Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 1 Downregulation of DUSP8 in LUAD correlated with poor OS. A mRNA expression analysis of DUSP8, DUSP10, and DUSP16 in samples obtained from patients with LUAD (n = 513) compared to non-tumor tissues (NTs) (n = 287) from the GTEx–TCGA patient cohort. B The clinical outcome associated with DUSP8, DUSP10, and DUSP16 expression in LUAD using KM Plotter [36]. C Expression of MAPK8 and MAPK9 in LUAD (n = 513) vs. NT (n = 287) respectively. D Correlation between OS of patients with LUAD and expressions of MAPK8 and MAPK9. E Protein interaction network using STRING. The edges indicate both functional and physical protein associations, line thickness indicates the strength of data support. F Correlation between OS of female and male patients in all stages of LUAD and DUSP8 expression. G DUSP8 protein level measured using ELISA (n = 5). H Western blot analysis of DUSP8 expression in NTs and tumor samples from the same patient (n = 3). I Representative images of DUSP8 (brown) co-staining with CD45 or pan-CK (red), in LUAD and healthy donor tissue samples, (n = 3), scale bar 50 µm. Data are presented as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. For all analyses, P-values ≤0.05 were considered statistically significant. *p ≤0.05, **p ≤0.01, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 2 Upregulation of DUSP8 leads to a suppressive phenotype, whereas down-regulation of DUSP8 is tumor-promoting in vitro. Validation of DUSP8 overexpression after transfection of A549 cells with empty vector (EV) and DUSP8 expression vector (OE) was quantified using (A) qRT-PCR (n = 3), (B) immunofluoresence staining, and (C) western blot (n = 3). Phosphorylation of JNK was performed using a western blot of A549-EV and A549-DUSP8 OE cells (n = 3). D Comparison of colony formation between DUSP8 OE cells and EV control cells (n = 3). E Comparison of cellular proliferation between DUSP8 OE cells and EV control cells using BrdU assay (n = 3). F Migratory ability of DUSP8 OE cells assessed using Boyden chamber assay (n = 3). G Apoptosis of DUSP8 OE cells compared to EV control cells (n = 3). H Representative immunofluorescence images of EMT markers expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. I mRNA expression of DUSP8 after siRNA transfection with DUSP8 siRNA and non-targeting siRNA control (siNT) (n = 6). J Representative immunocytochemistry images of DUSP8 after treatment with DUSP8 siRNA compared to a non-targeting control. Cells in panels B and J were labeled using DUSP8 antibody and revealed by Alexa Fluor 488 secondary antibody (green). DNA was stained with DAPI (blue) (scale bars: 50 µm). K Western blotting of A549 cells after treatment with DUSP8 siRNA (n = 3). Functional assessment via L colony formation, M cell proliferation, N migration and O apoptosis of A549 cells after treatment with DUSP8 siRNA. P Representative immunofluorescence images of EMT marker expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. Data are shown as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ***p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Staining, Western Blot, Phospho-proteomics, Comparison, Control, BrdU Staining, Boyden Chamber Assay, Immunocytochemistry, Labeling, Functional Assay, Migration, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 3 Impact of DUSP8 overexpression on cancer progression in vivo. A549-EV and A549-DUSP8 OE cells were injected into the right flank of immunodeficient mice. Tumors were harvested after 40 days. A Measurement of tumor size during tumor progression (n = 5). B Measurement of tumor mass after 40 days (n = 5). C Representative photographs of dissected DUSP8 overexpressing tumors, scale bar 5 mm. Validation of DUSP8 expression in mice tumor tissue samples via D mRNA (n = 5), E protein using ELISA (n = 5), and F western blot and G quantification of band intensity (n = 3). H Representative photomicrographs of TUNEL staining for apoptotic cells within the tumor counted per high power field (HPF) using Fiji Software (n = 5, 4 images per animal). I Representative photomicrographs of Ki67 staining of proliferating cells within the tumor counted per HPF using Fiji Software (n = 5, 5 images per animal). J Representative photomicrographs of vascular marker von Willebrand factor (vWF) quantified per HPF using Fiji (n = 5, 5 images per animal). Representative photomicrographs of EMT marker (K) CK18 and (L) VIM quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5, 3 images per animal). Ki67, vWF, CK18, and VIM staining were visualized using Alexa Flour 488 coupled secondary antibody (green). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (A) and two-tailed unpaired t-test with Welch’s correction (B–L). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01 and **p ≤0.001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, TUNEL Assay, Staining, Software, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 4 High expression of miR-147b promotes tumor progression in vitro and is correlated with low OS in lung cancer. A miR-147b - DUSP8 binding site predicted via RNA22. B Scatter plots of DUSP8 expression correlated with miR-147b expression in LUAD samples from the TCGA dataset (n = 34). The r-value and two-tailed, p-value were calculated using Pearson’s rank correlation coefficients. C In silico analysis of miR-147b expression in LUAD (n = 458) vs. non-tumor tissue (n = 46) from the same TCGA cohort. D Correlation between miR-147b expression and OS in patients with LUAD using Kaplan–Meier plotter. E Overexpression of miR-147b by transduced A549 cells compared to miR-SCR control (n = 3). F Luciferase reporter assay of 3’UTR DUSP8 transfected A549-miR-SCR vs. A549-miR-147b overexpressing cells (n = 3). G DUSP8 mRNA expression of A549-miR-147b OE vs. A549-miR-SCR transduced cells (n = 3). H Immunofluorescence staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. I Western blot analysis of DUSP8 compared to loading control ACTB (n = 3). J Volcano Plot depicting DUSP8 as top downregulated gene upon miR-147b overexpression using NanoString. Assessment of K colony formation, L proliferation, M migration, and (N) apoptosis of A549-miR- 147b overexpressing cells compared to miR-SCR transduced cells (n = 3). Representative photomicrographs O of CK18 and P VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, In Vitro, Binding Assay, Two Tailed Test, In Silico, Over Expression, Control, Luciferase, Reporter Assay, Transfection, Staining, Western Blot, Migration

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 5 Silencing miR-147b abrogates the oncogenic potential. Validation of miR-147b and DUSP8 after treatment of A549-miR-147b OE cells with miR-non-targeting control versus miR-147b inhibitor. A, B mRNA expression of miR-147b and DUSP8 (n = 6), C Immunocytochemistry staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. D Western blot of DUSP8 compared to loading control ACTB (n = 3). Inhibition of miR-147b in functional assays in vitro performed via E apoptosis, F proliferation, G colony formation and H migration (n = 3). Representative photomicrographs I of CK18 and J VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two-tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Biomarker Discovery, Control, Expressing, Immunocytochemistry, Staining, Western Blot, Inhibition, Functional Assay, In Vitro, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 6 High expression of DUSP8 in A549-miR-147b overexpressing cells rescues the tumor-promoting phenotype. A miR-147b expression in A549-miR-147b OE cells transfected with a DUSP8 overexpressing plasmid performed by qPCR (n = 3). Validation of DUSP8 in A549-miR- 147b-DUSP8 overexpressing cells at B mRNA level (n = 3) and C protein level, shown by immunocytochemistry staining of DUSP8 (green), counterstained with DAPI (blue), was quantified by calculating the mean fluorescence intensity (MFI) with Fiji (n = 5), scale bar 50 µm. D Western blot analysis of DUSP8 (n = 3). Quantification of E proliferation, F colony formation, G migration and H apoptosis of A549-miR-147b- DUSP8 overexpressing cells compared to A549-miR-147b transduced cells (n = 3). Representative photomicrographs of I CK18 and J VIM antibody staining were visualized with a secondary antibody (green) coupled to Alexa Flour 488. Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Quantification of the mean fluorescence intensity of CK18 and VIM (n = 5). P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Biomarker Discovery, Immunocytochemistry, Staining, Western Blot, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 7 miR-147b overexpression leads to increased tumor burden in vivo. A Subcutaneous injection of A549-miR-SCR and A549-miR-147b cells into the right flank of immunodeficient NSG mice. Tumors were harvested after 40 days. A Representative macroscopic pictures of subcutaneous tumors, scale bar 5 mm. B Measurement of tumor size during tumor progression (n = 5) and C tumor mass after 40 days. Validation of D miR-147b and E DUSP8 mRNA expression in mice tumor samples (n = 5). F DUSP8 level measured by ELISA (n = 5). G Western blot analysis of DUSP8 in miR-147b OE tumors compared to miR-SCR tumors (n = 3). H Intravenous injection of A549-miR-SCR and A549-miR- 147b cells into the tail vein of immunodeficient NSG mice. Representative images of micro-CT scans, extracted lung images, H&E-stained sections, and immunofluorescence staining for Ki67 and vWF (green) and DAPI (blue) in tumor sections. Scale bars, 50 μm. I Quantification of average lung intensity (n = 5). Quantification of proliferating J Ki67+cells (green) (n = 5, 5 images per animal) and K vWF (n = 5, 5 images per animal) counterstained with DAPI (blue) within the tumor counted per high power field (HPF) using Fiji Software. Validation of L miR-147b and M DUSP8 mRNA expression in mice lung tumor samples (n = 5). N DUSP8 level measured by ELISA (n = 5). O Western blot analysis of DUSP8 expression (n = 3). P Schematic representation of miR-147b-mediated tumor progression via suppression of DUSP8. Suppression of DUSP8 by miR-147b leads to inhibition of JNK de-phosphorylation resulting in activation of JNK signaling. Overexpression of miR-147b alters the phosphorylation of MAPKs and leads to increased proliferation and migration. The opposite effects were observed by silencing miR-147b expression and thereby restoring DUSP8 function with cancer-suppressive properties, leading to increased apoptosis. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (B) and two-tailed unpaired t-test with Welch’s correction (C–O). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Software, Inhibition, De-Phosphorylation Assay, Activation Assay, Phospho-proteomics, Migration, Two Tailed Test

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: Kinetic expression of MAPK activation and DUSPs during EBV immortalization. Human CD19+ B lymphocytes were purified from the buffy coat of a healthy blood donor. Cells were seeded in a 12-well plate at a density of 1 × 106 cells per well and infected with EBV. At 28 days postinfection, LCLs were established. RNA and proteins were harvested at the time points indicated. (A) Protein expression of MAPK activation, including phosphorylation of ERK (p-ERK), p38 (p-p38), and JNK (p-JNK), during EBV immortalization was detected by Western blotting. Total expression of MAPK (t-ERK, t-p38, and t-JNK) also is shown. β-Actin was the internal control. (B to E) Cells were also harvested for RT-Q-PCR for expression of DUSPs. The relative expression levels of DUSPs were compared to those of uninfected B lymphocytes after normalization with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression. The DUSPs analyzed included (B) typical type I DUSPs (DUSP1, DUSP2, DUSP4, and DUSP5), (C) typical type II DUSPs (DUSP6, DUSP7, and DUSP9), (D) typical type III DUSPs (DUSP8, DUSP10, and DUSP16), and (E) atypical DUSPs (DUSP14, DUSP19, and DUSP22).

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Expressing, Activation Assay, Purification, Infection, Phospho-proteomics, Western Blot, Control

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: DUSP6 and DUSP8 depletion caused by EBV infection. (A) Cells were infected with B95.8 strain EBV for 28 days to establish LCLs. Cell lysates of infected and uninfected B cells were analyzed for DUSP6, DUSP8, and LMP1 expression by Western blotting. β-Actin was the internal control. (B) B lymphocytes were infected with strain B95.8 EBV and harvested on the day postinfection indicated. Immunoblots of DUSP6, DUSP8, EBNA1, and LMP1 are shown. β-Actin served as an internal control. (C and D) B cells were infected with EBV or treated with anti-CD40/IL-4 for 3 days. Total RNA was extracted each day and subjected to RT-Q-PCR to detect transcripts of (C) DUSP6 and (D) DUSP8. The relative expression levels were compared to those of untreated B lymphocytes after normalization with GAPDH expression.

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Infection, Expressing, Western Blot, Control

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: DUSP6 and DUSP8 expression reduction caused by EBV LMP1. (A) EBV-negative Akata and BJAB lymphoma cells were infected with vector control lentivirus (pSIN) or lentiviruses that overexpressed LMP1, LMP2A, or Zta. DUSP6, DUSP8, LMP1, LMP2A, and Zta transcripts were analyzed by RT-PCR. β-Actin was detected as an internal control. (B) BJAB cells were electroporated with EBNA1, EBNA2, or EBNA3C. DUSP6, DUSP8, EBNA1, EBNA2, and EBNA3C transcripts were analyzed by RT-PCR. β-Actin was detected as an internal control. (C) BJAB cells were infected with LMP1 lentiviruses with increasing multiplicities of infection. Protein expression of DUSP6, DUSP8, and LMP1 was detected by Western blotting. β-Actin served as an internal control. (D) LCLs were infected with lentivirus containing shRNA targeting luciferase or LMP1. Data represent protein expression of DUSP6, DUSP8, and LMP1. β-Actin was detected as an internal control.

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Expressing, Infection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, shRNA, Luciferase

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: Apoptosis caused by overexpression of DUSP6 and DUSP8. (A and B) LCLs were overexpressed with wild-type DUSP6 (DUSP6 WT), wild-type DUSP8 (DUSP8 WT) or their catalytic mutants (DUSP6 CS and DUSP8 CS). Selected cells underwent PI staining, and cell cycle patterns were analyzed with flow cytometry. Cells were selected with G418, and their lysates were analyzed by Western blotting. (C) Protein expression of DUSP6, phosphorylated ERK (p-ERK), total ERK (t-ERK), caspase-3, cleaved caspase-3, PARP-1, and cleaved PARP-1 was detected by immunoblotting. β-Actin served as an internal control. (D) Protein expression of DUSP8, phosphorylated p38 (p-p38), total p38 (t-p38), phosphorylated JNK (p-JNK), and total JNK (t-JNK) was detected. β-Actin served as an internal control. (E) Protein expression of DUSP8, phosphorylated p38 (p-p38), total p38 (t-p38), and PUMA was detected by Western blotting. β-Actin served as the internal control. DUSP6 and DUSP8 were transduced into LCLs through lentiviral infection, and the cells were selected with G418 for 5 days. (A and B) Selected cells underwent PI staining, and cell cycle patterns were analyzed with flow cytometry. (C to E) LCLs were overexpressed with wild-type DUSP6 (DUSP6 WT) or wild-type DUSP8 (DUSP8 WT) or with their catalytic mutants (DUSP6 CS and DUSP8 CS). Cells were selected with G418, and their lysates were analyzed by Western blotting. (C) Protein expression of DUSP6, phosphorylated ERK (p-ERK), total ERK (t-ERK), caspase-3, cleaved caspase-3, PARP-1, and cleaved PARP-1 was detected by immunoblotting. β-Actin served as an internal control. (D) Protein expression of WT DUSP8, phosphorylated ERK (p-ERK), total ERK (t-ERK), phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated p38 (p-p38), and total p38 (t-p38) was detected. β-Actin served as an internal control. (E) Protein expression of wild-type DUSP8, DUSP8 CS, phosphorylated p38 (p-p38), total p38 (t-p38), and PUMA was detected by Western blotting. β-Actin served as the internal control.

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Over Expression, Staining, Flow Cytometry, Western Blot, Expressing, Control, Infection

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: Signaling pathways involved in LMP1-mediated DUSP6 and DUSP8 suppression. (A) LCLs were treated with dimethyl sulfoxide (DMSO), 20 μM U0126 (ERK inhibitor), 20 μM SB212090 (p38 inhibitor), 25 μM SP600125 (JNK inhibitor), 20 μM LY294002 (Akt inhibitor), or 10 μM Bay11-7082 (NF-κB inhibitor) for 2 days. Detection of DUSP6, DUSP8, phosphorylated ERK (p-ERK), phosphorylated p38 (p-p38), phosphorylated JNK (p-JNK), phosphorylated Akt (p-Akt), phosphorylated IκB (p-IκB), total ERK (t-ERK), total p38 (t-p38), total JNK (t-JNK), total Akt (t-Akt), total IκB (t-IκB), and LMP1 was carried out by Western blotting. β-Actin served as an internal control. The fold relative expression data were calculated by comparing the levels of β-actin-normalized DUSP expression of the inhibitor-treated LCLs with those of the untreated LCLs. (B) BJAB cells were transduced with vector control (pSIN) or LMP1 via lentiviral infection and treated with DMSO, 20 μM U0126, or 20 μM SB212090 for 2 days. Detection of DUSP6, DUSP8, p-ERK, p-p38, t-ERK, t-p38, and LMP1 was carried out by Western blotting. The related level of expression of DUSP in vector control-transfected, untreated cells was set as a value of 1 to calculate the fold relative expression levels of DUSP6 and DUSP8.

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Protein-Protein interactions, Western Blot, Control, Expressing, Transduction, Plasmid Preparation, Infection, Transfection

Journal: Journal of Virology

Article Title: Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival

doi: 10.1128/JVI.01837-19

Figure Lengend Snippet: Overexpression of DUSP6 and DUSP8 impaired the proliferation of LCLs. DUSP6 and DUSP8 were transduced into LCLs through lentiviral infection, and the cells were selected with G418 for 5 days. (A and B) Primary B cells or selected cells were analyzed by Western blotting to detect the indicated protein expression levels. (C and D) Selected cells were reseeded in 96-well plates at a density of 1 × 106 cells per ml. Photographs were taken under a bright-field microscope after 1 day of incubation. (E and F) Selected cells were reseeded in 96-well plates at a density of 1 × 105 cells per ml and incubated for 5 days. Proliferation rates of LCLs were determined by serial measurement of viable cell number every day via alamarBlue assay. (*, P＜0.05; **, P＜0.01 [Student's t test].)

Article Snippet: The antibodies used in this study were as follows: LMP1 (S12 or CS1 to CS4) and DUSP6 (Ab54940; Abcam), DUSP8 (sc-271250; Santa Cruz Biotechnology), phospho-ERK Thr202/Tyr204 (E-4; Santa Cruz Biotechnology), ERK (K-23; Santa Cruz Biotechnology), phospho-Akt Ser473 (9271; Cell Signaling Technology), Akt (H136; Santa Cruz Biotechnology), phospho-IκB Ser32/36 (9246; Cell Signaling Technology), IκB (sc-203; Santa Cruz Biotechnology), phospho-p38 Thr180/Tyr182 (9211; Cell Signaling Technology), p38 (9212; Cell Signaling Technology), caspase 3 (9662; Cell Signaling Technology), PARP-1 (sc-8007; Santa Cruz Biotechnology), phospho-JNK Thr183/Tyr185 (9251; Cell Signaling Technology), total JNK (06-748; Upstate), PUMA (4976; Cell Signaling Technology), and β-actin (AC-15; Sigma-Aldrich).

Techniques: Over Expression, Infection, Western Blot, Expressing, Microscopy, Incubation, Alamar Blue Assay

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 1 Downregulation of DUSP8 in LUAD correlated with poor OS. A mRNA expression analysis of DUSP8, DUSP10, and DUSP16 in samples obtained from patients with LUAD (n = 513) compared to non-tumor tissues (NTs) (n = 287) from the GTEx–TCGA patient cohort. B The clinical outcome associated with DUSP8, DUSP10, and DUSP16 expression in LUAD using KM Plotter [36]. C Expression of MAPK8 and MAPK9 in LUAD (n = 513) vs. NT (n = 287) respectively. D Correlation between OS of patients with LUAD and expressions of MAPK8 and MAPK9. E Protein interaction network using STRING. The edges indicate both functional and physical protein associations, line thickness indicates the strength of data support. F Correlation between OS of female and male patients in all stages of LUAD and DUSP8 expression. G DUSP8 protein level measured using ELISA (n = 5). H Western blot analysis of DUSP8 expression in NTs and tumor samples from the same patient (n = 3). I Representative images of DUSP8 (brown) co-staining with CD45 or pan-CK (red), in LUAD and healthy donor tissue samples, (n = 3), scale bar 50 µm. Data are presented as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. For all analyses, P-values ≤0.05 were considered statistically significant. *p ≤0.05, **p ≤0.01, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 2 Upregulation of DUSP8 leads to a suppressive phenotype, whereas down-regulation of DUSP8 is tumor-promoting in vitro. Validation of DUSP8 overexpression after transfection of A549 cells with empty vector (EV) and DUSP8 expression vector (OE) was quantified using (A) qRT-PCR (n = 3), (B) immunofluoresence staining, and (C) western blot (n = 3). Phosphorylation of JNK was performed using a western blot of A549-EV and A549-DUSP8 OE cells (n = 3). D Comparison of colony formation between DUSP8 OE cells and EV control cells (n = 3). E Comparison of cellular proliferation between DUSP8 OE cells and EV control cells using BrdU assay (n = 3). F Migratory ability of DUSP8 OE cells assessed using Boyden chamber assay (n = 3). G Apoptosis of DUSP8 OE cells compared to EV control cells (n = 3). H Representative immunofluorescence images of EMT markers expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. I mRNA expression of DUSP8 after siRNA transfection with DUSP8 siRNA and non-targeting siRNA control (siNT) (n = 6). J Representative immunocytochemistry images of DUSP8 after treatment with DUSP8 siRNA compared to a non-targeting control. Cells in panels B and J were labeled using DUSP8 antibody and revealed by Alexa Fluor 488 secondary antibody (green). DNA was stained with DAPI (blue) (scale bars: 50 µm). K Western blotting of A549 cells after treatment with DUSP8 siRNA (n = 3). Functional assessment via L colony formation, M cell proliferation, N migration and O apoptosis of A549 cells after treatment with DUSP8 siRNA. P Representative immunofluorescence images of EMT marker expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. Data are shown as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ***p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: In Vitro, Biomarker Discovery, Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Staining, Western Blot, Phospho-proteomics, Comparison, Control, BrdU Staining, Boyden Chamber Assay, Immunocytochemistry, Labeling, Functional Assay, Migration, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 3 Impact of DUSP8 overexpression on cancer progression in vivo. A549-EV and A549-DUSP8 OE cells were injected into the right flank of immunodeficient mice. Tumors were harvested after 40 days. A Measurement of tumor size during tumor progression (n = 5). B Measurement of tumor mass after 40 days (n = 5). C Representative photographs of dissected DUSP8 overexpressing tumors, scale bar 5 mm. Validation of DUSP8 expression in mice tumor tissue samples via D mRNA (n = 5), E protein using ELISA (n = 5), and F western blot and G quantification of band intensity (n = 3). H Representative photomicrographs of TUNEL staining for apoptotic cells within the tumor counted per high power field (HPF) using Fiji Software (n = 5, 4 images per animal). I Representative photomicrographs of Ki67 staining of proliferating cells within the tumor counted per HPF using Fiji Software (n = 5, 5 images per animal). J Representative photomicrographs of vascular marker von Willebrand factor (vWF) quantified per HPF using Fiji (n = 5, 5 images per animal). Representative photomicrographs of EMT marker (K) CK18 and (L) VIM quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5, 3 images per animal). Ki67, vWF, CK18, and VIM staining were visualized using Alexa Flour 488 coupled secondary antibody (green). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (A) and two-tailed unpaired t-test with Welch’s correction (B–L). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01 and **p ≤0.001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, TUNEL Assay, Staining, Software, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 4 High expression of miR-147b promotes tumor progression in vitro and is correlated with low OS in lung cancer. A miR-147b - DUSP8 binding site predicted via RNA22. B Scatter plots of DUSP8 expression correlated with miR-147b expression in LUAD samples from the TCGA dataset (n = 34). The r-value and two-tailed, p-value were calculated using Pearson’s rank correlation coefficients. C In silico analysis of miR-147b expression in LUAD (n = 458) vs. non-tumor tissue (n = 46) from the same TCGA cohort. D Correlation between miR-147b expression and OS in patients with LUAD using Kaplan–Meier plotter. E Overexpression of miR-147b by transduced A549 cells compared to miR-SCR control (n = 3). F Luciferase reporter assay of 3’UTR DUSP8 transfected A549-miR-SCR vs. A549-miR-147b overexpressing cells (n = 3). G DUSP8 mRNA expression of A549-miR-147b OE vs. A549-miR-SCR transduced cells (n = 3). H Immunofluorescence staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. I Western blot analysis of DUSP8 compared to loading control ACTB (n = 3). J Volcano Plot depicting DUSP8 as top downregulated gene upon miR-147b overexpression using NanoString. Assessment of K colony formation, L proliferation, M migration, and (N) apoptosis of A549-miR- 147b overexpressing cells compared to miR-SCR transduced cells (n = 3). Representative photomicrographs O of CK18 and P VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Binding Assay, Two Tailed Test, In Silico, Over Expression, Control, Luciferase, Reporter Assay, Transfection, Staining, Western Blot, Migration

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 5 Silencing miR-147b abrogates the oncogenic potential. Validation of miR-147b and DUSP8 after treatment of A549-miR-147b OE cells with miR-non-targeting control versus miR-147b inhibitor. A, B mRNA expression of miR-147b and DUSP8 (n = 6), C Immunocytochemistry staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. D Western blot of DUSP8 compared to loading control ACTB (n = 3). Inhibition of miR-147b in functional assays in vitro performed via E apoptosis, F proliferation, G colony formation and H migration (n = 3). Representative photomicrographs I of CK18 and J VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two-tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Control, Expressing, Immunocytochemistry, Staining, Western Blot, Inhibition, Functional Assay, In Vitro, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 6 High expression of DUSP8 in A549-miR-147b overexpressing cells rescues the tumor-promoting phenotype. A miR-147b expression in A549-miR-147b OE cells transfected with a DUSP8 overexpressing plasmid performed by qPCR (n = 3). Validation of DUSP8 in A549-miR- 147b-DUSP8 overexpressing cells at B mRNA level (n = 3) and C protein level, shown by immunocytochemistry staining of DUSP8 (green), counterstained with DAPI (blue), was quantified by calculating the mean fluorescence intensity (MFI) with Fiji (n = 5), scale bar 50 µm. D Western blot analysis of DUSP8 (n = 3). Quantification of E proliferation, F colony formation, G migration and H apoptosis of A549-miR-147b- DUSP8 overexpressing cells compared to A549-miR-147b transduced cells (n = 3). Representative photomicrographs of I CK18 and J VIM antibody staining were visualized with a secondary antibody (green) coupled to Alexa Flour 488. Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Quantification of the mean fluorescence intensity of CK18 and VIM (n = 5). P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Plasmid Preparation, Biomarker Discovery, Immunocytochemistry, Staining, Western Blot, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 7 miR-147b overexpression leads to increased tumor burden in vivo. A Subcutaneous injection of A549-miR-SCR and A549-miR-147b cells into the right flank of immunodeficient NSG mice. Tumors were harvested after 40 days. A Representative macroscopic pictures of subcutaneous tumors, scale bar 5 mm. B Measurement of tumor size during tumor progression (n = 5) and C tumor mass after 40 days. Validation of D miR-147b and E DUSP8 mRNA expression in mice tumor samples (n = 5). F DUSP8 level measured by ELISA (n = 5). G Western blot analysis of DUSP8 in miR-147b OE tumors compared to miR-SCR tumors (n = 3). H Intravenous injection of A549-miR-SCR and A549-miR- 147b cells into the tail vein of immunodeficient NSG mice. Representative images of micro-CT scans, extracted lung images, H&E-stained sections, and immunofluorescence staining for Ki67 and vWF (green) and DAPI (blue) in tumor sections. Scale bars, 50 μm. I Quantification of average lung intensity (n = 5). Quantification of proliferating J Ki67+cells (green) (n = 5, 5 images per animal) and K vWF (n = 5, 5 images per animal) counterstained with DAPI (blue) within the tumor counted per high power field (HPF) using Fiji Software. Validation of L miR-147b and M DUSP8 mRNA expression in mice lung tumor samples (n = 5). N DUSP8 level measured by ELISA (n = 5). O Western blot analysis of DUSP8 expression (n = 3). P Schematic representation of miR-147b-mediated tumor progression via suppression of DUSP8. Suppression of DUSP8 by miR-147b leads to inhibition of JNK de-phosphorylation resulting in activation of JNK signaling. Overexpression of miR-147b alters the phosphorylation of MAPKs and leads to increased proliferation and migration. The opposite effects were observed by silencing miR-147b expression and thereby restoring DUSP8 function with cancer-suppressive properties, leading to increased apoptosis. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (B) and two-tailed unpaired t-test with Welch’s correction (C–O). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Software, Inhibition, De-Phosphorylation Assay, Activation Assay, Phospho-proteomics, Migration, Two Tailed Test