Structured Review

OriGene open reading frame orf
<t>Lpo</t> protein expression detected by Western blotting and activity assay Panel A shows a Western blot of CMT-93 (CMT) cells, a CMT transfectant clone expressing Lpo <t>ORF</t> cDNA, colon of B6 DKO, B6 WT, 129 DKO and 129 WT 22-day-old mice, in this order. The upper panel shows the proteins recognized by anti-LPO antibody, and the lower panel shows β-actin. The open arrow points to ~90 kDa Lpo. Panel B shows the relative levels of Lpo protein in B6 and 129 DKO and WT colons, quantified from Western blots. Arrow bars are SEM from 4 colons in each group. Means that differ are shown with different letter, where a > b. Panel C shows the peroxidase activity in the colon of 10 B6 and five 129 DKO as well as 10 B6 and six 129 non-DKO control (Con) mice with at least 2 WT GPx alleles. The mean of total activity (open column) that differs is shown with different letter, where a > b > c. The mean of resorcinol-resistant activity (shaded column) that differs is shown with different number of asterisk, where *** > ** > *. The mean of NaCl-resistant activity (solid column) that differs is shown with different number of number sign, where ## > #. The error bars are standard deviations of the means.
Open Reading Frame Orf, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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open reading frame orf - by Bioz Stars, 2020-09
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1) Product Images from "Expression of lactoperoxidase in differentiated mouse colon epithelial cells"

Article Title: Expression of lactoperoxidase in differentiated mouse colon epithelial cells

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2012.02.009

Lpo protein expression detected by Western blotting and activity assay Panel A shows a Western blot of CMT-93 (CMT) cells, a CMT transfectant clone expressing Lpo ORF cDNA, colon of B6 DKO, B6 WT, 129 DKO and 129 WT 22-day-old mice, in this order. The upper panel shows the proteins recognized by anti-LPO antibody, and the lower panel shows β-actin. The open arrow points to ~90 kDa Lpo. Panel B shows the relative levels of Lpo protein in B6 and 129 DKO and WT colons, quantified from Western blots. Arrow bars are SEM from 4 colons in each group. Means that differ are shown with different letter, where a > b. Panel C shows the peroxidase activity in the colon of 10 B6 and five 129 DKO as well as 10 B6 and six 129 non-DKO control (Con) mice with at least 2 WT GPx alleles. The mean of total activity (open column) that differs is shown with different letter, where a > b > c. The mean of resorcinol-resistant activity (shaded column) that differs is shown with different number of asterisk, where *** > ** > *. The mean of NaCl-resistant activity (solid column) that differs is shown with different number of number sign, where ## > #. The error bars are standard deviations of the means.
Figure Legend Snippet: Lpo protein expression detected by Western blotting and activity assay Panel A shows a Western blot of CMT-93 (CMT) cells, a CMT transfectant clone expressing Lpo ORF cDNA, colon of B6 DKO, B6 WT, 129 DKO and 129 WT 22-day-old mice, in this order. The upper panel shows the proteins recognized by anti-LPO antibody, and the lower panel shows β-actin. The open arrow points to ~90 kDa Lpo. Panel B shows the relative levels of Lpo protein in B6 and 129 DKO and WT colons, quantified from Western blots. Arrow bars are SEM from 4 colons in each group. Means that differ are shown with different letter, where a > b. Panel C shows the peroxidase activity in the colon of 10 B6 and five 129 DKO as well as 10 B6 and six 129 non-DKO control (Con) mice with at least 2 WT GPx alleles. The mean of total activity (open column) that differs is shown with different letter, where a > b > c. The mean of resorcinol-resistant activity (shaded column) that differs is shown with different number of asterisk, where *** > ** > *. The mean of NaCl-resistant activity (solid column) that differs is shown with different number of number sign, where ## > #. The error bars are standard deviations of the means.

Techniques Used: Expressing, Western Blot, Activity Assay, Transfection, Mouse Assay

Related Articles

Clone Assay:

Article Title: Expression of lactoperoxidase in differentiated mouse colon epithelial cells
Article Snippet: .. Since these cells expressed extremely low levels of endogenous Lpo mRNA, we generated stable Lpo transfectant clones in CMT-93 cells from an open-reading frame (ORF) of Lpo cDNA subcloned from a full-length cDNA in pCMV6-Kan/Neo plasmid (MC201302, Origene Technologies, Inc., Rockville, MD). .. The forward primer contains a Kozak sequence and a Kpn I restriction site: 5′-CTGGATCCGGTACCGAGGAGATCTGCCGCC GCGATCGC GTGATGAAAGTGCTTCTGCAT-3′; the reverse primer has a Hind III site: 5′-GGGCAGA AGCTTCCTACTCCTTCACTGAGGCC-3′.

Generated:

Article Title: Expression of lactoperoxidase in differentiated mouse colon epithelial cells
Article Snippet: .. Since these cells expressed extremely low levels of endogenous Lpo mRNA, we generated stable Lpo transfectant clones in CMT-93 cells from an open-reading frame (ORF) of Lpo cDNA subcloned from a full-length cDNA in pCMV6-Kan/Neo plasmid (MC201302, Origene Technologies, Inc., Rockville, MD). .. The forward primer contains a Kozak sequence and a Kpn I restriction site: 5′-CTGGATCCGGTACCGAGGAGATCTGCCGCC GCGATCGC GTGATGAAAGTGCTTCTGCAT-3′; the reverse primer has a Hind III site: 5′-GGGCAGA AGCTTCCTACTCCTTCACTGAGGCC-3′.

Transfection:

Article Title: Expression of lactoperoxidase in differentiated mouse colon epithelial cells
Article Snippet: .. Since these cells expressed extremely low levels of endogenous Lpo mRNA, we generated stable Lpo transfectant clones in CMT-93 cells from an open-reading frame (ORF) of Lpo cDNA subcloned from a full-length cDNA in pCMV6-Kan/Neo plasmid (MC201302, Origene Technologies, Inc., Rockville, MD). .. The forward primer contains a Kozak sequence and a Kpn I restriction site: 5′-CTGGATCCGGTACCGAGGAGATCTGCCGCC GCGATCGC GTGATGAAAGTGCTTCTGCAT-3′; the reverse primer has a Hind III site: 5′-GGGCAGA AGCTTCCTACTCCTTCACTGAGGCC-3′.

Plasmid Preparation:

Article Title: Expression of lactoperoxidase in differentiated mouse colon epithelial cells
Article Snippet: .. Since these cells expressed extremely low levels of endogenous Lpo mRNA, we generated stable Lpo transfectant clones in CMT-93 cells from an open-reading frame (ORF) of Lpo cDNA subcloned from a full-length cDNA in pCMV6-Kan/Neo plasmid (MC201302, Origene Technologies, Inc., Rockville, MD). .. The forward primer contains a Kozak sequence and a Kpn I restriction site: 5′-CTGGATCCGGTACCGAGGAGATCTGCCGCC GCGATCGC GTGATGAAAGTGCTTCTGCAT-3′; the reverse primer has a Hind III site: 5′-GGGCAGA AGCTTCCTACTCCTTCACTGAGGCC-3′.

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  • 92
    OriGene pcmv tmem100
    <t>Tmem100</t> IHC profile and antibody preabsorption testing. Immunostained Tmem100 expression profile (A) with amplified images (B, B1) show the Tmem100 localization in intracellular (asterisks) and PM-like patterns (arrowheads) in DRG section from naive adult rat, using a goat anti-Tmem100 antibody, which detects a ∼17 KDa band on the lysates from DRGs (C, lanes 3 and 4) and <t>pCMV-Tmem100</t> transfected 293T cells (C, lane 5) but not in the lysate from sham-transfected 293T cells (C, lane 2). Preincubation with antigenic peptide eliminates the band in immunoblot, suggesting specificity of detection (D). GAPDH immunoblotting (bottom panels in panels C and D) was used as the loading control. IHC using this antibody detected Tmem100 in SDH (E), Tmem100-IR signals symmetrical in the region from DREZ to PKCγ-labeled laminae II inner ventral edge (IIiv) (E and F) and concentrated on the CGRP-positive laminae I afferents (G) and IB4-positive laminae II fibers (H). Scale bar: 100 μm for all. CGRP, calcitonin gene-related peptide; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; IHC, immunohistochemistry; PM, plasma membrane; SDH, superficial dorsal horn.
    Pcmv Tmem100, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    OriGene transient transfections human prdm1 tagged orf
    <t>PRDM1-</t> or NonO-deficient MO-DCs induce increased T FH differentiation. Allogenic culture of naïve CD4+ T cells and MO-DCs was set up to induce T FH differentiation. Expression levels of PRDM1 or NONO in MO-DCs were modulated by transfection with siRNAs before co-culture. After 6-days culture, T FH cell differentiation was measured by IL-21, IFNγ, CXCR5, and PD1 expression by flow cytometry. (A) A representative flow image. (B) Dead cells were excluded using Fixable Viability Dye eFluor 506, and the percentage of live CD4 + T cells was calculated. T FH -like cells (CXCR5+/PD1+), CXCR5- helper T cells (CXCR5-/PD1+), and cytokine expressing helper T cells (CXCR5-/PD1+/ IL21+/ IFN-γ-, and CXCR5-/PD1+/IL21+/ IFN-γ+) were calculated and plotted. Co-culture of T cells with LPS pre-stimulated MO-DCs was indicated with gray filled box and culture with unstipulated MO-DCs was indicated with open box. Negative control is naïve CD4+ T cell alone and Positive control is naïve CD4+ T cells with CD2/3/28 activation beads IL-6 (50 ng/ml) and IL-12 (20 ng/ml). In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Four independent experiments ( n = 9). Significance determined by Mann Whitney test. (C) BCL6 expression was quantified by qRT-PCR. Relative expression was calculated to the level of housekeeping gene, POLR2A . In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Three independent experiments ( n = 9). Significance determined by Mann Whitney test.
    Transient Transfections Human Prdm1 Tagged Orf, supplied by OriGene, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    OriGene pd l1 orf
    Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with <t>PD-L1</t> ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status
    Pd L1 Orf, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene mtp18 construct
    Fluorouracil treatment induces mitochondrial fission and apoptosis, and shows a similar trend of changes in <t>MTP18</t> expression as doxorubicin exposure (A) and (B) Fluorouracil (5-FU) induces mitochondrial fission in gastric cancer cells. (C) and (D) 5-FU induces apoptosis in gastric cancer cells. The cells were stimulated with 5-FU (4μmol/L) in AGS cell lines (A) and (C) or (10 μmol/L) in NCI-N87 cell lines (B) and (D) and then harvested at the indicated time for mitochondrial fission and apoptosis analysis. Percentage of apoptosis was analyzed by DNA fragmentation using the cell death detection ELISA. Data were expressed as the mean±SEM of three independent experiments. ** P
    Mtp18 Construct, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tmem100 IHC profile and antibody preabsorption testing. Immunostained Tmem100 expression profile (A) with amplified images (B, B1) show the Tmem100 localization in intracellular (asterisks) and PM-like patterns (arrowheads) in DRG section from naive adult rat, using a goat anti-Tmem100 antibody, which detects a ∼17 KDa band on the lysates from DRGs (C, lanes 3 and 4) and pCMV-Tmem100 transfected 293T cells (C, lane 5) but not in the lysate from sham-transfected 293T cells (C, lane 2). Preincubation with antigenic peptide eliminates the band in immunoblot, suggesting specificity of detection (D). GAPDH immunoblotting (bottom panels in panels C and D) was used as the loading control. IHC using this antibody detected Tmem100 in SDH (E), Tmem100-IR signals symmetrical in the region from DREZ to PKCγ-labeled laminae II inner ventral edge (IIiv) (E and F) and concentrated on the CGRP-positive laminae I afferents (G) and IB4-positive laminae II fibers (H). Scale bar: 100 μm for all. CGRP, calcitonin gene-related peptide; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; IHC, immunohistochemistry; PM, plasma membrane; SDH, superficial dorsal horn.

    Journal: Pain Reports

    Article Title: Transmembrane protein 100 is expressed in neurons and glia of dorsal root ganglia and is reduced after painful nerve injury

    doi: 10.1097/PR9.0000000000000703

    Figure Lengend Snippet: Tmem100 IHC profile and antibody preabsorption testing. Immunostained Tmem100 expression profile (A) with amplified images (B, B1) show the Tmem100 localization in intracellular (asterisks) and PM-like patterns (arrowheads) in DRG section from naive adult rat, using a goat anti-Tmem100 antibody, which detects a ∼17 KDa band on the lysates from DRGs (C, lanes 3 and 4) and pCMV-Tmem100 transfected 293T cells (C, lane 5) but not in the lysate from sham-transfected 293T cells (C, lane 2). Preincubation with antigenic peptide eliminates the band in immunoblot, suggesting specificity of detection (D). GAPDH immunoblotting (bottom panels in panels C and D) was used as the loading control. IHC using this antibody detected Tmem100 in SDH (E), Tmem100-IR signals symmetrical in the region from DREZ to PKCγ-labeled laminae II inner ventral edge (IIiv) (E and F) and concentrated on the CGRP-positive laminae I afferents (G) and IB4-positive laminae II fibers (H). Scale bar: 100 μm for all. CGRP, calcitonin gene-related peptide; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; IHC, immunohistochemistry; PM, plasma membrane; SDH, superficial dorsal horn.

    Article Snippet: Immunoblots The lysates from DRG tissues and HEK293T cells transfected with pCMV-Tmem100 (rat ORF, RN210875; OriGene, Rockville, MD) or pCMV5.0, as well as various cell lines (all from ATCC, Manassas, VA) of F11 (rat DRG neurons), BV2 (mouse microglia), C6 (rat astrocytes), A549 (human non–small-cell lung cancer [NSCLC]), and N2A (mouse brain neurons) were extracted using 1× RIPA buffer (20 mm Tris-HCl pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, with 0.1% Triton X100 and protease inhibitor cocktail).

    Techniques: Immunohistochemistry, Expressing, Amplification, Transfection, Labeling

    PRDM1- or NonO-deficient MO-DCs induce increased T FH differentiation. Allogenic culture of naïve CD4+ T cells and MO-DCs was set up to induce T FH differentiation. Expression levels of PRDM1 or NONO in MO-DCs were modulated by transfection with siRNAs before co-culture. After 6-days culture, T FH cell differentiation was measured by IL-21, IFNγ, CXCR5, and PD1 expression by flow cytometry. (A) A representative flow image. (B) Dead cells were excluded using Fixable Viability Dye eFluor 506, and the percentage of live CD4 + T cells was calculated. T FH -like cells (CXCR5+/PD1+), CXCR5- helper T cells (CXCR5-/PD1+), and cytokine expressing helper T cells (CXCR5-/PD1+/ IL21+/ IFN-γ-, and CXCR5-/PD1+/IL21+/ IFN-γ+) were calculated and plotted. Co-culture of T cells with LPS pre-stimulated MO-DCs was indicated with gray filled box and culture with unstipulated MO-DCs was indicated with open box. Negative control is naïve CD4+ T cell alone and Positive control is naïve CD4+ T cells with CD2/3/28 activation beads IL-6 (50 ng/ml) and IL-12 (20 ng/ml). In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Four independent experiments ( n = 9). Significance determined by Mann Whitney test. (C) BCL6 expression was quantified by qRT-PCR. Relative expression was calculated to the level of housekeeping gene, POLR2A . In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Three independent experiments ( n = 9). Significance determined by Mann Whitney test.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: PRDM1- or NonO-deficient MO-DCs induce increased T FH differentiation. Allogenic culture of naïve CD4+ T cells and MO-DCs was set up to induce T FH differentiation. Expression levels of PRDM1 or NONO in MO-DCs were modulated by transfection with siRNAs before co-culture. After 6-days culture, T FH cell differentiation was measured by IL-21, IFNγ, CXCR5, and PD1 expression by flow cytometry. (A) A representative flow image. (B) Dead cells were excluded using Fixable Viability Dye eFluor 506, and the percentage of live CD4 + T cells was calculated. T FH -like cells (CXCR5+/PD1+), CXCR5- helper T cells (CXCR5-/PD1+), and cytokine expressing helper T cells (CXCR5-/PD1+/ IL21+/ IFN-γ-, and CXCR5-/PD1+/IL21+/ IFN-γ+) were calculated and plotted. Co-culture of T cells with LPS pre-stimulated MO-DCs was indicated with gray filled box and culture with unstipulated MO-DCs was indicated with open box. Negative control is naïve CD4+ T cell alone and Positive control is naïve CD4+ T cells with CD2/3/28 activation beads IL-6 (50 ng/ml) and IL-12 (20 ng/ml). In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Four independent experiments ( n = 9). Significance determined by Mann Whitney test. (C) BCL6 expression was quantified by qRT-PCR. Relative expression was calculated to the level of housekeeping gene, POLR2A . In the Box-and-Whisker plot, horizontal bars indicate the median, boxes indicate 25–75th percentile, and the whiskers indicate 10 and 90th percentile. Three independent experiments ( n = 9). Significance determined by Mann Whitney test.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Expressing, Transfection, Co-Culture Assay, Cell Differentiation, Flow Cytometry, Negative Control, Positive Control, Activation Assay, Whisker Assay, MANN-WHITNEY, Quantitative RT-PCR

    The interaction of NonO and PRDM1 in the nucleus of MO-DCs. Binding of candidate proteins with PRDM1 in primary MO-DCs was measured by PLA. (A) MO-DCs were incubated with normal rabbit IgG (left column) or with anti-PRDM1 antibodies (right column) together with anti-TP53BP1, anti-hnRNPM, or anti-NonO antibodies. Their proximal interaction was assessed by PLA and visualized as red dots. Nucleus was stained with DAPI (blue). Each Z-Stacks maximum intensity projection image is a representative from three independent experiments and captured 60x magnification. Scale bar = 10 μm. (B) Quantification of the PLA signal on MO-DCs is represented. PLA signals in each nucleus were quantified by Imaris software. In the plot, horizontal bars indicate the mean with SEM and each dot represents counts of individual nucleus ( n = 40–50). Significance determined by unpaired t -test.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: The interaction of NonO and PRDM1 in the nucleus of MO-DCs. Binding of candidate proteins with PRDM1 in primary MO-DCs was measured by PLA. (A) MO-DCs were incubated with normal rabbit IgG (left column) or with anti-PRDM1 antibodies (right column) together with anti-TP53BP1, anti-hnRNPM, or anti-NonO antibodies. Their proximal interaction was assessed by PLA and visualized as red dots. Nucleus was stained with DAPI (blue). Each Z-Stacks maximum intensity projection image is a representative from three independent experiments and captured 60x magnification. Scale bar = 10 μm. (B) Quantification of the PLA signal on MO-DCs is represented. PLA signals in each nucleus were quantified by Imaris software. In the plot, horizontal bars indicate the mean with SEM and each dot represents counts of individual nucleus ( n = 40–50). Significance determined by unpaired t -test.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Binding Assay, Proximity Ligation Assay, Incubation, Staining, Software

    NonO-dependent regulation of IL-6 by PRDM1 in MO-DCs. (A) MO-DCs were stimulated with LPS for 6 h and stained with normal rabbit IgG + anti-NonO antibodies (left column) or with anti-PRDM1 antibodies + anti-NonO antibodies (right column). Their proximal interaction was assessed by PLA and visualized as red dots. Nucleus was stained with DAPI (blue). Each Z-Stacks maximum intensity projection image is a representative from three independent experiments and captured 60x magnification. Scale bar = 10 μm. (B) To knock down NonO, PRDM1, and both (NonO and PRDM1) expression, indicated siRNA or control siRNA was transfected into MO-DCs and NonO and PRDM1 expression level were quantified by qRT-PCR. Bar graph is a mean ± SEM ( n = 9). Significance determined by Man-Whitney test. (C) Indicated siRNA or control siRNA transfected MO-DCs were cultured with or without LPS (1 μg/ml) for 6 h, and total RNA was purified. Level of IL6 was measured by qRT-PCR and relative induction was calculated by normalization to the level of LPS stimulated control. Supernatant concentrations of IL-6 obtained from the cultures were measured using enzyme-linked immunosorbent assay (ELISA). Bar graph is a mean ± SEM ( n = 9). Significance determined by Mann Whitney test. (D) Diagram of human IL6 promoter region with indication of putative PRDM1 binding site (black bar #1–#8) and PCR primers (open arrow). Primer set for IL6 promoter cloning is designated as black arrows. ChIP was performed with anti-NonO antibodies or control IgG from MO-DCs. (E) PCR result was visualized in agarose gel. Binding of NonO to PRDM1 consensus sequences within the IL6 promoter were assessed by each primer set (indicated in C ). (F) To quantify the binding of NonO to #5 region, qPCR was performed and calculated by the percent of input. The graph is a mean ± SEM ( n = 5). Significance determined by Mann Whitney test.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: NonO-dependent regulation of IL-6 by PRDM1 in MO-DCs. (A) MO-DCs were stimulated with LPS for 6 h and stained with normal rabbit IgG + anti-NonO antibodies (left column) or with anti-PRDM1 antibodies + anti-NonO antibodies (right column). Their proximal interaction was assessed by PLA and visualized as red dots. Nucleus was stained with DAPI (blue). Each Z-Stacks maximum intensity projection image is a representative from three independent experiments and captured 60x magnification. Scale bar = 10 μm. (B) To knock down NonO, PRDM1, and both (NonO and PRDM1) expression, indicated siRNA or control siRNA was transfected into MO-DCs and NonO and PRDM1 expression level were quantified by qRT-PCR. Bar graph is a mean ± SEM ( n = 9). Significance determined by Man-Whitney test. (C) Indicated siRNA or control siRNA transfected MO-DCs were cultured with or without LPS (1 μg/ml) for 6 h, and total RNA was purified. Level of IL6 was measured by qRT-PCR and relative induction was calculated by normalization to the level of LPS stimulated control. Supernatant concentrations of IL-6 obtained from the cultures were measured using enzyme-linked immunosorbent assay (ELISA). Bar graph is a mean ± SEM ( n = 9). Significance determined by Mann Whitney test. (D) Diagram of human IL6 promoter region with indication of putative PRDM1 binding site (black bar #1–#8) and PCR primers (open arrow). Primer set for IL6 promoter cloning is designated as black arrows. ChIP was performed with anti-NonO antibodies or control IgG from MO-DCs. (E) PCR result was visualized in agarose gel. Binding of NonO to PRDM1 consensus sequences within the IL6 promoter were assessed by each primer set (indicated in C ). (F) To quantify the binding of NonO to #5 region, qPCR was performed and calculated by the percent of input. The graph is a mean ± SEM ( n = 5). Significance determined by Mann Whitney test.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Staining, Proximity Ligation Assay, Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Binding Assay, Polymerase Chain Reaction, Clone Assay, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    NonO regulates IL-6 promoter activity. (A) HEK-293 cells were transfected with NonO siRNA or PRDM1 expressing plasmid. The knockdown efficiency of siRNA and PRDM1 level was verified by immunoblott analysis. β- actin was used as a loading control. Gel image is a representative from two independent experiments. (B) The IL6 promoter activity under indicated conditions was determined by luciferase reporter gene analysis. pGL4.25 were used as control vectors. Mean values of relative luciferase unit (RLU; normalized on Renilla luciferase) from three independent experiments. Bar is a mean ± SEM ( n = 4). Significance determined by unpaired t -test.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: NonO regulates IL-6 promoter activity. (A) HEK-293 cells were transfected with NonO siRNA or PRDM1 expressing plasmid. The knockdown efficiency of siRNA and PRDM1 level was verified by immunoblott analysis. β- actin was used as a loading control. Gel image is a representative from two independent experiments. (B) The IL6 promoter activity under indicated conditions was determined by luciferase reporter gene analysis. pGL4.25 were used as control vectors. Mean values of relative luciferase unit (RLU; normalized on Renilla luciferase) from three independent experiments. Bar is a mean ± SEM ( n = 4). Significance determined by unpaired t -test.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    NonO binds with PRDM1 but does not regulate IL-6 in myeloma cells. (A) Binding between PRDM1 and NonO in human myeloma cells was visualized by PLA (red color) and nuclei were stained with DAPI (blue). Left columns are control groups of PLA (anti-NonO and control IgG) and right columns are experimental groups (anti-NonO and anti-PRDM1). Scale bar = 15 μm. Images were taken at 60x magnification. A representative image from three independent experiments. (B) PLA signals in each nucleus were quantified by Imaris software ( n = 30–40). Significance determined by Mann Whitney test. (C) 48 h NonO or control siRNA post transfection, cells were cultured with or without LPS (1 μg/ml) for last 6 h and level of IL-6 measured by qRT-PCR. Bar graph is a mean ± SEM ( n = 3). Significance determined by unpaired t -test.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: NonO binds with PRDM1 but does not regulate IL-6 in myeloma cells. (A) Binding between PRDM1 and NonO in human myeloma cells was visualized by PLA (red color) and nuclei were stained with DAPI (blue). Left columns are control groups of PLA (anti-NonO and control IgG) and right columns are experimental groups (anti-NonO and anti-PRDM1). Scale bar = 15 μm. Images were taken at 60x magnification. A representative image from three independent experiments. (B) PLA signals in each nucleus were quantified by Imaris software ( n = 30–40). Significance determined by Mann Whitney test. (C) 48 h NonO or control siRNA post transfection, cells were cultured with or without LPS (1 μg/ml) for last 6 h and level of IL-6 measured by qRT-PCR. Bar graph is a mean ± SEM ( n = 3). Significance determined by unpaired t -test.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Binding Assay, Proximity Ligation Assay, Staining, Software, MANN-WHITNEY, Transfection, Cell Culture, Quantitative RT-PCR

    Binding of PRDM1 and candidate proteins in HEK-293 cells. HEK-293 cells were transfected PRDM1 alone or together with Flag-NonO, Flag-TP53BP1 , or V5-hnRNPM expression vector. Binding between PRDM1 and each candidate proteins was detected by Co-IP and PLA. (A) Nuclear fraction was immunoprecipitated with anti- PRDM1 antibodies and immunoblotting was performed with anti-PRDM1, Flag-NonO, Flag-TP53BP1, or V5-hnRNPM antibody. A representative image from two independent experiments is shown. (B) Binding between PRDM1 and candidate proteins was visualized by PLA (red color) and nuclei were stained with DAPI (blue). Top panel; technical negative control PLA (PRDM1 antibody alone), other panels; detection of PLA (PRDM1 with Flag or V5 Ab). Scale bar = 10 μm. A representative image from three independent experiments. Co-IP, co-immunoprecipitation (Co-IP); PLA, proximity ligation assay.

    Journal: Frontiers in Immunology

    Article Title: NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells

    doi: 10.3389/fimmu.2020.01436

    Figure Lengend Snippet: Binding of PRDM1 and candidate proteins in HEK-293 cells. HEK-293 cells were transfected PRDM1 alone or together with Flag-NonO, Flag-TP53BP1 , or V5-hnRNPM expression vector. Binding between PRDM1 and each candidate proteins was detected by Co-IP and PLA. (A) Nuclear fraction was immunoprecipitated with anti- PRDM1 antibodies and immunoblotting was performed with anti-PRDM1, Flag-NonO, Flag-TP53BP1, or V5-hnRNPM antibody. A representative image from two independent experiments is shown. (B) Binding between PRDM1 and candidate proteins was visualized by PLA (red color) and nuclei were stained with DAPI (blue). Top panel; technical negative control PLA (PRDM1 antibody alone), other panels; detection of PLA (PRDM1 with Flag or V5 Ab). Scale bar = 10 μm. A representative image from three independent experiments. Co-IP, co-immunoprecipitation (Co-IP); PLA, proximity ligation assay.

    Article Snippet: Plasmids and Transient Transfections Human PRDM1 Tagged ORF Clone PRDM1 (RC217363L1V) and human small interfering Ribonuclic acid (siRNA) oligo duplex exogenous (SR300437; PRDM1 and SR321120; NonO ) were purchased from Origene.

    Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Immunoprecipitation, Staining, Negative Control

    Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with PD-L1 ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status

    Journal: Molecular Cancer

    Article Title: Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: Significance in claudin-low breast cancer cells

    doi: 10.1186/s12943-015-0421-2

    Figure Lengend Snippet: Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with PD-L1 ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status

    Article Snippet: PD-L1 expression in T-47D was induced by transfecting cells with PD-L1 ORF (pCMV6-AC-GFP vector from Origene) and the selection for PD-L1 ORF transfected cells were made using G418 (500 μg/mL).

    Techniques: Expressing, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Over Expression, Transfection, Clone Assay, Standard Deviation

    Fluorouracil treatment induces mitochondrial fission and apoptosis, and shows a similar trend of changes in MTP18 expression as doxorubicin exposure (A) and (B) Fluorouracil (5-FU) induces mitochondrial fission in gastric cancer cells. (C) and (D) 5-FU induces apoptosis in gastric cancer cells. The cells were stimulated with 5-FU (4μmol/L) in AGS cell lines (A) and (C) or (10 μmol/L) in NCI-N87 cell lines (B) and (D) and then harvested at the indicated time for mitochondrial fission and apoptosis analysis. Percentage of apoptosis was analyzed by DNA fragmentation using the cell death detection ELISA. Data were expressed as the mean±SEM of three independent experiments. ** P

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: Fluorouracil treatment induces mitochondrial fission and apoptosis, and shows a similar trend of changes in MTP18 expression as doxorubicin exposure (A) and (B) Fluorouracil (5-FU) induces mitochondrial fission in gastric cancer cells. (C) and (D) 5-FU induces apoptosis in gastric cancer cells. The cells were stimulated with 5-FU (4μmol/L) in AGS cell lines (A) and (C) or (10 μmol/L) in NCI-N87 cell lines (B) and (D) and then harvested at the indicated time for mitochondrial fission and apoptosis analysis. Percentage of apoptosis was analyzed by DNA fragmentation using the cell death detection ELISA. Data were expressed as the mean±SEM of three independent experiments. ** P

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    MTP18 requires DRP1 to induce mitochondrial fission and apoptosis during doxorubicin exposure (A) Mitochondrial protein 18 (MTP18)-induced mitochondrial fission upon doxorubicin (DOX) exposure is abolished when dynamic-related protein 1 (DRP1) is knocked down. Stable AGS cell line of overexpressed MTP18 was co-transfected with DRP1-siRNA and then treated with 1μmol/L DOX and percentages of cell undergoing mitochondrial fission was analyzed at 6hr. (A) Shows percentage of cells with mitochondrial fission. (B) MTP18-mediated apoptosis upon DOX exposure is abolished when DRP1 is knocked down. Stable AGS cell line of overexpressed MTP18 was co-transfected with DRP1-siRNA and treated with 1μmol/L DOX for apoptosis analysis at 24hr. The percentages of apoptosis were analyzed by cell death detection ELISA. (B) Shows percentages of apoptosis. Data were expressed as the mean±SEM of three independent experiments. * P

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: MTP18 requires DRP1 to induce mitochondrial fission and apoptosis during doxorubicin exposure (A) Mitochondrial protein 18 (MTP18)-induced mitochondrial fission upon doxorubicin (DOX) exposure is abolished when dynamic-related protein 1 (DRP1) is knocked down. Stable AGS cell line of overexpressed MTP18 was co-transfected with DRP1-siRNA and then treated with 1μmol/L DOX and percentages of cell undergoing mitochondrial fission was analyzed at 6hr. (A) Shows percentage of cells with mitochondrial fission. (B) MTP18-mediated apoptosis upon DOX exposure is abolished when DRP1 is knocked down. Stable AGS cell line of overexpressed MTP18 was co-transfected with DRP1-siRNA and treated with 1μmol/L DOX for apoptosis analysis at 24hr. The percentages of apoptosis were analyzed by cell death detection ELISA. (B) Shows percentages of apoptosis. Data were expressed as the mean±SEM of three independent experiments. * P

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay

    Knockdown of MTP18 interferes doxorubicin-induced mitochondrial fission and apoptosis (A) Analysis of mitochondrial protein 18 (MTP18) expression. Immunoblot shows MTP18 knockdown in AGS cells ( upper panel ). β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments ( lower panel ). * P

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: Knockdown of MTP18 interferes doxorubicin-induced mitochondrial fission and apoptosis (A) Analysis of mitochondrial protein 18 (MTP18) expression. Immunoblot shows MTP18 knockdown in AGS cells ( upper panel ). β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments ( lower panel ). * P

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Expressing

    Enforced expression of MTP18 sensitizes doxorubicin-induced mitochondrial fission and apoptosis in AGS cells (A) Analysis of MTP18 expression. Immunoblot shows mitochondrial protein 18 (MTP18) overexpression in AGS cells ( left panel ). β-actin served as a loading control. The densitometry data were expressed as the mean ± SEM of three independent experiments ( right panel ). * P

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: Enforced expression of MTP18 sensitizes doxorubicin-induced mitochondrial fission and apoptosis in AGS cells (A) Analysis of MTP18 expression. Immunoblot shows mitochondrial protein 18 (MTP18) overexpression in AGS cells ( left panel ). β-actin served as a loading control. The densitometry data were expressed as the mean ± SEM of three independent experiments ( right panel ). * P

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Expressing, Over Expression

    MTP18 promotes doxorubicin-induced DRP1 accumulation in mitochondria (A) and (B) Analysis of dynamic-related protein 1 (DRP1) expression. Immunoblot shows DRP1 expression in whole cell lysate upon modulation of mitochondrial protein 18 (MTP18) expression. Modulation of MTP18 does not significantly affect the expression level of DRP1 ( upper panel ). β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments ( lower panel ). (C) and (D) Analysis of DRP1 expression in mitochondria and cytosolic fraction. Immunoblot shows DRP1 expression in AGS cells. C shows an increase in DRP1 accumulation in mitochondria upon enforced expression of MTP18. D shows a reduction in DRP1 accumulation when MTP18 was knocked down. HM= mitochondria-enriched heavy membranes. Cytochrome c oxidase (COXIV) served as a loading control for HM and β-actin served as a loading control for whole cell lysate and cytosolic fraction. (E) MTP18 binds to endogenous DRP1. Stable cell line of AGS infected with MTP18 were treated with 1μmol/L DOX. Since doxorubicin (DOX) exposure could induce a time-dependent downregulation of MTP18, the cells were harvested within 1h of DOX treatment to capture the association of MTP18 and DRP1 timely. The association between MTP18 and DRP1 was analyzed by immunoprecipitation (IP) followed by immunoblot (IB). Figures presented are the representative of at least three independent experiments.

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: MTP18 promotes doxorubicin-induced DRP1 accumulation in mitochondria (A) and (B) Analysis of dynamic-related protein 1 (DRP1) expression. Immunoblot shows DRP1 expression in whole cell lysate upon modulation of mitochondrial protein 18 (MTP18) expression. Modulation of MTP18 does not significantly affect the expression level of DRP1 ( upper panel ). β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments ( lower panel ). (C) and (D) Analysis of DRP1 expression in mitochondria and cytosolic fraction. Immunoblot shows DRP1 expression in AGS cells. C shows an increase in DRP1 accumulation in mitochondria upon enforced expression of MTP18. D shows a reduction in DRP1 accumulation when MTP18 was knocked down. HM= mitochondria-enriched heavy membranes. Cytochrome c oxidase (COXIV) served as a loading control for HM and β-actin served as a loading control for whole cell lysate and cytosolic fraction. (E) MTP18 binds to endogenous DRP1. Stable cell line of AGS infected with MTP18 were treated with 1μmol/L DOX. Since doxorubicin (DOX) exposure could induce a time-dependent downregulation of MTP18, the cells were harvested within 1h of DOX treatment to capture the association of MTP18 and DRP1 timely. The association between MTP18 and DRP1 was analyzed by immunoprecipitation (IP) followed by immunoblot (IB). Figures presented are the representative of at least three independent experiments.

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Expressing, Stable Transfection, Infection, Immunoprecipitation

    Doxorubicin exposure induces mitochondrial fission and down-regulates the MTP18 expression in a dose- and time- dependent manner (A) and (B) AGS cells were stimulated with 1μmol/L doxorubicin (DOX) at indicated time points and mitochondrial morphology was analyzed. (A) Shows mitochondrial morphology of control and after 6hr of DOX exposure. (B) Shows percentage of cells undergone mitochondrial fission upon DOX exposure. (C) and (D) Analysis of mitochondrial protein 18 (MTP18) expression. AGS cells were stimulated with the indicated doses of DOX and harvested at 12h (C) , and cells were stimulated with 1μmol/L DOX and then harvested at the indicated time (D) for immunoblotting. (C) and (D) Upper panels show mitochondrial protein 18 (MTP18) expression on DOX exposure. (C) and (D) Lower panels show densitometry analysis. β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments. * P

    Journal: Oncotarget

    Article Title: Mitochondrial protein 18 (MTP18) plays a pro-apoptotic role in chemotherapy-induced gastric cancer cell apoptosis

    doi: 10.18632/oncotarget.17508

    Figure Lengend Snippet: Doxorubicin exposure induces mitochondrial fission and down-regulates the MTP18 expression in a dose- and time- dependent manner (A) and (B) AGS cells were stimulated with 1μmol/L doxorubicin (DOX) at indicated time points and mitochondrial morphology was analyzed. (A) Shows mitochondrial morphology of control and after 6hr of DOX exposure. (B) Shows percentage of cells undergone mitochondrial fission upon DOX exposure. (C) and (D) Analysis of mitochondrial protein 18 (MTP18) expression. AGS cells were stimulated with the indicated doses of DOX and harvested at 12h (C) , and cells were stimulated with 1μmol/L DOX and then harvested at the indicated time (D) for immunoblotting. (C) and (D) Upper panels show mitochondrial protein 18 (MTP18) expression on DOX exposure. (C) and (D) Lower panels show densitometry analysis. β-actin served as a loading control. The densitometry data were expressed as the mean±SEM of three independent experiments. * P

    Article Snippet: Lenti ORF clone of MTP18 construct and infection Lentiviral particle harboring the cDNA of MTP18 was purchased from Origene (Rockville, MD, USA).

    Techniques: Expressing