Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet: Acute treatment of H 2 O 2 induces changes in redox state of Prdx1 and Prdx2 and increased nuclear localization of redox sensitive transcription factors . Effects of different concentrations of H 2 O 2 on monomer (M)/dimer (D) ratio of Prdx1 (a) and Prdx2 (b) and Prdx SO 2 /SO 3 (c) in C2C12 myoblasts. Myoblasts were treated for 10 min with 25 μM H 2 O 2 , media was changed and % nuclear localization of transcription factors NRF2 (d), NF-κB (e), FOXO3a (f) and STAT3 (g) was analysed after 3 and 24 h; scale bar = 75 μm, n = 3–5. Protein extracts and Western blotting was performed from either Ctrl or 3 h and 24 h following H 2 O 2 treatment against PRDX1 (h), PRDX2 (i), PRDX3 (j), SOD2 (k), TRX1 (l), TRX2 (m), ULK1 (n) LC3b (o) and p62/SQSTM1 (p). Graphs are the normalized relative means ± SEM and all experiments were performed with n = 3 and p- value of <0.05 was considered as statistically significant *( p < 0.05), one-way ANOVA was used for significance between groups (a–p). p values (a: Ctrl vs 25 μM = 0.0386; b: Ctrl vs 25 μM < 0.0001; c: Ctrl vs 50 μM = 0.0277; d: Ctrl vs 3 h = 0.0066, Ctrl vs 24h = 0.0074; e: Ctrl vs 24 h < 0.0001; f: Ctrl vs 24 h < 0.0001; g: Ctrl vs 24 h < 0.0001; h: Ctrl vs 24 h = 0.0490; i: Ctrl vs 24 h = 0.0468; j: Ctrl vs 24 h = 0.0270; k: Ctrl vs 24 h = 0.0067; l: Ctrl vs 24 h = 0.0207; m: 3 h vs 24 h = 0.0355; n: Ctrl vs 24 h = 0.0128; o: Ctrl vs 3 h = 0.0091; p: Ctrl vs 24 h = 0.0061).

Article Snippet: rabbit anti-Peroxiredoxin 2 , Cell Signalling Technology , Cat# 46,855, RRID: AB_2799310.

Techniques: Western Blot

Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet: Hormesis effect of H 2 O 2 improves mitochondrial capacity of myoblasts resulting in enhanced myogenic differentiation . Myoblasts treated for 10 min with 25 μM H 2 O 2 were analysed for mitochondrial content using MitoTracker green and mitochondrial ROS using MitoSOX, n = 4, one-way ANOVA used for significance between groups (a). Seahorse analysis of oxygen consumption of cells 24 h following 10 min treatment with H 2 O 2 , n = 3 and a Student t -test used for analysis (b). Western blot analysis of proteins related to mitochondrial content TOM20 (c) and PGC1α (d) and TFAM (e) along with STAT3 abundance (f), n = 3–6 one-way ANOVA used for significance between groups significance *p < 0.05. MF 20 (antimyosin heavy chain; green) and DAPI (blue) immunostaining were performed for myogenic differentiation and nuclei identification, n = 4 one-way ANOVA used for analysis, scale bar = 75 μm. (g). Protein extracts for Western blots of proliferating and differentiating myoblasts at different time points, growth media (GM), differentiation media (DM) Western blots of PRDX1 and PRDX2 expression at different time points, two-way ANOVA analysis was used for significance between groups (h,i). Graphs are the mean ± SEM and all experiments were performed n = 3–4 and p- value of <0.05 was considered as statistically significant *( p < 0.05). p values (a: MitoTracker fluorescence intensity Ctrl vs 24h = 0.0417, MitoSOX fluorescence intensity Ctrl vs 24h = 0.4744; b: Basal respiration: Ctrl vs H 2 O2 = 0.0105, Maximal respiration: Ctrl vs H 2 O 2 = 0.0484, Spare respiration capacity: Ctrl vs H 2 O 2 = 0.0810; c: Ctrl vs 24h = 0.0010; d: Ctrl vs 24h = 0.0015; e: Ctrl vs 24h < 0.0001; f: Ctrl vs 24h = 0.0468; g: Myotube diameter: Ctrl vs 24h = 0.0004, Myotube area: Ctrl vs 24h = 0.0069, Fusion index: Ctrl vs 24h = 0.0273; h: Ctrl: PM1 vs Ctrl: D2 = 0.0098, Ctrl: PM2 vs H 2 O 2 : PM2 = 0.0019; i: Ctrl: PM1 vs Ctrl: D2 < 0.0001, Ctrl: PM2 vs H 2 O 2 : PM2 = 0.0127, H 2 O 2 : PM1 vs H 2 O 2 : PM2 < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: rabbit anti-Peroxiredoxin 2 , Cell Signalling Technology , Cat# 46,855, RRID: AB_2799310.

Techniques: Western Blot, Immunostaining, Expressing, Fluorescence

Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet: Knockdown of Prdx1 and/or Prdx2 inhibits hormesis effect of H 2 O 2 and increases oxidative stress . Western blot confirming decreased expression PRDX1 and PRDX2 in C2C12 by siPrdx1 and/or siPrdx2 and 24 h following 10 min 25 μM H 2 O 2 treatment, n = 3 (a,b). Increased expression of STAT3, Protein DJ-1, TFAM and BNIP3 24 h following H 2 O 2 treatment that was not detected with knockdown of Prdx1 and/or Prdx2 ( c –f), n = 3. MitoTracker green revealed increase in mitochondrial staining in H 2 O 2 treated cells but not with knockdown of siPrdx1 and/or Prdx2, increase in MitoSOX staining following H 2 O 2 treatment with siPrdx1+Prdx2 (g) n = 3, scale bar = 75 μm. MF 20 immunostaining for myogenic differentiation revealed PRDX1 and PRDX2 not required for differentiation in non H 2 O 2 treated cells but are required for hormesis effect and silencing results in disrupted myogenesis, scale bar = 75 μm (h). Graphs are the mean ± SEM and all experiments were performed with n = 3 and Two-way ANOVA analysis was used between groups and p- value of <0.05 was considered as statistically significant *( p < 0.05) (a–h). p values (a: Ctrl: Ctrl vs Ctrl: siPrdx1 = 0.0176, Ctrl: Ctrl vs Ctrl: siPrdx1 + 2 = 0.0110, H 2 O 2 : Ctrl vs H 2 O 2 : siPrdx1 < 0.0001, H 2 O 2 : Ctrl vs H 2 O 2 : siPrdx1 + 2 < 0.0001; b: Ctrl: Ctrl vs Ctrl: siPrdx2 = 0.0078, Ctrl: Ctrl vs Ctrl: siPrdx1 + 2 = 0.0050, Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0168, H 2 O 2 : Ctrl vs H 2 O 2 : siPrdx2 < 0.0001, H 2 O 2 : Ctrl vs H 2 O 2 : siPrdx1 + 2 < 0.0001; c: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0468; d: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0420; e: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0018; f: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0472; g: MitoTracker fluorescence intensity: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0445, Ctrl: Ctrl vs H 2 O 2 : siPrdx1 + 2 = 0.0083; h: Myotube diameter: Ctrl: Ctrl vs H 2 O 2 : Ctrl = 0.0414, Fusion index: Ctrl: Ctrl vs H 2 O 2 : Ctrl < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: rabbit anti-Peroxiredoxin 2 , Cell Signalling Technology , Cat# 46,855, RRID: AB_2799310.

Techniques: Western Blot, Expressing, Staining, Immunostaining, Fluorescence

Journal: Redox Biology

Article Title: Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

doi: 10.1016/j.redox.2023.102631

Figure Lengend Snippet:

Article Snippet: rabbit anti-Peroxiredoxin 2 , Cell Signalling Technology , Cat# 46,855, RRID: AB_2799310.

Techniques: Recombinant, Protease Inhibitor, SYBR Green Assay, Staining, Software

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) RT-PCR analysis of ZFP36L1 expression in human malignant B cells representing different stages of B cell differentiation. For comparison BLIMP1 expression was also measured. β ACTIN expression levels are shown as a loading control. (B) Comparison of ZFP36L1 protein levels in human Ramos B cells before and after 3 h PMA stimulation and in two myeloma cell lines RPMI-8226 cells and KMS-11. BLIMP1 expression is also shown in RPMI-8226 cells and KMS-11 cells. HSP90 levels are shown as a loading control. ZFP36L1/L2, HSP90 and BLIMP1 proteins were detected by anti-BRF1/2, anti-HSP90 and anti-BLIMP1 antibodies respectively. Anti-BRF1/2 antibody cross-reacts with ZFP36L2 (approx. 60 kDa) and ZFP36L1 appears typically as a constellation of induced bands (approx. 40 kDa) in human cells.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Differentiation

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: downregulation of ZFP36L1 is associated with plasmacytoid differentiation of B cells. (A) Western Blot analysis of ZFP36L1 expression in IL-2/5 treated murine leukemic BCL1 cells. Protein lysates were made from unstimulated cells (lane 1) or from cells 96 h after stimulation with cytokines (20 ng/ml IL-2 and 5 ng/ml IL-5) (lane 2). ZFP36L1 and HSP90 proteins were detected by anti-BRF1/2 and anti-HSP 90 antibodies respectively. (B) qRT-PCR analysis of zfp36l1 and blimp1 mRNA expression in day 0 versus 48 h IL-2/5 stimulated BCL1 cells. (C) qRT-PCR analysis of time course of zfp36l1 and blimp1 mRNA expression over 3 days in LPS (10 µg/ml) stimulated murine splenic B cells. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression. The results shown were normalized to β-actin mRNA expression. The results show mean ±SD from one representative experiment.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: IgM production. Levels of IgM production in ZFP36L1 transfected BCL1 stimulated with IL-2/5 (20 ng/ml IL-2 and 5 ng/ml IL-5) over 4 days compared to levels produced by empty vector BCL1 cells cultured under the same conditions. Cells were co-transfected with either pcDNA3.ZFP36L1 or empty pcDNA3 vector and pcDNA3.EGFP and then sorted on the basis of EGFP expression before been set up in culture in the absence or presence of cytokines. Cells were cultured at 4.0×10 4 /ml and ELISA measurements were made in duplicate. The results shown are representative of 3 similar experiments.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Transfection, Produced, Plasmid Preparation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) qRT-PCR analysis of zfp36l1 mRNA levels in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 lentivirus infected cells compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. The results represent mean ±SD (n = 3) of zfp36l1 mRNA levels in three independent cells lines generated by three independent rounds of lentiviral infection for each cell type (apart from wild-type). * = p<0.05 as determined by t-test. (B) Western blot analysis of ZFP36L1 protein expression in wild-type, empty vector, scramble, pSicoR.scramble.RNAi1and pSicoR.scramble.RNAi2 cells. HSP90 levels are shown as a loading control.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Generated, Western Blot, Expressing

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: Cell numbers in the absence (A) and presence (B) of cytokines (IL-2 and IL-5) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells numbers were counted in quadruplicate 4 days after seeding 2×10 5 /ml cells on day 0 in flasks in the absence or presence of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml). Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. IgM secretion (ng/ml) measured by ELISA, 4 days after the start of the culture, in the absence (C) and presence (D) of cytokines (IL-2 20 ng/ml and IL-5 5 ng/ml) in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. ** = p<0.01, * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Plasmid Preparation, Infection, Generated, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Heat map expression profile of known ZFP36L1 target genes in terminal B cell differentiation. The profiles are compared with the reference genes, ZFP36L1, BLIMP1 and XBP1 (first three rows). GMCSF, VEGFA and IL-3 are validated ZFP36L1 targets; the remaining genes have been validated for ZFP36 . Data was taken from GEO Accession number GSE 6691 . Red indicates high and blue, low expression. Key: CLL: B chronic lymphocytic leukemia, MM: multiple myeloma, WM-BL: Waldenstrom’s macroglobulinemia B cells, WM-PB: Waldenstrom’s macroglobulinemia plasma cells, NBC: normal B cells, PC: normal plasma cells. (B) Graphical representation of the ARACNe network for BLIMP1 and ZFP36L1. Nodes representing the BLIMP1 and ZFP36L1 hubs are shown enlarged. Only first neighbours of these hubs are shown in the module. Nodes representing inferred BLIMP1 targets (significantly up-regulated in normal B cells) are blue; nodes representing inferred ZFP36L1 targets (significantly up-regulated in normal plasma cells) are red. Network graphics were generated as group attribute layout using Cytoscape version 2.6.0. .

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Expressing, Cell Differentiation, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: qRT-PCR analysis of blimp1, xbp1, irf4 and bcl6 mRNA expression in pSicoR.zfp36l1.RNAi1 and pSicoR.zfp36l1.RNAi2 BCL1 cells compared to compared to wild-type, empty vector or pSicoR.scramble.RNAi infected cells. Cells were cultured in medium alone for 48h. Total RNA was extracted from 5×10 6 cells, 1 µg RNA was reverse transcribed and the resulting cDNA was used as template for qRT-PCR assay with mouse gene specific primers for blimp1, xbp1, irf4 and bcl6. The 2 –ΔΔCT method of relative quantification was used to determine the fold change in mRNA expression compared to levels in wild-type cells. Mean ±SD (n = 3) are shown for three independent cell lines generated for each of the lentivirus infected cells. * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Infection, Cell Culture, Generated

Journal: PLoS ONE

Article Title: ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA

doi: 10.1371/journal.pone.0052187

Figure Lengend Snippet: (A) Western blot analysis of BLIMP1 levels in control and ZFP36L1 knockdown cells. BLIMP1 expression levels are upregulated in ZFP36L1 knockdown cells compared to controls. βACTIN levels are shown as a loading control. (B) ZFP36L1 mediates degradation of the BLIMP1 3′UTR. HEK 293 T cells were transfected with pMIRBLIMP1 3′UTR construct alone (control) or with either ZFP36L1 or a zinc finger domain mutant, ZFP36L1 Mut. Renilla luciferase was also included in all transfections as a normalization control. 24 hours later cell lysates were harvested and firefly and renilla luciferase levels measured using a Fluorstar Optima plate reader. Relative levels of luciferase activity were measured. Mean ±SD, are shown for four independent experiments (n = 4), * = p<0.05 as determined by t-test.

Article Snippet: Nitrocellulose membranes were incubated with appropriate dilution of the following primary antibodies for 1h; rabbit polyclonal anti-BRF1/2 (ZFP36L1/2) (Cell Signalling, Danvers, MA, USA), goat anti-ZFP36 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BLIMP1 (Cell Signalling).

Techniques: Western Blot, Expressing, Transfection, Construct, Mutagenesis, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Mechanisms of GnRH-Induced Extracellular Signal-Regulated Kinase Nuclear Localization

doi: 10.1371/journal.pone.0040077

Figure Lengend Snippet: (A) HeLa cells were seeded in 12 well plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for varied periods with 100 nM GnRH or 1 µM PDBu. Whole cell protein extracts were then immunoblotted for phospho Ser217/221 MEK1/2 (ppMEK1/2), phospho Thr183/Tyr185 ERK (ppERK) and for total ERK (ERK1 and/or 2) as indicated. (B) HeLa cells were seeded in 96-well imaging plates, transduced with Ad mGnRHR and kept in reduced (0.1%) serum for 16 hours prior to stimulation for 5 min with 1 or 100 nM GnRH as indicated. Control cells (Ctrl.) were treated with medium alone. The cells were then fixed and stained for endogenous ppERK, ERK and DAPI before image acquisition and analysis (as described in the ). The figure shows representative images and the lower images show “segmented” ERK staining, with lines indicating the perimeters of the nuclei and cells obtained using automated image analysis algorithms, as described in the . Each of the image panels corresponds to a width of approximately 250 µm and represents approximately 1/20 th of the area captured in each field of view.

Article Snippet: Ser217/221 phosphorylated MEK1/2 were detected using a rabbit anti-ppMEK1/2 monoclonal antibody (clone 41G9, 1∶1000, Cell Signaling Technology).

Techniques: Transduction, Imaging, Staining