagenesis

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: Working model of the regulation of DUSP1, MAPKs, and innate immune response genes by m 6 A and the m 6 A-related proteins YTHDF2 and ALKBH5 during pathogenic infections.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques:

Journal: Life Sciences

Article Title: Vitamin D modulates systemic inflammation in patients with severe COVID-19

doi: 10.1016/j.lfs.2022.120909

Figure Lengend Snippet: VitD treatment decreased COVID-19 hyperinflammatory responses through attenuating STAT3 signaling, as well as JNK and AKT of the MAPK cascades in blood of severe COVID-19 patients. (A and B) mRNA and protein expression levels of VDR in whole blood of VitD treated compared to the VitD untreated COVID-19 patients. (C-E) Expression levels of pSTAT3, pJNK, and pAKT in whole blood of VitD treated compared to the VitD untreated COVID-19 patients. (F–I) mRNA and protein expression levels of DUSP1 and DUSP5 in whole blood of VitD treated compared to the VitD untreated COVID-19 patients. (J–M) The protein levels of IL-6, IL-17, IL-1β, and TNFα proinflammatory cytokines in plasma of VitD treated compared to VitD untreated patients. Correlation of IL-6, IL-17, IL-1β plasma levels with serum levels of D-dimer and CRP of these patients. Statistical tests; Comparison was done using unpaired t-test or Mann-Whitney U test, depending on the skewness of the data. Linear regression models adjusted for patient's age, male sex, BMI, and DM. P value of <0.05 was considered significant.

Article Snippet: The proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Ca, USA), blocked in skimmed milk for 1 h at room temperature, incubated overnight at 4 °C with antibodies specific to (cat#12550, Cell Signaling Technology, Beverly, MA, USA), p-STAT3 (cat# 9145, Cell Signaling Technology, Beverly, MA, USA), STAT3 (cat# 9132, Cell Signaling Technology, Beverly, MA, USA), p-JNK (cat# 9251, Cell Signaling Technology, Beverly, MA, USA), (cat# 9252, Cell Signaling Technology, Beverly, MA, USA), p- (cat# 9271, Cell Signaling Technology, Beverly, MA, USA), AKT (cat# 9272, Cell Signaling Technology, Beverly, MA, USA), DUSP1 (cat# 35217, Cell Signaling Technology, Beverly, MA, USA), DUSP5 (cat# 3483, Cell Signaling Technology, Beverly, MA, USA), (cat#54376, Cell Signaling Technology, Beverly, MA, USA), and β-Actin (cat#8457, Cell Signaling Technology, Beverly, MA, USA) was used as loading controls.

Techniques: Expressing, Clinical Proteomics, Comparison, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/JCI171063

Figure Lengend Snippet: ( A ) Heatmap of differential expression patterns of proliferative versus invasive gene signatures and transcriptional regulators associated with distinct melanoma phenotypes 22 (indicated by a red asterisk) in 451Lu-R and 1205Lu-R melanoma cells treated with 2.5 μM corin or DMSO for 24 hours. ( B ) Comparison of the corin-associated intermediate phenotype gene expression signature in 451Lu-R and 1205Lu-R melanoma cells treated with 2.5 μM corin or DMSO for 24 hours and the published intermediate phenotype defined by Wouters et al. . ( C ) Volcano plots of differentially expressed genes (DEGs) (log 2 FC >1, P adj < 0.01) in 451Lu-R (left) and 1205Lu-R (right) melanoma cells following 24 hours of treatment with 2.5 μM corin plus 5 μM PLX4032 versus 5 μM PLX4032 alone with highlighted changes in DUSP1 , DUSP5 , MITF , and AXL expression. ( D ) Heatmap of DUSP1/-4/-5/-6 expression in corin-treated BRAFi-S melanoma cells relative to DMSO treatment (2.5 μM, 24 hours). ( E ) Top known transcription factor–binding motifs enriched in corin-upregulated genes in 451Lu-R (left) and 1205Lu-R (right) melanoma cells.

Article Snippet: The DUSP1 human overexpression plasmid (Origene, NM_004417, catalog RC205220) was expanded and transfected into 451Lu BRAFi-R and 1205Lu BRAFi-R cells using jetPRIME Transfection Reagent (Polyplus Transfection).

Techniques: Expressing, Comparison, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/JCI171063

Figure Lengend Snippet: ( A ) Comparison of average RNA-Seq log 2 FC for genes that gained or lost H3K27ac, H3K9ac, or H3K4me2 peaks at the TSS with corin treatment in 1205Lu-R cells (2.5 μM, 24 hours). ( B and C ) GO plots of genes with H3K27ac ( B ) or H3K4me2 ( C ) ChIP-Seq peaks gained at their TSS in 1205Lu-R cells treated with 2.5 μM corin for 24 hours compared with DMSO control. ( D ) Integrative Genome Viewer track of input-subtracted H3K27ac, H3K9ac, and H3K4me2 ChIP signals in the genomic region upstream of the DUSP1 TSS for 1205Lu-R cells treated with 2.5 μM corin for 24 hours compared with DMSO control. ( E ) ChIP-qPCR validation of RCOR1, LSD1, H3K27ac, H3K9ac, and H3K4me2 peaks gained upstream of the DUSP1 TSS in 1205Lu-R cells treated with 2.5 μM corin for 24 hours compared with DMSO control. Data from 1 representative biological replicate are shown. * P < 0.05 and **** P < 0.0001, by 2-tailed, unpaired t test. ( F ) EMT and AP-1 family member transcription factors enriched in the HOMER motif analysis mapped to the DUSP1 promoter sequence. Coordinates relative to the TSS were obtained from the UCSC Genome Browser (GRCh38).

Article Snippet: The DUSP1 human overexpression plasmid (Origene, NM_004417, catalog RC205220) was expanded and transfected into 451Lu BRAFi-R and 1205Lu BRAFi-R cells using jetPRIME Transfection Reagent (Polyplus Transfection).

Techniques: Comparison, RNA Sequencing Assay, ChIP-sequencing, Control, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/JCI171063

Figure Lengend Snippet: ( A ) Western blot analysis of 1205Lu-R and 451Lu-R melanoma cells treated with DMSO, 5 μM PLX4032 alone, 2.5 μM corin alone, or 2.5 μM corin plus 5 μM PLX4032 for 24 hours. Western blots were run contemporaneously with the exception of DUSP1. DUSP1 expression in 1205Lu-R and 451Lu-R cells was evaluated on separate Western blots, as denoted by the separating dotted line. Quantification of the relative expression of p-p38 (active) versus p38 (total) protein expression relative to the DMSO control is shown below each p-p38 band. ( B and C ) Quantification of DUSP1 , -4 , -5 , and -6 mRNA expression levels (RT-qPCR) in BRAFi-S (S) versus BRAFi-R (R) 451Lu ( B ) and 1205Lu ( C ) melanoma cells ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, by 2-tailed, unpaired t test. ( D ) DUSP1 expression levels (RT-qPCR) in 451Lu-R, 1205Lu-R, SkMel28-R, and A375-R melanoma cells treated with DMSO or 2.5 μM corin for 2, 4, 8, or 24 hours. * P < 0.05 and *** P < 0.001, by 1-way ANOVA with Tukey’s test. ( E ) Proliferation of 1205Lu-R melanoma cells overexpressing DUSP1 versus control (Ctrl) vector, treated with increasing doses of PLX4032 for 72 hours. * P < 0.05 and ** P < 0.01, by 2-way ANOVA with Tukey’s test. The morphology of 1205Lu-R melanoma cells following vector control (top) or DUSP1 overexpression (bottom) is depicted on the right. Representative images shown. Scale bar: 100 μm. ( F ) Quantification of DUSP1 expression in normal skin ( n = 7), benign nevi ( n = 18), and malignant melanoma ( n = 44) tissues from patients. Data were generated using microarray data from Talantov et al. . *** P < 0.001 and **** P < 0.0001, by 1-way ANOVA with Tukey’s test. ( G ) Kaplan-Meier curves illustrating the correlation of DUSP1/RCOR1 expression in patients’ tumor specimens (split at the median) and overall survival using data obtained from TCGA melanoma database ( https://portal.gdc.cancer.gov ).

Article Snippet: The DUSP1 human overexpression plasmid (Origene, NM_004417, catalog RC205220) was expanded and transfected into 451Lu BRAFi-R and 1205Lu BRAFi-R cells using jetPRIME Transfection Reagent (Polyplus Transfection).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Plasmid Preparation, Over Expression, Generated, Microarray

Journal: The Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/JCI171063

Figure Lengend Snippet: ( A ) Proliferation assays of 451Lu-R and 1205Lu-R melanoma cells treated with increasing doses of p38i (BIRB-796) with or without 5 μM PLX4032 for 72 hours ( n = 3). ( B ) Proliferation assays of WM852, WM1361A, Sk-Mel-30, and IPC298 NRAS-mutant melanoma cell lines treated with increasing doses of corin for 72 hours ( n = 3). ( C ) Western blot data and quantification of the relative expression of p-p38 (active) versus p38 (total) protein in IPC298, WM1361A, MW852, and Sk-Mel-30 NRAS-mutant melanoma cell lines treated with DMSO or 2.5 μM corin for 24 hours. ( D ) DUSP1 and DUSP5 expression levels (RT-qPCR) in IPC298, Sk-Mel-30, WM852, and WM1361A NRAS-mutant melanoma cells treated with DMSO or 2.5 μM corin for 24 hours. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed, unpaired t test.

Article Snippet: The DUSP1 human overexpression plasmid (Origene, NM_004417, catalog RC205220) was expanded and transfected into 451Lu BRAFi-R and 1205Lu BRAFi-R cells using jetPRIME Transfection Reagent (Polyplus Transfection).

Techniques: Mutagenesis, Western Blot, Expressing, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/JCI171063

Figure Lengend Snippet: ( A and B ) Immunofluorescence staining for H3K4me2 ( A ) and H3K27ac ( B ) expression in 1205Lu-R melanoma tumor xenografts following corin treatment with or without PLX4032. ( C ) Expression (RT-qPCR) of DUSP1, DUSP5, MITF , and AXL in 1205Lu-R melanoma xenografts treated with corin with or without PLX4032 ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA with Dunnett’s test.

Article Snippet: The DUSP1 human overexpression plasmid (Origene, NM_004417, catalog RC205220) was expanded and transfected into 451Lu BRAFi-R and 1205Lu BRAFi-R cells using jetPRIME Transfection Reagent (Polyplus Transfection).

Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: m 6 A mediates DUSP1 transcript stability during bacterial infection in RAW264.7 cells. (A) Tracks of m 6 A peaks on the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa . (B) Expression levels of the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa quantified by transcriptome sequencing (RNA-seq). (C) m 6 A levels on the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa examined by MeRIP-qPCR. (D) Expression levels of the DUSP1 transcript 0, 2, 4, and 6 h after infection with P. aeruginosa quantified by RT-qPCR. (E) Examination of DUSP1 and ALKBH5 protein levels following ALKBH5 knockdown in RAW264.7 cells by Western blotting. (F) m 6 A levels on the DUSP1 transcript following ALKBH5 knockdown in RAW264.7 cells examined by MeRIP-qPCR. (G) Expression levels of the DUSP1 transcript following ALKBH5 knockdown examined by RT-qPCR. (H) Alterations in the half-lives (T1/2) of the DUSP1 transcript following ALKBH5 knockdown during P. aeruginosa infection examined by RT-qPCR at the indicated time points following the addition of 10 μg/mL actinomycin D. siALKBH5, siRNA targeting ALKBH5.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Infection, Expressing, Sequencing, RNA Sequencing Assay, Quantitative RT-PCR, Knockdown, Western Blot

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: YTHDF2 mediates m 6 A-dependent DUSP1 transcript stability during bacterial and viral infections in RAW264.7 cells. (A) Protein levels of the m 6 A writers METTL3, METTL14, and WTAP; the erasers ALKBH5 and FTO; and the readers YTHDF1 and YTHDF2 with or without infection by C. diphtheriae , P. aeruginosa , or HSV-1 at the indicated time points examined by Western blotting. (B) Examination of DUSP1 and YTHDF2 protein levels following YTHDF2 knockdown by Western blotting. (C) Expression levels of the DUSP1 transcript following YTHDF2 knockdown examined by RT-qPCR. (D) Alterations of the half-lives of the DUSP1 transcript following YTHDF2 knockdown in RAW264.7 cells during P. aeruginosa infection examined by RT-qPCR at the indicated time points following the addition of 10 μg/mL actinomycin D. (E) Binding of YTHDF2 to the DUSP1 transcript at the indicated time points following P. aeruginosa infection examined by RIP-qPCR.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Infection, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Binding Assay

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: DUSP1 regulates p38 and JNK phosphorylation and the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) DUSP1 knockdown enhanced p38 and JNK phosphorylation during infection by P. aeruginosa , C. diphtheriae , HSV-1, or the HSV-1 ICP34.5 mutant. (B) DUSP1 knockdown enhanced the expression of the IL-1β, CSF3, TGM2, and SRC genes during infection by P. aeruginosa , C. diphtheriae , HSV-1, or the HSV-1 ICP34.5 mutant. (C) DUSP1 knockdown enhanced the protein level of IL-1β during infection by P. aeruginosa , C. diphtheriae , or the HSV-1 ICP34.5 mutant.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques: Expressing, Knockdown, Infection, Mutagenesis

Journal: mBio

Article Title: N 6 -Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections

doi: 10.1128/mbio.03349-22

Figure Lengend Snippet: Working model of the regulation of DUSP1, MAPKs, and innate immune response genes by m 6 A and the m 6 A-related proteins YTHDF2 and ALKBH5 during pathogenic infections.

Article Snippet: The membrane was blocked with 5% milk and then incubated with primary antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number 5174S; Cell Signaling Technology [CST]), p38 (catalog number 8690S; CST), p-p38 (catalog number 4511S; CST), ERK (catalog number 4695S; CST), p-ERK (catalog number 4370S; CST), JNK (catalog number 9252S; CST), p-JNK (catalog number 4668S; CST), DUSP1 (catalog number NBP2-67909; Novus), IL-1β (catalog number AB-401-NA; R&D Systems), or ALKBH5 (catalog number HPA007196; Sigma) overnight at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Siglec-15 promotes DUSP1 expression in OS cells. (A) Identification of genes regulated by Siglec-15 in OS cells using RNA-Seq assay. (B, C) A heat map and a volcano plot were constructed from the DEGs between siSiglec-15 group and NC group. (D) The top 10 significantly downregulated genes were analyzed by RT-PCR. (E) DUSP1 gene expression in siSiglec-15 group and NC group. (F) Western blotting assay showed that compared with NC group, DUSP1 and p-DUSP1 expression deceased, but p-JNK/MAPK, JNK/MAPK, p-P38/MAPK and P38/MAPK activation increased in siSiglec-15 group. ** P < 0.01, *** P < 0.001, NS, no significance.

Article Snippet: DUSP1 (bs-1851R, rabbit, 1/300) was purchased from Bioss (Beijing, China).

Techniques: Expressing, RNA Sequencing Assay, Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activation Assay

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: DUSP1 reverses the effects of shSiglec-15 on the proliferation, migration and invasion of OS cells. (A–C) NC+DUSP1 group increased the proliferation and clone formation of OS cells compared with NC+Vector group, and shSiglec-15+DUSP1 group also increased the proliferation and clone formation OS cells compared with shSiglec-15+Vector group. (D–G) NC+DUSP1 group promoted the migration and invasion of OS cells compared with NC+Vector group, and shSiglec-15+DUSP1 group rescued the migration and invasion of OS cells compared with shSiglec-15+Vector group. * P < 0.05, ** P < 0.01, *** P < 0.001, NS, no significance. Scale bars, 400 µm and 200 µm.

Article Snippet: DUSP1 (bs-1851R, rabbit, 1/300) was purchased from Bioss (Beijing, China).

Techniques: Migration, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Siglec-15 promotes OS growth in vivo by inducing EMT. (A) The mice were sacrificed for tumor harvesting after 24 days and tumor image. (B) The tumor growth curves were plotted in shSiglec-15 group and shCtrl group. (C) Tumor weight was measured in shSiglec-15 group and shCtrl group. (D) Western blotting assay showed the expression of N-cadherin, Slug, Vimentin, and MMP-9 in shSiglec-15 group and shCtrl group. (E) Western blotting assay showed DUSP1, p-DUSP1, MAPK/JNK, p-MAPK/JNK, MAPK/P38, and p-MAPK/P38 in shSiglec-15 group and shCtrl group. (F) IHC staining for Siglec-15, DUSP1, Vimentin, P38/MAPK, PCNA and Ki-67 in in shSiglec-15 group and shCtrl group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DUSP1 (bs-1851R, rabbit, 1/300) was purchased from Bioss (Beijing, China).

Techniques: In Vivo, Western Blot, Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Both Siglec-15 and DUSP1 are expressed in OS specimens. (A) Representative image of Siglec-15 protein and DUSP1 protein in OS tissue at different stages. (B) The IHC scores of Siglec-15 and DUSP1 protein in high expression group and low expression group. (C, D) The IHC scores of DUSP1 for the Enneking stage and the IHC scores of Siglec-15 for lung metastasis. (E) Kaplan-Meier analysis between low Siglec-15 expression group and high Siglec-15 expression group. (F) The correlation between Siglec-15 expression and DUSP1 expression by the Pearson’s correlation analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001. Scale bars, 100 µm, in the lower right of photos.

Article Snippet: DUSP1 (bs-1851R, rabbit, 1/300) was purchased from Bioss (Beijing, China).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Relationship between clinicopathologic parameters and Siglec-15 and DUSP1 expression in human osteosarcoma tissues.

Article Snippet: DUSP1 (bs-1851R, rabbit, 1/300) was purchased from Bioss (Beijing, China).

Techniques: Expressing