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    Name:
    Ambion DNase I RNase free
    Description:
    Ambion DNase I RNase free E C 3 1 21 1 is a nonspecific endonuclease that degrades double and single stranded DNA and chromatin It functions by hydrolyzing phosphodiester linkages producing mono and oligonucleotides with a 5 phosphate and a 3 hydroxyl group RNase free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA For example DNase I is frequently used to remove template DNA following in vitro transcription and to remove contaminating DNA in total RNA preparations especially those from transfected cells that may contain plasmid DNA used for ribonuclease protection assays cDNA library contraction and RT PCR DNase I requires bivalent cations Mg2 and Ca2 at approximately 5 mM and 0 5 mM respectively for maximal activity and has a pH optimum of 7 8 RNase free DNase I outperforms the competitionA research report in BioTechniques Matthews et al 32 1412 1417 2002 compared RNase contamination in DNase I preparations from Sigma Roche Applied Science Qiagen and Ambion The results revealed that with the exception of Ambion s RNase free DNase I the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA Ambion s DNase was used for the remaining experiments requiring DNase digestion Ambion DNase I is tested for contaminating RNase and protease activity Functionality is determined by digestion of human genomic DNA followed by quantitative real time PCR to detect undigested DNA Unit definitionOne unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C and is equivalent to 0 04 Kunitz units Accessory productsFor an alternative to bovine DNase I please consider Recombinant DNase I Cat No AM2235 For a more active salt tolerant DNase please see the TURBO DNase products Cat Nos AM2239 and AM2238
    Catalog Number:
    am2222
    Price:
    None
    Applications:
    General Real-Time PCR Reagents|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher mirnas
    miR-218 inhibits CIP2A expression by specifically targeting its 3′-UTR. (A) Sequence alignment of the CIP2A WT and CIP2A MUT potential miR-218 targeting sites. (B) Luciferase reporter assay demonstrated decreased reporter activity following transfection with the CIP2A WT in <t>Caki-1</t> and 786-O cell lines overexpressing miR-218. (C) Inverse correlation between miR-218 expression level and CIP2A expression level in ccRCC tissues. (D) Alteration of the CIP2A protein expression levels in Caki-1 and 786-O cell lines transfected with miR-218 mimic or NC <t>miRNA.</t> Data are presented as the mean ± standard deviation. ***P
    Ambion DNase I RNase free E C 3 1 21 1 is a nonspecific endonuclease that degrades double and single stranded DNA and chromatin It functions by hydrolyzing phosphodiester linkages producing mono and oligonucleotides with a 5 phosphate and a 3 hydroxyl group RNase free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA For example DNase I is frequently used to remove template DNA following in vitro transcription and to remove contaminating DNA in total RNA preparations especially those from transfected cells that may contain plasmid DNA used for ribonuclease protection assays cDNA library contraction and RT PCR DNase I requires bivalent cations Mg2 and Ca2 at approximately 5 mM and 0 5 mM respectively for maximal activity and has a pH optimum of 7 8 RNase free DNase I outperforms the competitionA research report in BioTechniques Matthews et al 32 1412 1417 2002 compared RNase contamination in DNase I preparations from Sigma Roche Applied Science Qiagen and Ambion The results revealed that with the exception of Ambion s RNase free DNase I the integrity of cRNA from in vitro transcription reactions was compromised and was still contaminated with DNA Ambion s DNase was used for the remaining experiments requiring DNase digestion Ambion DNase I is tested for contaminating RNase and protease activity Functionality is determined by digestion of human genomic DNA followed by quantitative real time PCR to detect undigested DNA Unit definitionOne unit is the amount of enzyme required to completely degrade 1 µg DNA in 10 min at 37°C and is equivalent to 0 04 Kunitz units Accessory productsFor an alternative to bovine DNase I please consider Recombinant DNase I Cat No AM2235 For a more active salt tolerant DNase please see the TURBO DNase products Cat Nos AM2239 and AM2238
    https://www.bioz.com/result/mirnas/product/Thermo Fisher
    Average 90 stars, based on 61583 article reviews
    Price from $9.99 to $1999.99
    mirnas - by Bioz Stars, 2020-08
    90/100 stars

    Images

    1) Product Images from "MicroRNA-218 inhibits the cell proliferation and migration in clear cell renal cell carcinoma through targeting cancerous inhibitor of protein phosphatase 2A"

    Article Title: MicroRNA-218 inhibits the cell proliferation and migration in clear cell renal cell carcinoma through targeting cancerous inhibitor of protein phosphatase 2A

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.9986

    miR-218 inhibits CIP2A expression by specifically targeting its 3′-UTR. (A) Sequence alignment of the CIP2A WT and CIP2A MUT potential miR-218 targeting sites. (B) Luciferase reporter assay demonstrated decreased reporter activity following transfection with the CIP2A WT in Caki-1 and 786-O cell lines overexpressing miR-218. (C) Inverse correlation between miR-218 expression level and CIP2A expression level in ccRCC tissues. (D) Alteration of the CIP2A protein expression levels in Caki-1 and 786-O cell lines transfected with miR-218 mimic or NC miRNA. Data are presented as the mean ± standard deviation. ***P
    Figure Legend Snippet: miR-218 inhibits CIP2A expression by specifically targeting its 3′-UTR. (A) Sequence alignment of the CIP2A WT and CIP2A MUT potential miR-218 targeting sites. (B) Luciferase reporter assay demonstrated decreased reporter activity following transfection with the CIP2A WT in Caki-1 and 786-O cell lines overexpressing miR-218. (C) Inverse correlation between miR-218 expression level and CIP2A expression level in ccRCC tissues. (D) Alteration of the CIP2A protein expression levels in Caki-1 and 786-O cell lines transfected with miR-218 mimic or NC miRNA. Data are presented as the mean ± standard deviation. ***P

    Techniques Used: Expressing, Sequencing, Luciferase, Reporter Assay, Activity Assay, Transfection, Standard Deviation

    Influence of miR-218 on tumor cell proliferation and migration. (A) Alteration of the miR-218 expression levels in Caki-1 and 786-O cell lines by transfection with miR-218 mimic, miR-218 inhibitor or NC miRNA. ***P
    Figure Legend Snippet: Influence of miR-218 on tumor cell proliferation and migration. (A) Alteration of the miR-218 expression levels in Caki-1 and 786-O cell lines by transfection with miR-218 mimic, miR-218 inhibitor or NC miRNA. ***P

    Techniques Used: Migration, Expressing, Transfection

    2) Product Images from "Proteomic Profile of Carbonylated Proteins Screen Regulation of Apoptosis via CaMK Signaling in Response to Regular Aerobic Exercise"

    Article Title: Proteomic Profile of Carbonylated Proteins Screen Regulation of Apoptosis via CaMK Signaling in Response to Regular Aerobic Exercise

    Journal: BioMed Research International

    doi: 10.1155/2018/2828143

    mRNA expression levels of Hnrnpa2b1 and UCH-L1 as determined by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ∗ ρ
    Figure Legend Snippet: mRNA expression levels of Hnrnpa2b1 and UCH-L1 as determined by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ∗ ρ

    Techniques Used: Expressing, Quantitative RT-PCR

    mRNA expression levels of PI3K, Akt, and mTOR, as determined by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ρ
    Figure Legend Snippet: mRNA expression levels of PI3K, Akt, and mTOR, as determined by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ρ

    Techniques Used: Expressing, Quantitative RT-PCR

    mRNA expression levels of CaMKII α and Vdac1 by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ρ
    Figure Legend Snippet: mRNA expression levels of CaMKII α and Vdac1 by RT-qPCR. M-SED, middle-aged sedentary control group; M-EX, middle-aged aerobic exercise runner group; ∗ ρ

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "Dinitrosopiperazine‐decreased PKP3 through upregulating miR‐149 participates in nasopharyngeal carcinoma metastasis, et al. Dinitrosopiperazine‐decreased PKP3 through upregulating miR‐149 participates in nasopharyngeal carcinoma metastasis"

    Article Title: Dinitrosopiperazine‐decreased PKP3 through upregulating miR‐149 participates in nasopharyngeal carcinoma metastasis, et al. Dinitrosopiperazine‐decreased PKP3 through upregulating miR‐149 participates in nasopharyngeal carcinoma metastasis

    Journal: Molecular Carcinogenesis

    doi: 10.1002/mc.22895

    DNP‐mediated 6‐10B cell metastasis through miR‐149 in vivo. A, 5‐8F cells were transfected with miR‐149‐inhibitor or mimic, and then injected into nude mice through the tail vein. After 30 days, the mice were sacrificed and dissected, metastatic tumors were observed. The metastatic tumors were counted (a) and weighed (b). The tissue sections of metastatic tumors were made, and the sections were stained with hematoxylin and eosin (H E) (c, d). PKP3 in the metastatic tumor was detected using immunohistochemistry (IHC) (e, f), and miR‐149 was detected using qRT‐PCR (g). Scale bar, 100 μm (* P
    Figure Legend Snippet: DNP‐mediated 6‐10B cell metastasis through miR‐149 in vivo. A, 5‐8F cells were transfected with miR‐149‐inhibitor or mimic, and then injected into nude mice through the tail vein. After 30 days, the mice were sacrificed and dissected, metastatic tumors were observed. The metastatic tumors were counted (a) and weighed (b). The tissue sections of metastatic tumors were made, and the sections were stained with hematoxylin and eosin (H E) (c, d). PKP3 in the metastatic tumor was detected using immunohistochemistry (IHC) (e, f), and miR‐149 was detected using qRT‐PCR (g). Scale bar, 100 μm (* P

    Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR

    DNP‐induced NPC cell migration, invasion, and adhesion through miR‐149. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP, PKP3 expressions in the treated cells were detected using Western‐blotting (a), GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b), and miR‐149 expression was detected using qRT‐PCR (c). B, The migration (a‐d) and invasion (e‐h) of the treated 6‐10 cells were detected using Boden chamber assay, and the adhesion (i‐k) was detected using Cell adhesion assay. a, e, and i, Blank control; b, f, and j, miR‐149‐inhibitor treatment; c, g, and k, DNP treatment; d, h, and l, DNP plus miR‐149‐inhibitor. Cell migration (m), invasion (n), and adhesion (o) was quantitatively analyzed. C, 5‐8F cells were treated with mimic or miR‐149‐inhibitor. PKP3 in the treated cells was detected using Western‐blotting (a) and quantitatively analyzed (b), and miR‐149 mRNA were detected using qRT‐PCR (c). D, Migration (a‐c) and invasion (d‐f) of the treated 5‐8F cells were measured using Boden chamber assay. a and d, Blank control; b and e, Mimic treatment; c and f, miR‐149‐inhibitor. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. Scale bar, 5 μm; Original magnification, ×200. NC, Non‐targeting control; DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P
    Figure Legend Snippet: DNP‐induced NPC cell migration, invasion, and adhesion through miR‐149. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP, PKP3 expressions in the treated cells were detected using Western‐blotting (a), GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b), and miR‐149 expression was detected using qRT‐PCR (c). B, The migration (a‐d) and invasion (e‐h) of the treated 6‐10 cells were detected using Boden chamber assay, and the adhesion (i‐k) was detected using Cell adhesion assay. a, e, and i, Blank control; b, f, and j, miR‐149‐inhibitor treatment; c, g, and k, DNP treatment; d, h, and l, DNP plus miR‐149‐inhibitor. Cell migration (m), invasion (n), and adhesion (o) was quantitatively analyzed. C, 5‐8F cells were treated with mimic or miR‐149‐inhibitor. PKP3 in the treated cells was detected using Western‐blotting (a) and quantitatively analyzed (b), and miR‐149 mRNA were detected using qRT‐PCR (c). D, Migration (a‐c) and invasion (d‐f) of the treated 5‐8F cells were measured using Boden chamber assay. a and d, Blank control; b and e, Mimic treatment; c and f, miR‐149‐inhibitor. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. Scale bar, 5 μm; Original magnification, ×200. NC, Non‐targeting control; DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P

    Techniques Used: Migration, Western Blot, Expressing, Quantitative RT-PCR, Boyden Chamber Assay, Cell Adhesion Assay

    Cell apoptosis of 6‐10B cells treated with miR‐149 and DNP. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP, PKP3 expressions were detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b), and miR‐149 mRNA was detected using qRT‐PCR (c). B, The treated cells were stained with Annexin V/PI staining, and then analyzed with flow cytometry. The apoptotic cells were counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Apoptosis rates of the treated cells were compared. C, The treated cells were stained with PEA/FITC staining, and Δψm in the treated cells was detected with flow cytometry. JC‐1 monomer cells and JC‐1 polymer cells were respectively counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, JC‐1 monomer and JC‐1 polymer were compared. D, The treated 6‐10B cells were stained with TUNEL kit, the positive cells were counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Apoptosis cells were comparatively analyzed. E, 5‐8F cells were treated with miR‐149 mimic or its inhibitor. PKP3 expression (a) and miR‐149 mRNA (c) in the treated cells were detected, PKP3 expression was quantitatively compared (b). The treated cells were stained with FITC, apoptosis cells were detected. d, Blank control; e, Mimic; f, miR‐149‐inhibitor. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. Scale bar, 10 μm; DNP, Dinitrosopiperazine; PKP3, Plakophilin3; Original magnification, ×400.* P
    Figure Legend Snippet: Cell apoptosis of 6‐10B cells treated with miR‐149 and DNP. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP, PKP3 expressions were detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b), and miR‐149 mRNA was detected using qRT‐PCR (c). B, The treated cells were stained with Annexin V/PI staining, and then analyzed with flow cytometry. The apoptotic cells were counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Apoptosis rates of the treated cells were compared. C, The treated cells were stained with PEA/FITC staining, and Δψm in the treated cells was detected with flow cytometry. JC‐1 monomer cells and JC‐1 polymer cells were respectively counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, JC‐1 monomer and JC‐1 polymer were compared. D, The treated 6‐10B cells were stained with TUNEL kit, the positive cells were counted. a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Apoptosis cells were comparatively analyzed. E, 5‐8F cells were treated with miR‐149 mimic or its inhibitor. PKP3 expression (a) and miR‐149 mRNA (c) in the treated cells were detected, PKP3 expression was quantitatively compared (b). The treated cells were stained with FITC, apoptosis cells were detected. d, Blank control; e, Mimic; f, miR‐149‐inhibitor. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. Scale bar, 10 μm; DNP, Dinitrosopiperazine; PKP3, Plakophilin3; Original magnification, ×400.* P

    Techniques Used: Western Blot, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, TUNEL Assay, Expressing

    miR‐149 inhibits DNP‐induced proliferation and colony growt. A, 6‐10B cells were treated with DNP, DNP plus miR‐149‐inhibitor or miR‐149‐inhibitor, respectively. Viability of the treated cells was measured using MTT. The growth curves of the treated cells were calculated. NC, non‐targeting control. B, colony‐forming of 6‐10B cells treated with DNP, DNP plus miR‐149‐inhibitor, or miR‐149‐ inhibitor using colony formation assay. DNP, Dinitrosopiperazine. * P
    Figure Legend Snippet: miR‐149 inhibits DNP‐induced proliferation and colony growt. A, 6‐10B cells were treated with DNP, DNP plus miR‐149‐inhibitor or miR‐149‐inhibitor, respectively. Viability of the treated cells was measured using MTT. The growth curves of the treated cells were calculated. NC, non‐targeting control. B, colony‐forming of 6‐10B cells treated with DNP, DNP plus miR‐149‐inhibitor, or miR‐149‐ inhibitor using colony formation assay. DNP, Dinitrosopiperazine. * P

    Techniques Used: MTT Assay, Colony Assay

    Expressions of PKP3 and miR‐149 in 6‐10B cells with DNP treatment. A, 6‐10B cells were treated with the indicated concentration of DNP for dose‐course. B, 6‐10B cells were treated with DNP for the indicated time for time‐course. PKP3 in the treated cells was detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted for PKP3 quantitative analysis (b), and miR‐149 expression was detected using qRT‐PCR (c). DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P
    Figure Legend Snippet: Expressions of PKP3 and miR‐149 in 6‐10B cells with DNP treatment. A, 6‐10B cells were treated with the indicated concentration of DNP for dose‐course. B, 6‐10B cells were treated with DNP for the indicated time for time‐course. PKP3 in the treated cells was detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted for PKP3 quantitative analysis (b), and miR‐149 expression was detected using qRT‐PCR (c). DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P

    Techniques Used: Concentration Assay, Western Blot, Expressing, Quantitative RT-PCR

    DNP upregulates miR‐149 expression. A, Structure of DNP, a N‐nitroso compound. B, miRNA sequencing was used to construct miRNAs expression profiles of DNP‐treated 6‐10B cells and the untreated. Red represents high expression, and green is low expression. C, miR‐149 was detected using qRT‐PCR in 6‐10B cells treated with DNP and the untreated cells, and 5‐8F cells served as a positive control. D, The expression of miR‐149 was detected in the tumors and paired adjacent tissues from NPC patients. DNP, Dinitrosopiperazine. * P
    Figure Legend Snippet: DNP upregulates miR‐149 expression. A, Structure of DNP, a N‐nitroso compound. B, miRNA sequencing was used to construct miRNAs expression profiles of DNP‐treated 6‐10B cells and the untreated. Red represents high expression, and green is low expression. C, miR‐149 was detected using qRT‐PCR in 6‐10B cells treated with DNP and the untreated cells, and 5‐8F cells served as a positive control. D, The expression of miR‐149 was detected in the tumors and paired adjacent tissues from NPC patients. DNP, Dinitrosopiperazine. * P

    Techniques Used: Expressing, Sequencing, Construct, Quantitative RT-PCR, Positive Control

    Tube formation and F‐actin expression in 6‐10B cells treated with miR‐149 and DNP. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP. PKP3 expressions were detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b). miR‐149 mRNA was detected using qRT‐PCR (c). B, Tube information of the treated cells was detected with the tube formation assay (a–d). The tubular structures formed in the matrigel were counted (e). a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Tube formation was quantitively analyzed (e). scale bar, 5 μm; Original magnification, ×200. The treated cells were stained with F‐actin immunostaining (f‐i). The intensity of F‐actin fluorescence was determined in 10 fields/well and divided by the cells stained with DAPI (j). f, Blank control; g, Treatment with miR‐149‐inhibitor; h, Treatment with DNP; i, Treatment with miR‐149‐inhibitor plus DNP; j, Tube formation was quantitively analyzed. scale bar, 20 μm; Original magnification, ×1000. C, 5‐8F cells were treated with miR‐149 mimic or its inhibitor. PKP3 (a) and miR‐149 mRNA (c) were detected in the treated cells, PKP3 expression was quantitatively compared (b). D. Tube information of the treated 5‐8F cells was detected (a, b, c). The tubular structures formed in the matrigel were counted (e). The treated 5‐8F cells were stained with F‐actin immunostaining (e‐g). The intensity of F‐actin fluorescence was determined in 10 fields/well and divided by the cells stained with DAPI (h). a, Blank control; b, Mimic; c, miR‐149‐inhibitor; d, Tube formation was quantitively analyzed; e, Blank control; f, Mimic; g, miR‐149‐inhibitor; h, Tube formation was quantitively analyzed. The experiments were performed in triplicate, and five fields from each chamber were counted and averaged. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P
    Figure Legend Snippet: Tube formation and F‐actin expression in 6‐10B cells treated with miR‐149 and DNP. A, 6‐10B cells were treated with miR‐149‐inhibitor and/or DNP. PKP3 expressions were detected using Western‐blotting (a). GAPDH was used as an internal control. The abundance ratio of PKP3 to GAPDH was counted (b). miR‐149 mRNA was detected using qRT‐PCR (c). B, Tube information of the treated cells was detected with the tube formation assay (a–d). The tubular structures formed in the matrigel were counted (e). a, Blank control; b, Treatment with miR‐149‐inhibitor; c, Treatment with DNP; d, Treatment with miR‐149‐inhibitor plus DNP; e, Tube formation was quantitively analyzed (e). scale bar, 5 μm; Original magnification, ×200. The treated cells were stained with F‐actin immunostaining (f‐i). The intensity of F‐actin fluorescence was determined in 10 fields/well and divided by the cells stained with DAPI (j). f, Blank control; g, Treatment with miR‐149‐inhibitor; h, Treatment with DNP; i, Treatment with miR‐149‐inhibitor plus DNP; j, Tube formation was quantitively analyzed. scale bar, 20 μm; Original magnification, ×1000. C, 5‐8F cells were treated with miR‐149 mimic or its inhibitor. PKP3 (a) and miR‐149 mRNA (c) were detected in the treated cells, PKP3 expression was quantitatively compared (b). D. Tube information of the treated 5‐8F cells was detected (a, b, c). The tubular structures formed in the matrigel were counted (e). The treated 5‐8F cells were stained with F‐actin immunostaining (e‐g). The intensity of F‐actin fluorescence was determined in 10 fields/well and divided by the cells stained with DAPI (h). a, Blank control; b, Mimic; c, miR‐149‐inhibitor; d, Tube formation was quantitively analyzed; e, Blank control; f, Mimic; g, miR‐149‐inhibitor; h, Tube formation was quantitively analyzed. The experiments were performed in triplicate, and five fields from each chamber were counted and averaged. Data are presented as means ± S.D. from three independent experiments statistically using the Student's t test. DNP, Dinitrosopiperazine; PKP3, Plakophilin3. * P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Tube Formation Assay, Staining, Immunostaining, Fluorescence

    4) Product Images from "Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis"

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4086

    Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.
    Figure Legend Snippet: Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Techniques Used: Next-Generation Sequencing, Indirect Immunoperoxidase Assay, Functional Assay, Generated, Expressing

    5) Product Images from "Molecular Mechanisms of Drug Resistance in Clinical Candida Species Isolated from Tunisian Hospitals"

    Article Title: Molecular Mechanisms of Drug Resistance in Clinical Candida Species Isolated from Tunisian Hospitals

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00555-13

    Sterol profiles of azole-susceptible and azole-resistant isolates. Sterols were extracted from cells grown in YPD, and the spectral profiles between 240 and 310 nm were determined by using a NanoDrop ND-1000 UV-Vis spectrophotometer. (A) VSY2 and SC5314, C. albicans isolates with nonfunctional and functional ERG3 alleles, respectively; JEY311, azole-susceptible C. tropicalis clinical isolate with wild-type CtERG3 ; JEY162, FLC- and AMB-resistant isolate with nonfunctional CtERG11 and CtERG3 alleles. (B) JEY424 and JEY425, isolates with nonfunctional and functional ERG3 alleles, respectively.
    Figure Legend Snippet: Sterol profiles of azole-susceptible and azole-resistant isolates. Sterols were extracted from cells grown in YPD, and the spectral profiles between 240 and 310 nm were determined by using a NanoDrop ND-1000 UV-Vis spectrophotometer. (A) VSY2 and SC5314, C. albicans isolates with nonfunctional and functional ERG3 alleles, respectively; JEY311, azole-susceptible C. tropicalis clinical isolate with wild-type CtERG3 ; JEY162, FLC- and AMB-resistant isolate with nonfunctional CtERG11 and CtERG3 alleles. (B) JEY424 and JEY425, isolates with nonfunctional and functional ERG3 alleles, respectively.

    Techniques Used: Spectrophotometry, Functional Assay

    6) Product Images from "RSK1 Activation Promotes Invasion in Nodular Melanoma"

    Article Title: RSK1 Activation Promotes Invasion in Nodular Melanoma

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2014.11.021

    Genetic or pharmacological inhibition of protein S6 kinase, 90 kDa, polypeptide 1 (RSK1) inhibits nodular melanoma (NM) but not superficial spreading melanoma (SSM) cell proliferation. A: Western blot analysis showing RSK1 levels after transfection with nontargeting (N.T.), RSK1-targeting, or RSK2-targeting siRNA in WM 1552 melanoma cells (SSM). B: Crystal violet proliferation assay of SSM cells 48 hours after transient transfection with RSK1 or RSK2 siRNA or 72 hours after treatment with escalating doses of the RSK inhibitor BI-D1870. Data were normalized by setting N.T. siRNA-transfected or dimethyl sulfoxide (DMSO)–treated cells to 100%. C: Same as in A but using WM 3248 melanoma cells (NM). D: Same as in B but using NM cell lines. Data are given as means ± SEM. ∗∗ P
    Figure Legend Snippet: Genetic or pharmacological inhibition of protein S6 kinase, 90 kDa, polypeptide 1 (RSK1) inhibits nodular melanoma (NM) but not superficial spreading melanoma (SSM) cell proliferation. A: Western blot analysis showing RSK1 levels after transfection with nontargeting (N.T.), RSK1-targeting, or RSK2-targeting siRNA in WM 1552 melanoma cells (SSM). B: Crystal violet proliferation assay of SSM cells 48 hours after transient transfection with RSK1 or RSK2 siRNA or 72 hours after treatment with escalating doses of the RSK inhibitor BI-D1870. Data were normalized by setting N.T. siRNA-transfected or dimethyl sulfoxide (DMSO)–treated cells to 100%. C: Same as in A but using WM 3248 melanoma cells (NM). D: Same as in B but using NM cell lines. Data are given as means ± SEM. ∗∗ P

    Techniques Used: Inhibition, Western Blot, Transfection, Proliferation Assay

    p90 ribosomal S6 kinase (RSK) inhibition decreases nodular melanoma (NM) cell migration and invasion. A: WM 278 and WM 3248 cells treated with equal amounts of dimethyl sulfoxide (DMSO) or 5 μmol/L BI-D1870 migrated through 8-μm migration chambers for 6 hours and then were fixed and stained with crystal violet. Five high-power fields from each chamber were photographed, and cells were then counted. Images shown are representative of one high-power field from each condition. B: Same as A but chamber coated with Matrigel and cells allowed to invade for 20 hours. For A and B, data were normalized by setting DMSO-treated cells to 100%. C: Same as B but using siRNA against RSK1. Data are given as means ± SEM. ∗∗∗ P
    Figure Legend Snippet: p90 ribosomal S6 kinase (RSK) inhibition decreases nodular melanoma (NM) cell migration and invasion. A: WM 278 and WM 3248 cells treated with equal amounts of dimethyl sulfoxide (DMSO) or 5 μmol/L BI-D1870 migrated through 8-μm migration chambers for 6 hours and then were fixed and stained with crystal violet. Five high-power fields from each chamber were photographed, and cells were then counted. Images shown are representative of one high-power field from each condition. B: Same as A but chamber coated with Matrigel and cells allowed to invade for 20 hours. For A and B, data were normalized by setting DMSO-treated cells to 100%. C: Same as B but using siRNA against RSK1. Data are given as means ± SEM. ∗∗∗ P

    Techniques Used: Inhibition, Migration, Staining

    7) Product Images from "Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells"

    Article Title: Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-12-15

    Anti-HPV and anti-cancer effect of B. pinnata crude leaf extract and fraction F4 in cervical cancer cells . A . Northern blot analysis of HPV18 positive HeLa cells incubated with indicated concentrations of crude leaf extract and fraction F4. Quantity and quality of total RNA (15 μg/lane) extracted was examined on agarose gel by assessing 28S 18S ribosomal RNAs (upper panels). The membranes were hybridized with HPV18-specific probe (middle panel), stripped and rehybridized with β-actin -specific DNA probe as an internal control to assess equal loading (lower panels). B . Aggregated mean (± S.D.) abundance ratio of HPV18 transcript w.r.t. to β-actin following treatment with indicated doses of crude extract (a) and fraction F4 (b) in three independent experiments. The abundance ratio HPV18 transcript to β-actin mRNA was analyzed by densitometry as described in
    Figure Legend Snippet: Anti-HPV and anti-cancer effect of B. pinnata crude leaf extract and fraction F4 in cervical cancer cells . A . Northern blot analysis of HPV18 positive HeLa cells incubated with indicated concentrations of crude leaf extract and fraction F4. Quantity and quality of total RNA (15 μg/lane) extracted was examined on agarose gel by assessing 28S 18S ribosomal RNAs (upper panels). The membranes were hybridized with HPV18-specific probe (middle panel), stripped and rehybridized with β-actin -specific DNA probe as an internal control to assess equal loading (lower panels). B . Aggregated mean (± S.D.) abundance ratio of HPV18 transcript w.r.t. to β-actin following treatment with indicated doses of crude extract (a) and fraction F4 (b) in three independent experiments. The abundance ratio HPV18 transcript to β-actin mRNA was analyzed by densitometry as described in "Methods" and their ratio in untreated control was used as reference. C . Immunoblotting analysis of Bcl-2, and Bax, caspase-3, poly (ADP-ribose) polymerase-1 (PARP- 1), in fraction F4 treated HeLa cells at different time-intervals. D . The abundance ratio of indicated proteins to β-actin was analyzed by densitometry and their ratio in untreated control was used as reference. The values indicate aggregated mean ± S.D. of three independent experiments. *p

    Techniques Used: Northern Blot, Incubation, Agarose Gel Electrophoresis

    8) Product Images from "Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle"

    Article Title: Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-263

    Effects of saikosaponin-d and radiation on apoptosis in hepatocellular carcinoma cell SMMC-7721. A . Flow cytometry shows apoptotic changes before and after treatment under oxia and hypoxia; B . Apoptotic fraction of cells under oxia and hypoxia; SSd: saikosaponin-d; IR: radiation. *p
    Figure Legend Snippet: Effects of saikosaponin-d and radiation on apoptosis in hepatocellular carcinoma cell SMMC-7721. A . Flow cytometry shows apoptotic changes before and after treatment under oxia and hypoxia; B . Apoptotic fraction of cells under oxia and hypoxia; SSd: saikosaponin-d; IR: radiation. *p

    Techniques Used: Flow Cytometry, Cytometry

    Effect of saikosaponin-d and radiation on the mRNA levels of p53, bcl2 and BAX in SMMC-7721 human hepatocellular carcinoma cells under normoxia and hypoxia. A . Fold changes in p53 mRNA; B . Folds changes in bcl-2 mRNA; C . Fold changes in BAX mRNA; SSd: saikosaponin-d; IR: radiation; *p
    Figure Legend Snippet: Effect of saikosaponin-d and radiation on the mRNA levels of p53, bcl2 and BAX in SMMC-7721 human hepatocellular carcinoma cells under normoxia and hypoxia. A . Fold changes in p53 mRNA; B . Folds changes in bcl-2 mRNA; C . Fold changes in BAX mRNA; SSd: saikosaponin-d; IR: radiation; *p

    Techniques Used:

    Effects of saikosaponin-d and radiation on the growth of SMMC-7721 hepatocellular carcinoma cells. All data are presented as mean ± SD. SSd: saikosaponin-d; IR: radiation. *P
    Figure Legend Snippet: Effects of saikosaponin-d and radiation on the growth of SMMC-7721 hepatocellular carcinoma cells. All data are presented as mean ± SD. SSd: saikosaponin-d; IR: radiation. *P

    Techniques Used:

    Effect of saikosaponin-d and radiation on the cell cycle distribution of SMMC-7721 human hepatocellular carcinoma cells under oxia and hypoxia. A . Changes in cell cycle distribution under oxia; B . changes in cell cycle distribution under hypoxia; C . Statistical analysis showing the changes in cell cycle progression before and after intervention under oxia; D . Statistical analysis showing the changes in cell cycle progression before and after intervention under hypoxia. SSd: saikosaponin-d; IR: radiation. *p
    Figure Legend Snippet: Effect of saikosaponin-d and radiation on the cell cycle distribution of SMMC-7721 human hepatocellular carcinoma cells under oxia and hypoxia. A . Changes in cell cycle distribution under oxia; B . changes in cell cycle distribution under hypoxia; C . Statistical analysis showing the changes in cell cycle progression before and after intervention under oxia; D . Statistical analysis showing the changes in cell cycle progression before and after intervention under hypoxia. SSd: saikosaponin-d; IR: radiation. *p

    Techniques Used:

    Effect of saikosaponin-d and radiation on the p53 levels and the bcl2/BAX ratio in SMMC-7721 human hepatocellular carcinoma cells under oxia and hypoxia. A . Western blot analysis of p53, Bax, and Bcl-2 levels under oxia and hypoxia. B . Relative expression of p53. C . Change of Bax/Bcl-2 ratio. SSd: saikosaponin-d; IR: radiation; *p
    Figure Legend Snippet: Effect of saikosaponin-d and radiation on the p53 levels and the bcl2/BAX ratio in SMMC-7721 human hepatocellular carcinoma cells under oxia and hypoxia. A . Western blot analysis of p53, Bax, and Bcl-2 levels under oxia and hypoxia. B . Relative expression of p53. C . Change of Bax/Bcl-2 ratio. SSd: saikosaponin-d; IR: radiation; *p

    Techniques Used: Western Blot, Expressing

    9) Product Images from "TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice"

    Article Title: TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083987

    Inhibiting TWEAK/Fn14 signaling attenuates the induction of mitogens associated with liver regeneration. QRT-PCR analysis of whole liver expression of TNFα, IL6 and HGF mRNA in (A) WT and Fn14 KO mice (B) WT, TWEAK KO and WT mice treated with Anti-TWEAK antibodies after PH. Mean +/− SEM results from all mice (n = 3–4/time point) are graphed relative to baseline, pre-PH (time 0) in WT group. * p
    Figure Legend Snippet: Inhibiting TWEAK/Fn14 signaling attenuates the induction of mitogens associated with liver regeneration. QRT-PCR analysis of whole liver expression of TNFα, IL6 and HGF mRNA in (A) WT and Fn14 KO mice (B) WT, TWEAK KO and WT mice treated with Anti-TWEAK antibodies after PH. Mean +/− SEM results from all mice (n = 3–4/time point) are graphed relative to baseline, pre-PH (time 0) in WT group. * p

    Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

    Post- PH growth of Fn14(+) cells is Fn14-dependent and requires TWEAK. (A) Liver sections from WT mice were double-stained for Fn14 (brown) and Ki67 (green), a proliferation marker. Representative image of liver from 6 h after PH is displayed. Arrow indicates double positive cells. Mean +/− SEM numbers of positive cells in 10 random 40× fields/section in 5 mice/group/time point. (B) qRT-PCR analysis of TWEAK mRNA expression in whole liver RNA from WT and Fn14 KO mice at various time points after PH; * p
    Figure Legend Snippet: Post- PH growth of Fn14(+) cells is Fn14-dependent and requires TWEAK. (A) Liver sections from WT mice were double-stained for Fn14 (brown) and Ki67 (green), a proliferation marker. Representative image of liver from 6 h after PH is displayed. Arrow indicates double positive cells. Mean +/− SEM numbers of positive cells in 10 random 40× fields/section in 5 mice/group/time point. (B) qRT-PCR analysis of TWEAK mRNA expression in whole liver RNA from WT and Fn14 KO mice at various time points after PH; * p

    Techniques Used: Mouse Assay, Staining, Marker, Quantitative RT-PCR, Expressing

    10) Product Images from "Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes"

    Article Title: Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201209135

    The first 30 amino acids of TFEB are sufficient for binding to active Rag heterodimers. (A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm. (B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out. (C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1–30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm. (D) Relative RT-PCR analysis of the mRNA expression of autophagy ( ATG9B and UVRAG ) and lysosomal ( MCOLN1 ) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P
    Figure Legend Snippet: The first 30 amino acids of TFEB are sufficient for binding to active Rag heterodimers. (A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm. (B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out. (C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1–30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm. (D) Relative RT-PCR analysis of the mRNA expression of autophagy ( ATG9B and UVRAG ) and lysosomal ( MCOLN1 ) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P

    Techniques Used: Binding Assay, Staining, Construct, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection

    11) Product Images from "A New Mouse Model for Mania Shares Genetic Correlates with Human Bipolar Disorder"

    Article Title: A New Mouse Model for Mania Shares Genetic Correlates with Human Bipolar Disorder

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038128

    RT-qPCR confirmation results for seven genes from the microarray. Ratio distribution is graphed as a box-and-whiskers plot. Ratios greater than 1 represent genes with higher expression in the MSN strain and ratios less than 1 represent genes with lower expression in the MSN strain relative to the ICR strain. *P
    Figure Legend Snippet: RT-qPCR confirmation results for seven genes from the microarray. Ratio distribution is graphed as a box-and-whiskers plot. Ratios greater than 1 represent genes with higher expression in the MSN strain and ratios less than 1 represent genes with lower expression in the MSN strain relative to the ICR strain. *P

    Techniques Used: Quantitative RT-PCR, Microarray, Expressing

    Heatmap of normalized values for the top 100 well-annotated genes from the microarray experiment listed by P-value in order from top to bottom, then left to right. The lowest P-values are at the top left corner, and the highest are at the bottom right. Blue values represent lower expression and orange values represent higher expression. The names of the genes discussed in the text of this manuscript are highlighted in light orange.
    Figure Legend Snippet: Heatmap of normalized values for the top 100 well-annotated genes from the microarray experiment listed by P-value in order from top to bottom, then left to right. The lowest P-values are at the top left corner, and the highest are at the bottom right. Blue values represent lower expression and orange values represent higher expression. The names of the genes discussed in the text of this manuscript are highlighted in light orange.

    Techniques Used: Microarray, Expressing

    12) Product Images from "Stem Cell Marker (Nanog) and Stat-3 Signaling Promote MicroRNA-21 Expression and Chemoresistance in Hyaluronan/CD44-activated Head and Neck Squamous Cell Carcinoma Cells"

    Article Title: Stem Cell Marker (Nanog) and Stat-3 Signaling Promote MicroRNA-21 Expression and Chemoresistance in Hyaluronan/CD44-activated Head and Neck Squamous Cell Carcinoma Cells

    Journal: Oncogene

    doi: 10.1038/onc.2011.222

    Interaction between Nanog, Stat-3 and the upstream promoter/enhancer region of miR-21 promoter in HSC-3 cells A: In vivo binding of Nanog and Stat-3 (or phosphorylated Stat-3) to the miR-21 upstream promoter/enhancer region in HSC-3 cells: ChIP assay was performed in HSC-3 cells following protocols described in the Materials and Methods using the Stat-3 binding site-containing miR-21 promoter (upstream promoter/enhancer region)-specific primers by PCR. Identical volumes from the final precipitated materials were used for the PCR reactions [untreated cells (lane 1); cells treated with HA for 30min (lane 2); cells pretreated with anti-CD44 antibody for 1h plus 30min HA addition (lane 3); cells pretreated with scrambled siRNA with no HA (lane 4); cells pretreated with scrambled siRNA plus 30min HA addition (lane 5); cells pretreated with Nanog siRNA plus 30min HA addition (lane 6); cells pretreated with Stat-3 siRNA plus 30min HA addition) (lane 7). (a: anti-Stat-3-mediated immunoprecipitated material; b: anti-phospho-Stat-3 (pY 705 )-mediated immunoprecipitated material; c: anti-Nanog-mediated immunoprecipitated material; d: IgG isotype control-mediated precipitated material; e: total input materials). B: Verification of the specificity of Nanog siRNA or Stat-3 siRNA used in the study. Cell lysates isolated from HSC-3 cells [transfected with Nanog siRNA or Stat-3 siRNA-target or scrambled siRNA] were solubilized by 1% Nonidet P-40 (NP-40) buffer followed by immunoblotting with anti-Stat-3 antibody (a), anti-Nanog antibody (b) or anti-actin (a loading control) (c).
    Figure Legend Snippet: Interaction between Nanog, Stat-3 and the upstream promoter/enhancer region of miR-21 promoter in HSC-3 cells A: In vivo binding of Nanog and Stat-3 (or phosphorylated Stat-3) to the miR-21 upstream promoter/enhancer region in HSC-3 cells: ChIP assay was performed in HSC-3 cells following protocols described in the Materials and Methods using the Stat-3 binding site-containing miR-21 promoter (upstream promoter/enhancer region)-specific primers by PCR. Identical volumes from the final precipitated materials were used for the PCR reactions [untreated cells (lane 1); cells treated with HA for 30min (lane 2); cells pretreated with anti-CD44 antibody for 1h plus 30min HA addition (lane 3); cells pretreated with scrambled siRNA with no HA (lane 4); cells pretreated with scrambled siRNA plus 30min HA addition (lane 5); cells pretreated with Nanog siRNA plus 30min HA addition (lane 6); cells pretreated with Stat-3 siRNA plus 30min HA addition) (lane 7). (a: anti-Stat-3-mediated immunoprecipitated material; b: anti-phospho-Stat-3 (pY 705 )-mediated immunoprecipitated material; c: anti-Nanog-mediated immunoprecipitated material; d: IgG isotype control-mediated precipitated material; e: total input materials). B: Verification of the specificity of Nanog siRNA or Stat-3 siRNA used in the study. Cell lysates isolated from HSC-3 cells [transfected with Nanog siRNA or Stat-3 siRNA-target or scrambled siRNA] were solubilized by 1% Nonidet P-40 (NP-40) buffer followed by immunoblotting with anti-Stat-3 antibody (a), anti-Nanog antibody (b) or anti-actin (a loading control) (c).

    Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Isolation, Transfection

    Detection of HA/CD44-induced miR-21 production in HSC-3 cells Detection of miR-21 in HSC-3 cells using RNase protection assay as described in the Materials and methods. A: Autoradiogram of miR-21 detected in HSC-3 cells incubated with scrambled sequence siRNA [without HA (lane 1) or with 2h HA treatment (lane 2) or pretreated with anti-CD44 antibody for 1h followed by HA addition for 2h (lane 3) or incubated with Nanog siRNA plus 2h HA treatment (lane 4) or incubated with Stat-3 siRNA plus 2h HA treatment (lane 5) or incubated with miRNA-negative control [without HA (lane 6) or with 2h HA treatment (lane 7)] or incubated with an anti-miR-21 inhibitor plus 2h HA treatment (lane 8). (Autoradiogram of miR-191 in each gel lane was used as a loading control).
    Figure Legend Snippet: Detection of HA/CD44-induced miR-21 production in HSC-3 cells Detection of miR-21 in HSC-3 cells using RNase protection assay as described in the Materials and methods. A: Autoradiogram of miR-21 detected in HSC-3 cells incubated with scrambled sequence siRNA [without HA (lane 1) or with 2h HA treatment (lane 2) or pretreated with anti-CD44 antibody for 1h followed by HA addition for 2h (lane 3) or incubated with Nanog siRNA plus 2h HA treatment (lane 4) or incubated with Stat-3 siRNA plus 2h HA treatment (lane 5) or incubated with miRNA-negative control [without HA (lane 6) or with 2h HA treatment (lane 7)] or incubated with an anti-miR-21 inhibitor plus 2h HA treatment (lane 8). (Autoradiogram of miR-191 in each gel lane was used as a loading control).

    Techniques Used: Rnase Protection Assay, Incubation, Sequencing, Negative Control

    Analyses of HA/CD44-mediated PDCD4 and IAP expression in HSC-3 cells Detection of HA/CD44-induced PDCD4 and IAP (cIAP-1, cIAP-2 and XIAP) expression in HSC-3 cells was performed by solubilizing cells with 1% Nonidet P-40 (NP-40) buffer followed by immunobloting with anti-PDCD4 antibody or anti-cIAP-1 antibody or anti-cIAP-2 antibody or anti-XIAP antibody, respectively as described in the Materials and Methods. First, cell lysates were prepared from HSC-3 cells treated with scrambled sequence siRNA [without HA (lane 1) or with HA for 24h (lane 2)] or treated with anti-CD44 antibody for 1h followed by 24h HA addition (lane 3) or treated with Nanog siRNA plus HA for 24h (lane 4) or treated with Stat-3 siRNA plus HA for 24h (lane 5) or treated with miRNA-negative control [without HA (lane 6) or with HA for 24h (lane 7)] or treated with anti-miR-21 inhibitor plus HA for 24h (lane 8). These samples were then immunoblotted with anti-PDCD4 antibody (a) or anti-cIAP-1 antibody (b) or anti-cIAP-2 antibody (c) or anti-XIAP antibody (d), respectively. The amount of actin detected by anti-actin-mediated immunoblot (e) in each gel lane was used as a loading control.
    Figure Legend Snippet: Analyses of HA/CD44-mediated PDCD4 and IAP expression in HSC-3 cells Detection of HA/CD44-induced PDCD4 and IAP (cIAP-1, cIAP-2 and XIAP) expression in HSC-3 cells was performed by solubilizing cells with 1% Nonidet P-40 (NP-40) buffer followed by immunobloting with anti-PDCD4 antibody or anti-cIAP-1 antibody or anti-cIAP-2 antibody or anti-XIAP antibody, respectively as described in the Materials and Methods. First, cell lysates were prepared from HSC-3 cells treated with scrambled sequence siRNA [without HA (lane 1) or with HA for 24h (lane 2)] or treated with anti-CD44 antibody for 1h followed by 24h HA addition (lane 3) or treated with Nanog siRNA plus HA for 24h (lane 4) or treated with Stat-3 siRNA plus HA for 24h (lane 5) or treated with miRNA-negative control [without HA (lane 6) or with HA for 24h (lane 7)] or treated with anti-miR-21 inhibitor plus HA for 24h (lane 8). These samples were then immunoblotted with anti-PDCD4 antibody (a) or anti-cIAP-1 antibody (b) or anti-cIAP-2 antibody (c) or anti-XIAP antibody (d), respectively. The amount of actin detected by anti-actin-mediated immunoblot (e) in each gel lane was used as a loading control.

    Techniques Used: Expressing, Western Blot, Sequencing, Negative Control

    13) Product Images from "Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone"

    Article Title: Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-495

    Silencing of specific microRNAs in GCTSCs. Total RNA including microRNAs was extracted from cultured GCTSCs (n = 10) and MSCs (n = 10) and expression of microRNAs was quantified relative to the expression of the small nuclear RNA RNU6B. The white lines indicate the median, the lower and upper boundaries of the box indicate the 25th and 75th percentile. The whiskers indicate the highest and lowest values. (**p
    Figure Legend Snippet: Silencing of specific microRNAs in GCTSCs. Total RNA including microRNAs was extracted from cultured GCTSCs (n = 10) and MSCs (n = 10) and expression of microRNAs was quantified relative to the expression of the small nuclear RNA RNU6B. The white lines indicate the median, the lower and upper boundaries of the box indicate the 25th and 75th percentile. The whiskers indicate the highest and lowest values. (**p

    Techniques Used: Cell Culture, Expressing

    14) Product Images from "Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics"

    Article Title: Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

    Journal: Toxins

    doi: 10.3390/toxins6051644

    HRP as a reporter for endo/lysosomal escape in isolated organelles. Lysosomes were isolated by cell fractionation form ECV-304 cells (3 × 10 7 cells). ( A ) First, the permeabilizing effects of SA1641 on the lysosomal membranes were evaluated by the β - N -acetylglucosaminidase (NAG) release assay.The permeabilizing effects of digitonin (highly lytic saponin) and α-hederin (non-lytic saponin) were simultaneously determined as controls; ( B ) Thereafter, the endo/lysosomal escape enhancement of saporin-HRP observed in the cytotoxicity assay in Figure 1 F was tried, to monitor in isolated lysosomes loaded with saporin-HRP (from cells previously treated with the conjugate at 100 nM and 37 °C for 6 h) by determination of the peroxidase activity. Saponin concentrations are shown in µM to allow a better comparison. Each data point represents the mean ± SD, n = 3.
    Figure Legend Snippet: HRP as a reporter for endo/lysosomal escape in isolated organelles. Lysosomes were isolated by cell fractionation form ECV-304 cells (3 × 10 7 cells). ( A ) First, the permeabilizing effects of SA1641 on the lysosomal membranes were evaluated by the β - N -acetylglucosaminidase (NAG) release assay.The permeabilizing effects of digitonin (highly lytic saponin) and α-hederin (non-lytic saponin) were simultaneously determined as controls; ( B ) Thereafter, the endo/lysosomal escape enhancement of saporin-HRP observed in the cytotoxicity assay in Figure 1 F was tried, to monitor in isolated lysosomes loaded with saporin-HRP (from cells previously treated with the conjugate at 100 nM and 37 °C for 6 h) by determination of the peroxidase activity. Saponin concentrations are shown in µM to allow a better comparison. Each data point represents the mean ± SD, n = 3.

    Techniques Used: Isolation, Cell Fractionation, Release Assay, Cytotoxicity Assay, Activity Assay

    Chemical conjugation, purification and characterization of saporin-HRP. ( A ) Cross-linkage of saporin and HRP. The reaction mixture was directly analysed by SDS-PAGE. Saporin and HRP served as unconjugated controls; ( B ) Purification of saporin-HRP by Ni-NTA chromatography with increasing concentrations of imidazole in the elution buffer. All fractions were assessed by SDS-PAGE. Saporin-HRP was eluted in Fractions 1–3 at 62 mM imidazole (see arrow); ( C ) Validation of the chemical conjugation of saporin and HRP. Saporin-HRP was analysed by SDS-PAGE, and the conjugate was visualized in the gel (see arrow). Saporin-HRP was analysed by Western blot with a primary polyclonal antibody against saporin. Saporin and the conjugate (see arrow) were specifically detected in the membrane; ( D ) Peroxidase activity of saporin-HRP. A directly proportional correlation between absorbance and concentration was observed from 0.1 to 10 nM saporin-HRP. Each data point is the mean ± SD, n = 3; Comparison of the cytotoxicity of ( E ) saporin and ( F ) saporin-HRP in the presence of SA1641. ECV-304 cells (4000 cells/well) were treated with saporin or saporin-HRP in a concentration range from 0.01 to 100 nM alone or in combination with SA1641 (final concentration of 5 µg/mL) for 48 h. Cell viability was determined by the MTT assay. Data represents the mean ± SD, n = 4.
    Figure Legend Snippet: Chemical conjugation, purification and characterization of saporin-HRP. ( A ) Cross-linkage of saporin and HRP. The reaction mixture was directly analysed by SDS-PAGE. Saporin and HRP served as unconjugated controls; ( B ) Purification of saporin-HRP by Ni-NTA chromatography with increasing concentrations of imidazole in the elution buffer. All fractions were assessed by SDS-PAGE. Saporin-HRP was eluted in Fractions 1–3 at 62 mM imidazole (see arrow); ( C ) Validation of the chemical conjugation of saporin and HRP. Saporin-HRP was analysed by SDS-PAGE, and the conjugate was visualized in the gel (see arrow). Saporin-HRP was analysed by Western blot with a primary polyclonal antibody against saporin. Saporin and the conjugate (see arrow) were specifically detected in the membrane; ( D ) Peroxidase activity of saporin-HRP. A directly proportional correlation between absorbance and concentration was observed from 0.1 to 10 nM saporin-HRP. Each data point is the mean ± SD, n = 3; Comparison of the cytotoxicity of ( E ) saporin and ( F ) saporin-HRP in the presence of SA1641. ECV-304 cells (4000 cells/well) were treated with saporin or saporin-HRP in a concentration range from 0.01 to 100 nM alone or in combination with SA1641 (final concentration of 5 µg/mL) for 48 h. Cell viability was determined by the MTT assay. Data represents the mean ± SD, n = 4.

    Techniques Used: Conjugation Assay, Purification, SDS Page, Chromatography, Western Blot, Activity Assay, Concentration Assay, MTT Assay

    15) Product Images from "Developmental gene expression profiles of the human pathogen Schistosoma japonicum"

    Article Title: Developmental gene expression profiles of the human pathogen Schistosoma japonicum

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-128

    The complex lifecycle of Schistosoma japonicum involves distinct free-living and parasitic stages (see text for details) . The numbers indicate the seven developmental stages investigated by microarray and real time PCR analysis.
    Figure Legend Snippet: The complex lifecycle of Schistosoma japonicum involves distinct free-living and parasitic stages (see text for details) . The numbers indicate the seven developmental stages investigated by microarray and real time PCR analysis.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction

    Gene Ontology (GO) analysis showing the correlation between GO categories and microarray expression data calculated using ErmineJ software . The GO annotations with parent-child analysis are presented on the left. Contributions to each of the categories from each lifecycle stage (2-fold or higher gene expression) are shaded. The overall number of genes in each category represented on the microarray and the correlation p-value associated with the entire lifecycle are shown on the right. NA, the parent category is significant but a child category is not. Genes expressed in adult parasites from weeks 6–7.
    Figure Legend Snippet: Gene Ontology (GO) analysis showing the correlation between GO categories and microarray expression data calculated using ErmineJ software . The GO annotations with parent-child analysis are presented on the left. Contributions to each of the categories from each lifecycle stage (2-fold or higher gene expression) are shaded. The overall number of genes in each category represented on the microarray and the correlation p-value associated with the entire lifecycle are shown on the right. NA, the parent category is significant but a child category is not. Genes expressed in adult parasites from weeks 6–7.

    Techniques Used: Microarray, Expressing, Software

    Validation of selected transcripts in different developmental stages of S. japonicum . The real time PCR analysis is presented as bar graphs and is shown in copy number for each gene and stage. The corresponding microarray gene expression data are presented below the bar graphs as heat maps, with up-regulated genes shown in red, down-regulated genes shown in green and unchanged genes shown in black. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.
    Figure Legend Snippet: Validation of selected transcripts in different developmental stages of S. japonicum . The real time PCR analysis is presented as bar graphs and is shown in copy number for each gene and stage. The corresponding microarray gene expression data are presented below the bar graphs as heat maps, with up-regulated genes shown in red, down-regulated genes shown in green and unchanged genes shown in black. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, Expressing

    16) Product Images from "Do anesthetics and sampling strategies affect transcription analysis of fish tissues?"

    Article Title: Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-48

    NanoDrop RNA quality assessment (Abs 260/280 nm ratio) of A) liver, B) gills, C) head kidney and D) brain tissues of Atlantic salmon either flash frozen in liquefied nitrogen (N 2 ) or preserved in RNA later immediately after sampling in the six treatment groups. n = 6 in all groups except 120 min. metacaine and 120 min. isoeugenol groups, n = 5. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). Overall Kruskal-Wallis P-values are shown in the figures.
    Figure Legend Snippet: NanoDrop RNA quality assessment (Abs 260/280 nm ratio) of A) liver, B) gills, C) head kidney and D) brain tissues of Atlantic salmon either flash frozen in liquefied nitrogen (N 2 ) or preserved in RNA later immediately after sampling in the six treatment groups. n = 6 in all groups except 120 min. metacaine and 120 min. isoeugenol groups, n = 5. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). Overall Kruskal-Wallis P-values are shown in the figures.

    Techniques Used: Sampling

    17) Product Images from "Do anesthetics and sampling strategies affect transcription analysis of fish tissues?"

    Article Title: Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-48

    NanoDrop RNA quality assessment (Abs 260/280 nm ratio) of A) liver, B) gills, C) head kidney and D) brain tissues of Atlantic salmon either flash frozen in liquefied nitrogen (N 2 ) or preserved in RNA later immediately after sampling in the six treatment groups. n = 6 in all groups except 120 min. metacaine and 120 min. isoeugenol groups, n = 5. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). Overall Kruskal-Wallis P-values are shown in the figures.
    Figure Legend Snippet: NanoDrop RNA quality assessment (Abs 260/280 nm ratio) of A) liver, B) gills, C) head kidney and D) brain tissues of Atlantic salmon either flash frozen in liquefied nitrogen (N 2 ) or preserved in RNA later immediately after sampling in the six treatment groups. n = 6 in all groups except 120 min. metacaine and 120 min. isoeugenol groups, n = 5. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). Overall Kruskal-Wallis P-values are shown in the figures.

    Techniques Used: Sampling

    18) Product Images from "Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas"

    Article Title: Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-2-23

    Bioanalyzer profiles of total RNA (A) and cRNA (B) of matched FF and FFPE ovarian serous adenocarcinoma samples 3136, 3138, 3194, 3207 and 390 . The method used for RNA extraction (Qiagen, Agencourt, Ambion) is indicated for each sample type. The RNA Integrity Number (RIN) is shown next to each total RNA profile.
    Figure Legend Snippet: Bioanalyzer profiles of total RNA (A) and cRNA (B) of matched FF and FFPE ovarian serous adenocarcinoma samples 3136, 3138, 3194, 3207 and 390 . The method used for RNA extraction (Qiagen, Agencourt, Ambion) is indicated for each sample type. The RNA Integrity Number (RIN) is shown next to each total RNA profile.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction

    19) Product Images from "Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair-infarcted myocardium"

    Article Title: Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair-infarcted myocardium

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01568.x

    Pre-conditioning of Aged MSCs. (A) RT-PCR analysis for the expression of AKT, IGF-1 and SIRT-1 in aged control and pre-treated MSCs. (B) Gel quantification of RT-PCR bands. (C) Gene expression of VEGF , SDF-1 and FGF-2 after 1 hr of pre-conditioning. (D) Gel quantification of all genes using Image J software. (E) Effect of caloric restriction on cell proliferation of aged MSCs. Absorbance was measured at 450nm and was higher in 1 hr pre-treated group. (F) Cell viability assay performed in a luminometer. The results showed a significant increase in viability of aged MSCs after 1 and 6hr pre-treatment compared with untreated aged MSCs. (G–I) Senescence associated β-galactosidase staining after 8, 16 and 24 days. (G1 and G2) after 8 days, (H1 and H2) after 16 hrs, (I1 and I2) after 24 hrs. (J) Percentage expression of senescence associated β-gal. The number of β-gal positive cells was significantly higher in aged untreated MSCs than aged pre-treated MSCs. The values are expressed in mean ± SEM. * P
    Figure Legend Snippet: Pre-conditioning of Aged MSCs. (A) RT-PCR analysis for the expression of AKT, IGF-1 and SIRT-1 in aged control and pre-treated MSCs. (B) Gel quantification of RT-PCR bands. (C) Gene expression of VEGF , SDF-1 and FGF-2 after 1 hr of pre-conditioning. (D) Gel quantification of all genes using Image J software. (E) Effect of caloric restriction on cell proliferation of aged MSCs. Absorbance was measured at 450nm and was higher in 1 hr pre-treated group. (F) Cell viability assay performed in a luminometer. The results showed a significant increase in viability of aged MSCs after 1 and 6hr pre-treatment compared with untreated aged MSCs. (G–I) Senescence associated β-galactosidase staining after 8, 16 and 24 days. (G1 and G2) after 8 days, (H1 and H2) after 16 hrs, (I1 and I2) after 24 hrs. (J) Percentage expression of senescence associated β-gal. The number of β-gal positive cells was significantly higher in aged untreated MSCs than aged pre-treated MSCs. The values are expressed in mean ± SEM. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Viability Assay, Staining

    Enhanced paracrine signalling, angiogenesis and cardiac transcription factors in senescent hearts after transplantation of pre-conditioned aged MSCs. (A) RT-PCR shows increased expression of IGF-1 , FGF-2 , VEGF and SDF-1α 7 and 30 days after transplantation in group III hearts. (B) Quantification of gel bands with Image J software. (C) Increased expression of cardiac transcription factors in senescent hearts of group III animals, 7 and 30 days after transplantation as measured by qRT-PCR. (D–F) Blood vessel density analysis by fluorescent immunostaining for the expression of endothelial marker CD31 in all three groups. Nuclei were stained with DAPI. (G) Quantification of CD31 expression in all three groups. (H–J) SMA expression in group I, II and III transplanted hearts. (K) Percentage blood vessel density as measured by SMA expression in all three groups. * P
    Figure Legend Snippet: Enhanced paracrine signalling, angiogenesis and cardiac transcription factors in senescent hearts after transplantation of pre-conditioned aged MSCs. (A) RT-PCR shows increased expression of IGF-1 , FGF-2 , VEGF and SDF-1α 7 and 30 days after transplantation in group III hearts. (B) Quantification of gel bands with Image J software. (C) Increased expression of cardiac transcription factors in senescent hearts of group III animals, 7 and 30 days after transplantation as measured by qRT-PCR. (D–F) Blood vessel density analysis by fluorescent immunostaining for the expression of endothelial marker CD31 in all three groups. Nuclei were stained with DAPI. (G) Quantification of CD31 expression in all three groups. (H–J) SMA expression in group I, II and III transplanted hearts. (K) Percentage blood vessel density as measured by SMA expression in all three groups. * P

    Techniques Used: Transplantation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Quantitative RT-PCR, Immunostaining, Marker, Staining

    20) Product Images from " Application of Novel PCR-Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances"

    Article Title: Application of Novel PCR-Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances

    Journal: mBio

    doi: 10.1128/mBio.00261-11

    PCR-based detection of Sutterella 16S rRNA gene sequences (V6–V8 region and C4–V8 region) in biopsies from AUT-GI and Control-GI patients. (A) Agarose gel detection of 260-bp Sutterella products in ileal (4 biopsy samples/patient) and cecal (4 biopsy samples/patient) biopsy DNA using SuttFor and SuttRev primers (V6–V8 region) in conventional PCR assays. (B) Agarose gel detection of 715-bp Sutterella products in ileal and cecal biopsy DNA using pan-bacterial primer 515For and SuttRev primer (C4–V8) in conventional PCR assays. The negative control is PCR reagents with water substituted for DNA. The positive control is DNA isolated from cultured S. wadsworthensis (ATCC 51579).
    Figure Legend Snippet: PCR-based detection of Sutterella 16S rRNA gene sequences (V6–V8 region and C4–V8 region) in biopsies from AUT-GI and Control-GI patients. (A) Agarose gel detection of 260-bp Sutterella products in ileal (4 biopsy samples/patient) and cecal (4 biopsy samples/patient) biopsy DNA using SuttFor and SuttRev primers (V6–V8 region) in conventional PCR assays. (B) Agarose gel detection of 715-bp Sutterella products in ileal and cecal biopsy DNA using pan-bacterial primer 515For and SuttRev primer (C4–V8) in conventional PCR assays. The negative control is PCR reagents with water substituted for DNA. The positive control is DNA isolated from cultured S. wadsworthensis (ATCC 51579).

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Positive Control, Isolation, Cell Culture

    Western immunoblot analysis of AUT-GI and Control-GI patients’ plasma antibody immunoreactivity against S. wadsworthensis antigens. (A) Patients’ plasma IgG antibody immunoreactivity against S. wadsworthensis antigens. (B) Patients’ IgM antibody immunoreactivity against S. wadsworthensis antigens. 2°, secondary antibody control.
    Figure Legend Snippet: Western immunoblot analysis of AUT-GI and Control-GI patients’ plasma antibody immunoreactivity against S. wadsworthensis antigens. (A) Patients’ plasma IgG antibody immunoreactivity against S. wadsworthensis antigens. (B) Patients’ IgM antibody immunoreactivity against S. wadsworthensis antigens. 2°, secondary antibody control.

    Techniques Used: Western Blot

    21) Product Images from "Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region"

    Article Title: Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007215

    Mapping of IgM regions required for allotype-restricted binding to gp41 MPER. A) Surface plasmon resonance analysis of IgM a and IgM b binding to gp41 MPER. Monomeric or F(ab) 2 fragments derived from IgM a and IgM b mAbs were injected over biotinylated (B) gp41 MPER nominal epitope peptide, anchored to a SA-coated sensor chip, as shown in the top left-hand schema, and described in materials and methods . mAbs 17b and 2F5 were run as negative and positive controls for gp41 MPER binding, respectively. Results are representative of two independent experiments. B) V H family usage in gp41 MPER-sorted IgM + B cells from naïve BALB/c mice. Independent 5′ RACE clones (n) were derived from purified BALB/c splenic B cell populations, either unsorted or sorted into gp41 MPER + fractions and subjected to IgM-specific 5′ RACE analysis, as described in Materials and Methods . V H family frequencies within expressed V H repertoires was determined by Ig Blast analysis of sequences. Statistical comparisons between unsorted and gp41 MPER+ fractions were performed using the Fisher Exact test (*, p
    Figure Legend Snippet: Mapping of IgM regions required for allotype-restricted binding to gp41 MPER. A) Surface plasmon resonance analysis of IgM a and IgM b binding to gp41 MPER. Monomeric or F(ab) 2 fragments derived from IgM a and IgM b mAbs were injected over biotinylated (B) gp41 MPER nominal epitope peptide, anchored to a SA-coated sensor chip, as shown in the top left-hand schema, and described in materials and methods . mAbs 17b and 2F5 were run as negative and positive controls for gp41 MPER binding, respectively. Results are representative of two independent experiments. B) V H family usage in gp41 MPER-sorted IgM + B cells from naïve BALB/c mice. Independent 5′ RACE clones (n) were derived from purified BALB/c splenic B cell populations, either unsorted or sorted into gp41 MPER + fractions and subjected to IgM-specific 5′ RACE analysis, as described in Materials and Methods . V H family frequencies within expressed V H repertoires was determined by Ig Blast analysis of sequences. Statistical comparisons between unsorted and gp41 MPER+ fractions were performed using the Fisher Exact test (*, p

    Techniques Used: Binding Assay, SPR Assay, Derivative Assay, Injection, Chromatin Immunoprecipitation, Mouse Assay, Purification

    22) Product Images from "Expression of Xhdsi-1VOC, a novel member of the vicinal oxygen chelate (VOC) metalloenzyme superfamily, is up-regulated in leaves and roots during desiccation in the resurrection plant Xerophyta humilis (Bak) Dur and Schinz"

    Article Title: Expression of Xhdsi-1VOC, a novel member of the vicinal oxygen chelate (VOC) metalloenzyme superfamily, is up-regulated in leaves and roots during desiccation in the resurrection plant Xerophyta humilis (Bak) Dur and Schinz

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ern226

    Phylogenetic relationship amongst plant dsi-1 VOC orthologues including X . humilis , rice ( Oryza sativa ), wheat ( Triticum aestivum ), barley ( Hordeum vulgare ), maize ( Zea mays ), guar ( Cyamopsis tetragonoloba ), runner bean ( Phaseolus coccineus ), soybean ( Glycine max ), walnut ( Juglans regia ), spotted knapweed ( Centaurea maculosa ), oilseed rape ( Brassica napus ), Arabidopsis thaliana , and Loblolly pine ( Pinus taeda ). A Neighbor–Joining tree based on the alignment of full-length amino acid sequences shown in Fig. 2 , was constructed using the Jones–Taylor–Thornton matrix. Bootstrap values are given at each node. The Loblolly pine ( Pinus taeda ) sequence was used to root the tree. Genbank and TIGR EST database accession numbers are indicated.
    Figure Legend Snippet: Phylogenetic relationship amongst plant dsi-1 VOC orthologues including X . humilis , rice ( Oryza sativa ), wheat ( Triticum aestivum ), barley ( Hordeum vulgare ), maize ( Zea mays ), guar ( Cyamopsis tetragonoloba ), runner bean ( Phaseolus coccineus ), soybean ( Glycine max ), walnut ( Juglans regia ), spotted knapweed ( Centaurea maculosa ), oilseed rape ( Brassica napus ), Arabidopsis thaliana , and Loblolly pine ( Pinus taeda ). A Neighbor–Joining tree based on the alignment of full-length amino acid sequences shown in Fig. 2 , was constructed using the Jones–Taylor–Thornton matrix. Bootstrap values are given at each node. The Loblolly pine ( Pinus taeda ) sequence was used to root the tree. Genbank and TIGR EST database accession numbers are indicated.

    Techniques Used: Construct, Sequencing

    RT-PCR analysis of HC205/At1g07645 mRNA transcript abundance in seed and leaf tissues of X . humilis and A . thaliana . (A) Products of amplification of HC205 mRNA transcripts in X . humilis leaf tissue (100% and 36% RWC) and in mature dry seeds, and amplification of At1g07645 mRNA transcripts in 3-week-old seedlings and mature dry seeds of A . thaliana are indicated. Amplification of 18S rRNA transcripts are shown as a control for relative abundance of sample RNA. (B) Amplification of At1g07645 , LEA2, and ubiquitin mRNA transcripts in mature seeds and 2-week-old A . thaliana seedlings exposed to mannitol, salt, and dehydration stress for 4 h. Controls include A . thaliana seedlings prior to stress treatments. Amplification of LEA 2 mRNA transcripts was included as a positive control to show activation of the abiotic stress response. Ubiquitin mRNA transcripts are expected to be expressed at similar levels in all samples, and were included to control for starting sample RNA concentrations. For each RT-PCR reaction, (+) indicates the presence, and (–) the absence, of reverse transcriptase in the cDNA synthesis reactions.
    Figure Legend Snippet: RT-PCR analysis of HC205/At1g07645 mRNA transcript abundance in seed and leaf tissues of X . humilis and A . thaliana . (A) Products of amplification of HC205 mRNA transcripts in X . humilis leaf tissue (100% and 36% RWC) and in mature dry seeds, and amplification of At1g07645 mRNA transcripts in 3-week-old seedlings and mature dry seeds of A . thaliana are indicated. Amplification of 18S rRNA transcripts are shown as a control for relative abundance of sample RNA. (B) Amplification of At1g07645 , LEA2, and ubiquitin mRNA transcripts in mature seeds and 2-week-old A . thaliana seedlings exposed to mannitol, salt, and dehydration stress for 4 h. Controls include A . thaliana seedlings prior to stress treatments. Amplification of LEA 2 mRNA transcripts was included as a positive control to show activation of the abiotic stress response. Ubiquitin mRNA transcripts are expected to be expressed at similar levels in all samples, and were included to control for starting sample RNA concentrations. For each RT-PCR reaction, (+) indicates the presence, and (–) the absence, of reverse transcriptase in the cDNA synthesis reactions.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Activation Assay

    ClustalW alignment of predicted amino acid sequences of plant dsi-1 VOC orthologues identified in the Genbank and TIGR EST databases, including X . humilis , rice ( Oryza sativa ), wheat ( Triticum aestivum ), barley ( Hordeum vulgare ), maize ( Zea mays ), guar ( Cyamopsis tetragonoloba ), runner bean ( Phaseolus coccineus ), soybean ( Glycine max ), walnut ( Juglans regia ), spotted knapweed ( Centaurea maculosa ), oilseed rape ( Brassica napus ), Arabidopsis thaliana , and Loblolly pine ( Pinus taeda ). Boxed amino acids demarcate conserved phosphorylation sites for protein kinase C (PKC) and protein casein kinase II (CK2) identified by PROSITE, and a potential disulphide bonding site predicted by DISULFIND. Amino acids in the X . humilis and A . thaliana dsi-1 VOC orthologues which are predicted to fold into the βαβββ structural repeats characteristic of the VOC superfamily, are underlined.
    Figure Legend Snippet: ClustalW alignment of predicted amino acid sequences of plant dsi-1 VOC orthologues identified in the Genbank and TIGR EST databases, including X . humilis , rice ( Oryza sativa ), wheat ( Triticum aestivum ), barley ( Hordeum vulgare ), maize ( Zea mays ), guar ( Cyamopsis tetragonoloba ), runner bean ( Phaseolus coccineus ), soybean ( Glycine max ), walnut ( Juglans regia ), spotted knapweed ( Centaurea maculosa ), oilseed rape ( Brassica napus ), Arabidopsis thaliana , and Loblolly pine ( Pinus taeda ). Boxed amino acids demarcate conserved phosphorylation sites for protein kinase C (PKC) and protein casein kinase II (CK2) identified by PROSITE, and a potential disulphide bonding site predicted by DISULFIND. Amino acids in the X . humilis and A . thaliana dsi-1 VOC orthologues which are predicted to fold into the βαβββ structural repeats characteristic of the VOC superfamily, are underlined.

    Techniques Used:

    ClustalW alignment of amino acid sequences of X . humilis HC205 (AY570978) and the A . thaliana orthologue (At1g07645), with known glyoxalase I genes, including L07837 ( Homo sapiens ), Y13239 ( Brassica juncea ), At1g08110 ( A . thaliana ), and Z48183 ( Lycopersicon esculentum ). The asterisks represent amino acids that are identical in all of the six sequences. The βαβββ structural repeat determined in the X-ray crystal structure of the human glyoxalase I protein is indicated. This structural repeat is represented by β 1 α 2 β 2 β 3 β 4 in the first domain (α 1 −β 4 ), and by β 5 α 4 β 6 β 7 β 8 in the second domain (β 5 −α 6 ) ( Cameron et al. , 1997 ). Conserved amino acids that constitute the glutathione binding sites of glyoxalase I are highlighted in grey. Conserved amino acids which form the zinc binding sites are in bold and are boxed.
    Figure Legend Snippet: ClustalW alignment of amino acid sequences of X . humilis HC205 (AY570978) and the A . thaliana orthologue (At1g07645), with known glyoxalase I genes, including L07837 ( Homo sapiens ), Y13239 ( Brassica juncea ), At1g08110 ( A . thaliana ), and Z48183 ( Lycopersicon esculentum ). The asterisks represent amino acids that are identical in all of the six sequences. The βαβββ structural repeat determined in the X-ray crystal structure of the human glyoxalase I protein is indicated. This structural repeat is represented by β 1 α 2 β 2 β 3 β 4 in the first domain (α 1 −β 4 ), and by β 5 α 4 β 6 β 7 β 8 in the second domain (β 5 −α 6 ) ( Cameron et al. , 1997 ). Conserved amino acids that constitute the glutathione binding sites of glyoxalase I are highlighted in grey. Conserved amino acids which form the zinc binding sites are in bold and are boxed.

    Techniques Used: Binding Assay

    23) Product Images from "Selected imprinting of INS in the marsupial"

    Article Title: Selected imprinting of INS in the marsupial

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-5-14

    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.
    Figure Legend Snippet: INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Techniques Used: Sequencing, Expressing, Produced

    Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.
    Figure Legend Snippet: Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Techniques Used: Methylation, Rapid Amplification of cDNA Ends, Derivative Assay, Amplification

    24) Product Images from "Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis"

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123904

    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value
    Figure Legend Snippet: Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Techniques Used: Mouse Assay, Infection, Injection, Isolation, RNA Sequencing Assay

    Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p
    Figure Legend Snippet: Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Techniques Used: Expressing, Infection, Mouse Assay, Isolation, Quantitative RT-PCR

    Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p
    Figure Legend Snippet: Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Techniques Used: Expressing, Infection, Mouse Assay, Injection, Isolation, RNA Sequencing Assay, Quantitative RT-PCR

    25) Product Images from "3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways"

    Article Title: 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2015.03.035

    Experimental design to compare 2D versus 3D hypoxic response A) Schematic illustrating the fabrication of microscale alginate scaffolds. Cells were suspended within alginate and cast onto an array of 200 μm-deep × 4 mm-diameter wells. CaCl 2 solution was applied through a filtration membrane to crosslink the gels. Scaffolds were removed from the mold and cultured in suspension. B) Experimental design comprised culturing OSCC-3 cells on conventional tissue culture polystyrene (2D) or embedded within microfabricated alginate discs (3D) inside ambient (17% O 2 ) or hypoxic (1% O 2 ) incubators for 6 days. Subsequently, total RNA was isolated and transcriptional changes analyzed by Affymetrix GeneChip microarray.
    Figure Legend Snippet: Experimental design to compare 2D versus 3D hypoxic response A) Schematic illustrating the fabrication of microscale alginate scaffolds. Cells were suspended within alginate and cast onto an array of 200 μm-deep × 4 mm-diameter wells. CaCl 2 solution was applied through a filtration membrane to crosslink the gels. Scaffolds were removed from the mold and cultured in suspension. B) Experimental design comprised culturing OSCC-3 cells on conventional tissue culture polystyrene (2D) or embedded within microfabricated alginate discs (3D) inside ambient (17% O 2 ) or hypoxic (1% O 2 ) incubators for 6 days. Subsequently, total RNA was isolated and transcriptional changes analyzed by Affymetrix GeneChip microarray.

    Techniques Used: Filtration, Cell Culture, Isolation, Microarray

    26) Product Images from "Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5"

    Article Title: Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20111569

    NFAT5-dependent activation of the Nos2 promoter and iNOS induction. Activity of the hypertonicity-responsive ORE-Luc reporter (A) and the LPS-responsive mouse Nos2 promoter (iNOS-Luc; B) in RAW 264.7 cells cotransfected with a short hairpin RNA (shRNA) vector specific for GFP or two independent NFAT5-specific shRNA vectors (N5-1 and N5-2), together with the control reporter plasmid TK-Renilla. Luciferase was measured 20 h after hypertonicity treatment (500 mOsm/kg) or LPS stimulation (25 µg/ml), normalized to TK-Renilla, and represented as percentage of reporter activity with respect to cells transfected with shGFP and stimulated (100%). Graphs show the mean ± SEM of four independent experiments. Bottom panels show the Western blot for NFAT5 done in parallel to the reporter assays. Pyruvate kinase (PyrK) is shown as loading control. Results are representative of three independent experiments. (C) Activity of WT mouse Nos2 promoter construct (WT) or an NFAT5-binding site mutant (NFAT5 mut) in RAW 264.7 cells after 20 h of LPS stimulation. Luciferase activity normalized to TK-Renilla is represented as fold induction relative to the reporter activity in unstimulated cells. Graphs show the mean ± SEM of three independent experiments. (D) Activation of the Nos2 promoter in response to different TLR agonists was measured in RAW 264.7 cells cotransfected with the iNOS-Luc and TK-Renilla reporters plus either shGFP or shN5-1 vectors. Transfected cells were stimulated for 20 h with 1 µg/ml Pam3CSK4 (P3C), 300 µg/ml zymosan A (Zym), 100 µg/ml poly I:C (pIC), 25 µg/ml LPS, 1 mM loxoribine (Lox), or 1 µM CpG oligodeoxynucleotide (CpG). Luciferase activity normalized to TK-Renilla is represented as fold induction over the reporter activity in unstimulated cells (−). Graphics show the mean ± SEM of three independent experiments. (E) RAW 264.7 cells transfected with either shGFP or shN5-1 vectors were left untreated or stimulated with different TLR ligands as in D. Expression of NFAT5, iNOS, and pyruvate kinase (normalization control) was detected by Western blotting. The experiment shown is representative of three independently performed. (F) Nitric oxide production upon 24 h of stimulation with 100 µg/ml pIC or 25 µg/ml LPS in RAW 264.7 cells transfected with either shGFP or shN5-1 vectors. Graphics show the mean ± SEM of three independent experiments.
    Figure Legend Snippet: NFAT5-dependent activation of the Nos2 promoter and iNOS induction. Activity of the hypertonicity-responsive ORE-Luc reporter (A) and the LPS-responsive mouse Nos2 promoter (iNOS-Luc; B) in RAW 264.7 cells cotransfected with a short hairpin RNA (shRNA) vector specific for GFP or two independent NFAT5-specific shRNA vectors (N5-1 and N5-2), together with the control reporter plasmid TK-Renilla. Luciferase was measured 20 h after hypertonicity treatment (500 mOsm/kg) or LPS stimulation (25 µg/ml), normalized to TK-Renilla, and represented as percentage of reporter activity with respect to cells transfected with shGFP and stimulated (100%). Graphs show the mean ± SEM of four independent experiments. Bottom panels show the Western blot for NFAT5 done in parallel to the reporter assays. Pyruvate kinase (PyrK) is shown as loading control. Results are representative of three independent experiments. (C) Activity of WT mouse Nos2 promoter construct (WT) or an NFAT5-binding site mutant (NFAT5 mut) in RAW 264.7 cells after 20 h of LPS stimulation. Luciferase activity normalized to TK-Renilla is represented as fold induction relative to the reporter activity in unstimulated cells. Graphs show the mean ± SEM of three independent experiments. (D) Activation of the Nos2 promoter in response to different TLR agonists was measured in RAW 264.7 cells cotransfected with the iNOS-Luc and TK-Renilla reporters plus either shGFP or shN5-1 vectors. Transfected cells were stimulated for 20 h with 1 µg/ml Pam3CSK4 (P3C), 300 µg/ml zymosan A (Zym), 100 µg/ml poly I:C (pIC), 25 µg/ml LPS, 1 mM loxoribine (Lox), or 1 µM CpG oligodeoxynucleotide (CpG). Luciferase activity normalized to TK-Renilla is represented as fold induction over the reporter activity in unstimulated cells (−). Graphics show the mean ± SEM of three independent experiments. (E) RAW 264.7 cells transfected with either shGFP or shN5-1 vectors were left untreated or stimulated with different TLR ligands as in D. Expression of NFAT5, iNOS, and pyruvate kinase (normalization control) was detected by Western blotting. The experiment shown is representative of three independently performed. (F) Nitric oxide production upon 24 h of stimulation with 100 µg/ml pIC or 25 µg/ml LPS in RAW 264.7 cells transfected with either shGFP or shN5-1 vectors. Graphics show the mean ± SEM of three independent experiments.

    Techniques Used: Activation Assay, Activity Assay, shRNA, Plasmid Preparation, Luciferase, Transfection, Western Blot, Construct, Binding Assay, Mutagenesis, Expressing

    27) Product Images from "PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability"

    Article Title: PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1713912115

    The RGG motif of CIRBP is involved in PAR polymer binding. ( A ) Key residues in RRM domain that are responsible for RNA binding. The structure was adapted from Protein Data Bank ID code 1X5S. The key RNA-interacting residues, including F15, F49, and F51, are labeled in magenta. In 3F/A mutant, the three F residues were mutated to A. ( B ) Sequences of CIRBP WT and R to A mutants. In 4R/A mutant, R91, R94, R116, and R121 were mutated to A. In 9R/A mutant, R91, R94, R101, R105, R 108, R110, R112, R116, and R121 were mutated to A. ( C ) Arginines in RGG motifs mediate the recruitment of CIRBP to laser-microirradiated regions. EGFP-tagged WT, 3F/A, 4R/A, and 9R/A CIRBP were overexpressed in U2OS cells, and the fluorescent signals at laser microirradiation sites were recorded. ( D ) Quantification of the results in C . ( E ) Dose-dependent PAR binding of WT CIRBP. Increasing amounts of recombinant His-tagged WT CIRBP (2.5, 5, 10, 20, and 40 ng) were dot-blotted on nitrocellulose membrane and then incubated with PAR polymers. Binding of CIRBP to PAR polymers was visualized by anti-PAR antibody. ( F ) Quantification of the results in E . The signal intensity of PAR binding of each dot was analyzed using Fuji ImageJ software and normalized to that of 40 ng of membrane-bound CIRBP. Data are presented as mean ± SD from duplicate experiments. ( G ) In vitro PAR binding of CIRBP WT and mutants. His-tagged CIRBP WT, 3F/A, and 9R/A mutants (8 ng) were used to perform an in vitro PAR-binding assay. Histone 1 (H1) and BSA served as positive and negative controls, respectively. ( H ) Quantification of the results in G . The intensities of PAR-binding signals of different proteins were normalized to the WT CIRBP. Data are presented as mean ± SD from experiments done in triplicate.
    Figure Legend Snippet: The RGG motif of CIRBP is involved in PAR polymer binding. ( A ) Key residues in RRM domain that are responsible for RNA binding. The structure was adapted from Protein Data Bank ID code 1X5S. The key RNA-interacting residues, including F15, F49, and F51, are labeled in magenta. In 3F/A mutant, the three F residues were mutated to A. ( B ) Sequences of CIRBP WT and R to A mutants. In 4R/A mutant, R91, R94, R116, and R121 were mutated to A. In 9R/A mutant, R91, R94, R101, R105, R 108, R110, R112, R116, and R121 were mutated to A. ( C ) Arginines in RGG motifs mediate the recruitment of CIRBP to laser-microirradiated regions. EGFP-tagged WT, 3F/A, 4R/A, and 9R/A CIRBP were overexpressed in U2OS cells, and the fluorescent signals at laser microirradiation sites were recorded. ( D ) Quantification of the results in C . ( E ) Dose-dependent PAR binding of WT CIRBP. Increasing amounts of recombinant His-tagged WT CIRBP (2.5, 5, 10, 20, and 40 ng) were dot-blotted on nitrocellulose membrane and then incubated with PAR polymers. Binding of CIRBP to PAR polymers was visualized by anti-PAR antibody. ( F ) Quantification of the results in E . The signal intensity of PAR binding of each dot was analyzed using Fuji ImageJ software and normalized to that of 40 ng of membrane-bound CIRBP. Data are presented as mean ± SD from duplicate experiments. ( G ) In vitro PAR binding of CIRBP WT and mutants. His-tagged CIRBP WT, 3F/A, and 9R/A mutants (8 ng) were used to perform an in vitro PAR-binding assay. Histone 1 (H1) and BSA served as positive and negative controls, respectively. ( H ) Quantification of the results in G . The intensities of PAR-binding signals of different proteins were normalized to the WT CIRBP. Data are presented as mean ± SD from experiments done in triplicate.

    Techniques Used: Binding Assay, RNA Binding Assay, Labeling, Mutagenesis, Recombinant, Incubation, Software, In Vitro

    28) Product Images from "MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA"

    Article Title: MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-016-0070-z

    Knockdown SMG5 expression repressed NMD activity. a Quantitative polymerase chain reaction (qPCR) analysis of SMG5 mRNA expression in C2C12 cells transfected for 24 h with either three RNA interference fragments or negative control, positive control and Mock respectively. b Western Blot analysis of endogenous SMG5 protein levels in C2C12 cells 24 h after transfection with the RNA interference fragment or a negative control fragment. β -actin was used as the internal control. c – f showed the qPCR and western blot analysis of NMD substrates, TBL2 and GADD45B , mRNA and protein level in C2C12 cells 24 h after transfection with the RNA interference fragment or a negative control fragment. β -actin was used as the internal control. *P
    Figure Legend Snippet: Knockdown SMG5 expression repressed NMD activity. a Quantitative polymerase chain reaction (qPCR) analysis of SMG5 mRNA expression in C2C12 cells transfected for 24 h with either three RNA interference fragments or negative control, positive control and Mock respectively. b Western Blot analysis of endogenous SMG5 protein levels in C2C12 cells 24 h after transfection with the RNA interference fragment or a negative control fragment. β -actin was used as the internal control. c – f showed the qPCR and western blot analysis of NMD substrates, TBL2 and GADD45B , mRNA and protein level in C2C12 cells 24 h after transfection with the RNA interference fragment or a negative control fragment. β -actin was used as the internal control. *P

    Techniques Used: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Positive Control, Western Blot

    29) Product Images from "Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro"

    Article Title: Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro

    Journal: Journal of Orthopaedic Surgery and Research

    doi: 10.1186/s13018-018-0800-7

    Effect of alcohol on primary cultured MSC integrin β1 mRNA and protein expression. Primary rat MSC were cultured in media alone or media plus 50 mM ethanol for 24 h. Cells were harvested and used for a mRNA or b total protein isolation as described. Int β1 mRNA levels were assessed by qRT-PCR as described. Int β1 protein levels were assessed by western blot analysis as described. a mRNA: media vs EtOH p = 0.0021. b Protein: media vs EtOH p = 0.0030. Each experiment was repeated at least three times utilizing unique primary MSC cultures. * p
    Figure Legend Snippet: Effect of alcohol on primary cultured MSC integrin β1 mRNA and protein expression. Primary rat MSC were cultured in media alone or media plus 50 mM ethanol for 24 h. Cells were harvested and used for a mRNA or b total protein isolation as described. Int β1 mRNA levels were assessed by qRT-PCR as described. Int β1 protein levels were assessed by western blot analysis as described. a mRNA: media vs EtOH p = 0.0021. b Protein: media vs EtOH p = 0.0030. Each experiment was repeated at least three times utilizing unique primary MSC cultures. * p

    Techniques Used: Cell Culture, Expressing, Isolation, Quantitative RT-PCR, Western Blot

    30) Product Images from "ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue"

    Article Title: ICOS signaling limits regulatory T cell accumulation and function in visceral adipose tissue

    Journal: bioRxiv

    doi: 10.1101/2020.06.01.128504

    Increased VAT-T R accumulation in the absence of ICOS signaling is associated with elevated CCR3 expression. (A) ST2 expression in splenic T R in mice aged 8-16 weeks. (B) ST2 expression in CD45.1 + and CD45.2 + donor splenic T R in WT:YF and WT:KO chimeric mice. Line in graph connects CD45.1 + and CD45.2 + donor splenic T R within the same chimera. (C) Expression of CCR3 in VAT-T R in mice ≤8 wk and > 8 wk of age. (D) Expression of CCR2 and CCR3 by VAT-T R . (E) Expression of indicated CCR3 ligands in total VAT normalized to Tbp as measured by qPCR. (F) (Left) Schematic of in vitro culture experiments examining the impact of ICOS signaling on CCR3 expression. (Middle) Graphs indicating fold change in T R frequency of CD4 + cells (top) and %CCR3 + of T R (bottom) between individual culture samples stimulated with or without αICOS for 2 d. (Right) Representative flow cytometry plots with frequency of CCR3 + T R after 2 d in specified culture conditions. (G) Groups of WT and YF mice were treated intraperitoneally with blocking antibodies against CCL11 and CCL24 every 5 days for 2 wk. (Left) Representative flow cytometry plots indicating VAT-T R frequency with or without CCL11/24 blockade. Graphs summarize T R frequencies in indicated tissues after 2 wk. Data in (G) are from 1 experiment with n = 3-4 mice per group. For all other experiments, flow cytometry plots are representative of data from 2 or more independent experiments. Graphical data are compiled from 2 or more independent experiments with n = 3-4 per group per experiment. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A, C) , two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (B) , and two-tailed Student’s t test (F, G) . All data are presented as mean values ± SD.
    Figure Legend Snippet: Increased VAT-T R accumulation in the absence of ICOS signaling is associated with elevated CCR3 expression. (A) ST2 expression in splenic T R in mice aged 8-16 weeks. (B) ST2 expression in CD45.1 + and CD45.2 + donor splenic T R in WT:YF and WT:KO chimeric mice. Line in graph connects CD45.1 + and CD45.2 + donor splenic T R within the same chimera. (C) Expression of CCR3 in VAT-T R in mice ≤8 wk and > 8 wk of age. (D) Expression of CCR2 and CCR3 by VAT-T R . (E) Expression of indicated CCR3 ligands in total VAT normalized to Tbp as measured by qPCR. (F) (Left) Schematic of in vitro culture experiments examining the impact of ICOS signaling on CCR3 expression. (Middle) Graphs indicating fold change in T R frequency of CD4 + cells (top) and %CCR3 + of T R (bottom) between individual culture samples stimulated with or without αICOS for 2 d. (Right) Representative flow cytometry plots with frequency of CCR3 + T R after 2 d in specified culture conditions. (G) Groups of WT and YF mice were treated intraperitoneally with blocking antibodies against CCL11 and CCL24 every 5 days for 2 wk. (Left) Representative flow cytometry plots indicating VAT-T R frequency with or without CCL11/24 blockade. Graphs summarize T R frequencies in indicated tissues after 2 wk. Data in (G) are from 1 experiment with n = 3-4 mice per group. For all other experiments, flow cytometry plots are representative of data from 2 or more independent experiments. Graphical data are compiled from 2 or more independent experiments with n = 3-4 per group per experiment. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A, C) , two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (B) , and two-tailed Student’s t test (F, G) . All data are presented as mean values ± SD.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, In Vitro, Flow Cytometry, Blocking Assay, Two Tailed Test

    31) Product Images from "Complex regional pain syndrome patient IgM has pronociceptive effects in the skin and spinal cord of tibia fracture mice"

    Article Title: Complex regional pain syndrome patient IgM has pronociceptive effects in the skin and spinal cord of tibia fracture mice

    Journal: Pain

    doi: 10.1097/j.pain.0000000000001765

    CRPS patient serum and IgM had pronociceptive effects in B cell deficient fracture mice. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited unilateral hindpaw von Frey allodynia ( A ) and unweighting ( B ). After intraperitoneal injection of early (1–12 months post injury) CRPS patient serum (0.5ml, I.P) or IgM (500ug/1ml, I.P.) into 3 weeks post FX muMT mice, the mice gradually developed increased allodynia and unweighting over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 2 weeks post-injection. The pronociceptive effects of the CRPS serum were restricted to the FX limb and there was no serum effect on post FX hindpaw edema or warmth (data not shown). No pronociceptive effects were observed after intraperitoneal injection of early CRPS patient IgG (5mg/1ml, I.P.) or after injection of control subject serum (0.5ml, I.P.) in 3 weeks post-FX mice. Measurements for ( A ) represent the difference between the FX side and contralateral paw, thus a negative value represents a decrease in mechanical withdrawal thresholds on the affected side. Measurements for ( B ) represent weight-bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SEM, n = 5 patients per cohort and each patients serum or immunoglobulin was injected into 3 mice for a total n of 15 mice. #P
    Figure Legend Snippet: CRPS patient serum and IgM had pronociceptive effects in B cell deficient fracture mice. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited unilateral hindpaw von Frey allodynia ( A ) and unweighting ( B ). After intraperitoneal injection of early (1–12 months post injury) CRPS patient serum (0.5ml, I.P) or IgM (500ug/1ml, I.P.) into 3 weeks post FX muMT mice, the mice gradually developed increased allodynia and unweighting over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 2 weeks post-injection. The pronociceptive effects of the CRPS serum were restricted to the FX limb and there was no serum effect on post FX hindpaw edema or warmth (data not shown). No pronociceptive effects were observed after intraperitoneal injection of early CRPS patient IgG (5mg/1ml, I.P.) or after injection of control subject serum (0.5ml, I.P.) in 3 weeks post-FX mice. Measurements for ( A ) represent the difference between the FX side and contralateral paw, thus a negative value represents a decrease in mechanical withdrawal thresholds on the affected side. Measurements for ( B ) represent weight-bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SEM, n = 5 patients per cohort and each patients serum or immunoglobulin was injected into 3 mice for a total n of 15 mice. #P

    Techniques Used: Mouse Assay, Injection

    32) Product Images from "MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells"

    Article Title: MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells

    Journal: Biology Open

    doi: 10.1242/bio.20147765

    RT-PCR validation of candidate miR-153-regulated mRNAs. Bar graphs depict the real time RT-PCR quantification of mRNAs in vector control and miR-153 over-expression conditions for candidate mRNA transcripts identified from the microarray experiment that achieved the adjusted p-value cut-off of 0.1 (a) and raw p-value of 0.05 (b), respectively. The y -axis indicates normalized mRNA expression (expressed as 1/2ΔCt relative to 18s RNA). Data were expressed as mean±SEM and quantified from 6 independent replicates (*p
    Figure Legend Snippet: RT-PCR validation of candidate miR-153-regulated mRNAs. Bar graphs depict the real time RT-PCR quantification of mRNAs in vector control and miR-153 over-expression conditions for candidate mRNA transcripts identified from the microarray experiment that achieved the adjusted p-value cut-off of 0.1 (a) and raw p-value of 0.05 (b), respectively. The y -axis indicates normalized mRNA expression (expressed as 1/2ΔCt relative to 18s RNA). Data were expressed as mean±SEM and quantified from 6 independent replicates (*p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Over Expression, Microarray, Expressing

    Identification and gene ontology classification of mRNAs that are down-regulated following miR-153 over-expression. (a) Schematic structure of the pre-miR-153/GFP-puromycin expression vector (Cell Biolabs, CA). (b) Sample flow-cytometry frequency histograms documenting transfection efficiency. The upper panel depicts mock-transfected controls, whereas the lower two panels (s1 and s2) show GFP expression following transfection. GFP-transfected cells exhibit a bi-modal distribution, with a mean transfection efficiency of 68±2%. Cells were cultured for 24 hours before labeling with anti-GFP antibody, followed by flow cytometry. (c) Bar graph shows that transfection of neurosphere-derived cells with pre-miR-153 expression vector results in a 30-fold increase in miR-153 expression compared to transfection with vector control. Data from six independent replicates (n = 6) are expressed as mean±SEM. (d) Frequency histogram of miRNA expression (ΔCT relative to U6 snRNA) in neurosphere cultures showing the relative baseline and transfection-induced expression of miR-153. Smaller ΔCT values indicate increased expression. Data show that baseline miR-153 expression is within the upper 12th percentile of all expressed miRNAs, and that over-expression results in a shift to the 1st percentile. However, another ethanol-sensitive miRNA, miR-9 (indicated with arrow), is expressed at a higher baseline level compared to miR-153 over-expression.
    Figure Legend Snippet: Identification and gene ontology classification of mRNAs that are down-regulated following miR-153 over-expression. (a) Schematic structure of the pre-miR-153/GFP-puromycin expression vector (Cell Biolabs, CA). (b) Sample flow-cytometry frequency histograms documenting transfection efficiency. The upper panel depicts mock-transfected controls, whereas the lower two panels (s1 and s2) show GFP expression following transfection. GFP-transfected cells exhibit a bi-modal distribution, with a mean transfection efficiency of 68±2%. Cells were cultured for 24 hours before labeling with anti-GFP antibody, followed by flow cytometry. (c) Bar graph shows that transfection of neurosphere-derived cells with pre-miR-153 expression vector results in a 30-fold increase in miR-153 expression compared to transfection with vector control. Data from six independent replicates (n = 6) are expressed as mean±SEM. (d) Frequency histogram of miRNA expression (ΔCT relative to U6 snRNA) in neurosphere cultures showing the relative baseline and transfection-induced expression of miR-153. Smaller ΔCT values indicate increased expression. Data show that baseline miR-153 expression is within the upper 12th percentile of all expressed miRNAs, and that over-expression results in a shift to the 1st percentile. However, another ethanol-sensitive miRNA, miR-9 (indicated with arrow), is expressed at a higher baseline level compared to miR-153 over-expression.

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Transfection, Cell Culture, Labeling, Derivative Assay

    MiR-153 prevents and partly reverses ethanol's effects on miR-153-regulated gene transcripts. Bar graphs represent real-time RT-PCR analysis for mRNA expression of miR-153 sensitive genes in control neurosphere cultures (untreated or transfection control), ethanol (320 mg/dl) alone, miR-153 over-expression with ethanol exposure (prevention paradigm), or miR-153 over-expression for 48 hours after 5 days of ethanol exposure (reversal paradigm) of NSCs. The y -axis indicates normalized mRNA expression (normalized to 18s) relative to control samples. Data were expressed as mean±SEM. n = 4 independent experiments. *Significant difference from control. #Significant difference from ethanol-exposed. See Results section for p-values.
    Figure Legend Snippet: MiR-153 prevents and partly reverses ethanol's effects on miR-153-regulated gene transcripts. Bar graphs represent real-time RT-PCR analysis for mRNA expression of miR-153 sensitive genes in control neurosphere cultures (untreated or transfection control), ethanol (320 mg/dl) alone, miR-153 over-expression with ethanol exposure (prevention paradigm), or miR-153 over-expression for 48 hours after 5 days of ethanol exposure (reversal paradigm) of NSCs. The y -axis indicates normalized mRNA expression (normalized to 18s) relative to control samples. Data were expressed as mean±SEM. n = 4 independent experiments. *Significant difference from control. #Significant difference from ethanol-exposed. See Results section for p-values.

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Over Expression

    Relationship between miR-153 over-expression and the expression of the neuronal differentiation marker DCX, and the neuronal lineage stem cell marker, CD24 and Map2A, in the cerebral cortical VZ and SVZ. In each row, panel ‘i’ depicts miR-153-GFP or control GFP expression, panel ‘ii’ depicts immunofluorescence for DCX, CD24 or Map2A, panel ‘iii’ depicts combined immunofluorescence and panel ‘iv’ depicts DAPI labeling of cell nuclei. (a,b) DCX-immunofluorescence is localized to the SVZ, but not VZ (b.ii,c.ii). Control-GFP over-expression (a.i–a.iv) does not alter DCX expression in the SVZ; however, miR-153 over-expression (b.i–b.iv, circled areas) results in loss of DCX expression in the SVZ. (c–d) CD24-immunofluorescence localizes to VZ and SVZ in GFP-control (c.i–c.iv) and following miR-153 over-expression (d.i–d.iv). miR-153 over-expression does not result in a loss of CD24 immunofluorescence (white circles). (e.i–e.iv) MiR-153 over-expression does not result in a loss of MAP2a/b expression in newly generated neurons of the VZ (white circles). VZ: ventricular zone; SVZ: subventricular zone; CP: cortical plate. Scale bars: 50 µm.
    Figure Legend Snippet: Relationship between miR-153 over-expression and the expression of the neuronal differentiation marker DCX, and the neuronal lineage stem cell marker, CD24 and Map2A, in the cerebral cortical VZ and SVZ. In each row, panel ‘i’ depicts miR-153-GFP or control GFP expression, panel ‘ii’ depicts immunofluorescence for DCX, CD24 or Map2A, panel ‘iii’ depicts combined immunofluorescence and panel ‘iv’ depicts DAPI labeling of cell nuclei. (a,b) DCX-immunofluorescence is localized to the SVZ, but not VZ (b.ii,c.ii). Control-GFP over-expression (a.i–a.iv) does not alter DCX expression in the SVZ; however, miR-153 over-expression (b.i–b.iv, circled areas) results in loss of DCX expression in the SVZ. (c–d) CD24-immunofluorescence localizes to VZ and SVZ in GFP-control (c.i–c.iv) and following miR-153 over-expression (d.i–d.iv). miR-153 over-expression does not result in a loss of CD24 immunofluorescence (white circles). (e.i–e.iv) MiR-153 over-expression does not result in a loss of MAP2a/b expression in newly generated neurons of the VZ (white circles). VZ: ventricular zone; SVZ: subventricular zone; CP: cortical plate. Scale bars: 50 µm.

    Techniques Used: Over Expression, Expressing, Marker, Immunofluorescence, Labeling, Generated

    Effects of miR-153 over-expression on differentiation, apoptosis and cell proliferation. (a,b) Photomicrographs depicting GFP immunofluorescent cells cultured under mitogen-withdrawal-induced differentiation conditions on a laminin substrate 48 hours after transfection with GFP control vector (a.i–a.iii) or with GFP-premiR-153 (b.i–b.iv). MiR-153 over-expressing cells exhibited deficient morphological transformation compared to control cells. (c,d) Differentiating control-GFP (c.i–c.iii) and miR-153/GFP (d.i–d.iii) over-expressing cells exhibit co-localized expression of the early neuronal marker Map2a/b (white arrows). Yellow arrowheads show that the GFP label fills the cellular processes and completely overlaps the expression of Map2a/b. Map2a/b immunolabeling also shows deficient morphological transformation following miR-153/GFP over-expression. (e) Bar graph depicts Sholl analysis of neurite length expressed as number of intersections ( y -axis) as a function of distance from soma ( x -axis) per cell. MiR-153 over-expressing cells have shorter neurites compared to controls. Data based on analysis of 34 control and 26 miR-153-over-expressing cells. Photomicrographs were obtained from all four quadrants of each culture dish, and cells whose processes showed no overlap with those of an adjacent cell were selected for analysis. Asterisks, all p-values
    Figure Legend Snippet: Effects of miR-153 over-expression on differentiation, apoptosis and cell proliferation. (a,b) Photomicrographs depicting GFP immunofluorescent cells cultured under mitogen-withdrawal-induced differentiation conditions on a laminin substrate 48 hours after transfection with GFP control vector (a.i–a.iii) or with GFP-premiR-153 (b.i–b.iv). MiR-153 over-expressing cells exhibited deficient morphological transformation compared to control cells. (c,d) Differentiating control-GFP (c.i–c.iii) and miR-153/GFP (d.i–d.iii) over-expressing cells exhibit co-localized expression of the early neuronal marker Map2a/b (white arrows). Yellow arrowheads show that the GFP label fills the cellular processes and completely overlaps the expression of Map2a/b. Map2a/b immunolabeling also shows deficient morphological transformation following miR-153/GFP over-expression. (e) Bar graph depicts Sholl analysis of neurite length expressed as number of intersections ( y -axis) as a function of distance from soma ( x -axis) per cell. MiR-153 over-expressing cells have shorter neurites compared to controls. Data based on analysis of 34 control and 26 miR-153-over-expressing cells. Photomicrographs were obtained from all four quadrants of each culture dish, and cells whose processes showed no overlap with those of an adjacent cell were selected for analysis. Asterisks, all p-values

    Techniques Used: Over Expression, Cell Culture, Transfection, Plasmid Preparation, Expressing, Transformation Assay, Marker, Immunolabeling

    Matn2 and Vegfa are not direct targets of miR-153. Firefly activity relative to RLuc luciferase activity was measured in NSCs 24 hours after transfection with control or miR-153 mimetics and the luciferase construct containing 3′UTR of Matn2 or Vegfa. Bars are normalized to the relative firefly units of samples treated with the transfected control. The x -axis depicts treatment conditions (control or miR-153). The y -axis indicates normalized luciferase activity. Data were expressed as mean ± SEM (n = 5).
    Figure Legend Snippet: Matn2 and Vegfa are not direct targets of miR-153. Firefly activity relative to RLuc luciferase activity was measured in NSCs 24 hours after transfection with control or miR-153 mimetics and the luciferase construct containing 3′UTR of Matn2 or Vegfa. Bars are normalized to the relative firefly units of samples treated with the transfected control. The x -axis depicts treatment conditions (control or miR-153). The y -axis indicates normalized luciferase activity. Data were expressed as mean ± SEM (n = 5).

    Techniques Used: Activity Assay, Luciferase, Transfection, Construct

    Model for the hypothesis that a network of miR-153 and the NF1 neurogenic transcription factor family (Nfia/b/c) mediate the effects of ethanol on NSC maturation. (a) miR-153 is a direct and negative regulator of NF1 expression. Our data on miR-153 regulation of gene expression and published data on NF-1 suggest that suppressing NF1 will in turn retard NSC maturation, and be predicted to be permissive of continued NSC renewal. (b) Ethanol suppresses miR-153, resulting in release of NF1 expression, potentially explaining findings that ethanol promotes NSC maturation. (c) Nicotinic (nAChR) activation prevents ethanol suppression of miR-153 and may serve to mitigate premature NSC differentiation. Green arrows depict positive regulation while red bars indicate negative regulation. Dashed lines indicate diminished regulation, while gray text indicates diminished expression or function.
    Figure Legend Snippet: Model for the hypothesis that a network of miR-153 and the NF1 neurogenic transcription factor family (Nfia/b/c) mediate the effects of ethanol on NSC maturation. (a) miR-153 is a direct and negative regulator of NF1 expression. Our data on miR-153 regulation of gene expression and published data on NF-1 suggest that suppressing NF1 will in turn retard NSC maturation, and be predicted to be permissive of continued NSC renewal. (b) Ethanol suppresses miR-153, resulting in release of NF1 expression, potentially explaining findings that ethanol promotes NSC maturation. (c) Nicotinic (nAChR) activation prevents ethanol suppression of miR-153 and may serve to mitigate premature NSC differentiation. Green arrows depict positive regulation while red bars indicate negative regulation. Dashed lines indicate diminished regulation, while gray text indicates diminished expression or function.

    Techniques Used: Expressing, Activation Assay

    In silico analysis of the RNA folding structure of Nfia and Nfib. Predicted secondary structure conformation of the 3′UTR of (a) Nfia and (b) Nfib. Locations of the 5′ and 3′ends of the 3′UTR sequences are marked with blue arrows. (a) Two miR-153 binding sites, target_7285 and target_9451, validated from luciferase assay above are shown on Nfia 3′UTR. Target_7285 is located close to complex stem–loop structures, while target_9451 localizes to a linear portion, of the Nfia 3′UTR. MiR-153 sequences are shown in black while the matching binding site sequences are illustrated in blue (target_7285) and red (target_9451). (b) One miR-153 binding site, target_6559, validated from luciferase analysis is located on the linear portion of Nfib 3′UTR and is labeled in red, whereas miR-153 sequence is shown in black.
    Figure Legend Snippet: In silico analysis of the RNA folding structure of Nfia and Nfib. Predicted secondary structure conformation of the 3′UTR of (a) Nfia and (b) Nfib. Locations of the 5′ and 3′ends of the 3′UTR sequences are marked with blue arrows. (a) Two miR-153 binding sites, target_7285 and target_9451, validated from luciferase assay above are shown on Nfia 3′UTR. Target_7285 is located close to complex stem–loop structures, while target_9451 localizes to a linear portion, of the Nfia 3′UTR. MiR-153 sequences are shown in black while the matching binding site sequences are illustrated in blue (target_7285) and red (target_9451). (b) One miR-153 binding site, target_6559, validated from luciferase analysis is located on the linear portion of Nfib 3′UTR and is labeled in red, whereas miR-153 sequence is shown in black.

    Techniques Used: In Silico, Binding Assay, Luciferase, Labeling, Sequencing

    Identification of Nfia 3′UTR as a direct target of miR-153. (a) Schematic structure of the luciferase reporter constructs containing murine Nfia 3′UTR fragments (GeneCopoeia, Rockville, MD). (b) Schematic of the full length of Nfia 3′UTR depicting the location of the three Nfia 3′UTR fragments (Nfia 3′UTR_a in blue, Nfia 3′UTR_b in orange, Nfia 3′UTR_c in pink) that were cloned into luciferase reporters for studies. Green triangles indicate the predicted miR-153 binding sites that are conserved among vertebrates, whereas the blue triangle illustrates the predicted miR-153 binding site that shares conservation between mouse and human. Purple bars represent the morpholinos used to protect the predicted miR-153 binding sites. (c) We assayed firefly activity relative to RLuc luciferase activity in NSCs 24 h after transfection with the Luc_miR153 binding site reporter construct as the positive control and transfected control or miR-153 mimetics. Bar graphs represent luciferase activity normalized to the mean activity of samples transfected with the miR-153 control vector. The x -axis depicts treatment conditions. The y -axis indicates normalized luciferase activity. (d–f) NSCs were transfected with luciferase reporter constructs containing different fragments of Nfia 3′UTR, (d) 3′UTR_a, (e) 3′UTR_b, (f) 3′UTR_c, with control or miR-153 mimetics for 24 hours. Additional control or antisense morpholinos, (e) mask_i, mask_ii, and (f) mask_iii, used to protect the miR-153 binding sites were co-transfected along with other constructs as indicated on the x -axis. Bars are normalized to the relative firefly units of samples treated with the transfected control. Data were expressed as mean±SEM (n = 5).
    Figure Legend Snippet: Identification of Nfia 3′UTR as a direct target of miR-153. (a) Schematic structure of the luciferase reporter constructs containing murine Nfia 3′UTR fragments (GeneCopoeia, Rockville, MD). (b) Schematic of the full length of Nfia 3′UTR depicting the location of the three Nfia 3′UTR fragments (Nfia 3′UTR_a in blue, Nfia 3′UTR_b in orange, Nfia 3′UTR_c in pink) that were cloned into luciferase reporters for studies. Green triangles indicate the predicted miR-153 binding sites that are conserved among vertebrates, whereas the blue triangle illustrates the predicted miR-153 binding site that shares conservation between mouse and human. Purple bars represent the morpholinos used to protect the predicted miR-153 binding sites. (c) We assayed firefly activity relative to RLuc luciferase activity in NSCs 24 h after transfection with the Luc_miR153 binding site reporter construct as the positive control and transfected control or miR-153 mimetics. Bar graphs represent luciferase activity normalized to the mean activity of samples transfected with the miR-153 control vector. The x -axis depicts treatment conditions. The y -axis indicates normalized luciferase activity. (d–f) NSCs were transfected with luciferase reporter constructs containing different fragments of Nfia 3′UTR, (d) 3′UTR_a, (e) 3′UTR_b, (f) 3′UTR_c, with control or miR-153 mimetics for 24 hours. Additional control or antisense morpholinos, (e) mask_i, mask_ii, and (f) mask_iii, used to protect the miR-153 binding sites were co-transfected along with other constructs as indicated on the x -axis. Bars are normalized to the relative firefly units of samples treated with the transfected control. Data were expressed as mean±SEM (n = 5).

    Techniques Used: Luciferase, Construct, Clone Assay, Binding Assay, Activity Assay, Transfection, Positive Control, Plasmid Preparation

    Nfib is a direct target of miR-153. (a) Schematic shows the full length Nfib 3′UTR in relation to the three 3′UTR fragments (Nfib 3′UTR_a in blue, 3′UTR_b in green, 3′UTR_c in purple) that were cloned into luciferase constructs for studies. Green triangles indicate the predicted miR-153 binding sites that are conserved among vertebrates. Purple bars represent the morpholinos used to mask the miR-153 binding sites. (b–d) Firefly activity relative to RLuc luciferase activity is determined in NSCs 24 h after transfection with luciferase reporter constructs containing different fragments of Nfib 3′UTR, (b) 3′UTR_a, (c) 3′UTR_b, (d) 3′UTR_c, with control or miR-153 mimetics. Additional control or antisense morpholinos, (d) Nfib-mask_i, used to protect the predicted miR-153 binding sites in Nfib 3′UTR were co-transfected into same samples as indicated. Data were normalized to the samples treated with the transfected control. The x -axis depicts treatment conditions. The y -axis indicates normalized luciferase activity. Data were expressed as mean±SEM (n = 5).
    Figure Legend Snippet: Nfib is a direct target of miR-153. (a) Schematic shows the full length Nfib 3′UTR in relation to the three 3′UTR fragments (Nfib 3′UTR_a in blue, 3′UTR_b in green, 3′UTR_c in purple) that were cloned into luciferase constructs for studies. Green triangles indicate the predicted miR-153 binding sites that are conserved among vertebrates. Purple bars represent the morpholinos used to mask the miR-153 binding sites. (b–d) Firefly activity relative to RLuc luciferase activity is determined in NSCs 24 h after transfection with luciferase reporter constructs containing different fragments of Nfib 3′UTR, (b) 3′UTR_a, (c) 3′UTR_b, (d) 3′UTR_c, with control or miR-153 mimetics. Additional control or antisense morpholinos, (d) Nfib-mask_i, used to protect the predicted miR-153 binding sites in Nfib 3′UTR were co-transfected into same samples as indicated. Data were normalized to the samples treated with the transfected control. The x -axis depicts treatment conditions. The y -axis indicates normalized luciferase activity. Data were expressed as mean±SEM (n = 5).

    Techniques Used: Clone Assay, Luciferase, Construct, Binding Assay, Activity Assay, Transfection

    Varenicline prevents and reverses the effects of ethanol on miR-153 target gene expression. Bar graph depicts real-time RT-PCR analysis of mRNA expression of miR-153-regulated genes in control neurospheres, or neurospheres treated with varenicline (1 µM) alone, varenicline in combination with ethanol (prevention paradigm), and varenicline treatment for 48 hours following 5 days of ethanol exposure (reversal paradigm). The striped bars show reference ethanol exposure data from Fig. 9 . The y -axis indicates normalized mRNA expression (normalized to 18s) relative to control samples. Data were expressed as mean±SEM. n = 4 independent replicates. *Significant difference from control. #Significant difference from ethanol-exposed. See Results section for p-values.
    Figure Legend Snippet: Varenicline prevents and reverses the effects of ethanol on miR-153 target gene expression. Bar graph depicts real-time RT-PCR analysis of mRNA expression of miR-153-regulated genes in control neurospheres, or neurospheres treated with varenicline (1 µM) alone, varenicline in combination with ethanol (prevention paradigm), and varenicline treatment for 48 hours following 5 days of ethanol exposure (reversal paradigm). The striped bars show reference ethanol exposure data from Fig. 9 . The y -axis indicates normalized mRNA expression (normalized to 18s) relative to control samples. Data were expressed as mean±SEM. n = 4 independent replicates. *Significant difference from control. #Significant difference from ethanol-exposed. See Results section for p-values.

    Techniques Used: Expressing, Quantitative RT-PCR

    Microarray analysis of gene expression following miR-153 over-expression. (a) Volcano-plot illustrates relationship between log 2 (mRNA expression ratio) and log 2 (FDR-corrected p-value) in miR-153 over-expressing cultures compared to controls. Filled red and blue circles indicate mRNA transcripts that are suppressed or induced, respectively, by more than 1.3-fold following miR-153 over-expression, at an FDR (Benjamini and Hochberg)-adjusted p
    Figure Legend Snippet: Microarray analysis of gene expression following miR-153 over-expression. (a) Volcano-plot illustrates relationship between log 2 (mRNA expression ratio) and log 2 (FDR-corrected p-value) in miR-153 over-expressing cultures compared to controls. Filled red and blue circles indicate mRNA transcripts that are suppressed or induced, respectively, by more than 1.3-fold following miR-153 over-expression, at an FDR (Benjamini and Hochberg)-adjusted p

    Techniques Used: Microarray, Expressing, Over Expression

    Nicotine and the nAChR partial agonist varenicline induce miR-153 expression. Bar graph depicts real-time RT-PCR expression of miR-153 in control, nicotine and varenicline-exposed neurosphere cultures. MiR-153 expression is significantly induced in nicotine and varenicline-treated groups. The y -axis indicates normalized miR-153 expression (normalized to U6) relative to control samples. Data were expressed as mean±SEM. n = 4 independent replicates. Asterisk indicates significant difference from control.
    Figure Legend Snippet: Nicotine and the nAChR partial agonist varenicline induce miR-153 expression. Bar graph depicts real-time RT-PCR expression of miR-153 in control, nicotine and varenicline-exposed neurosphere cultures. MiR-153 expression is significantly induced in nicotine and varenicline-treated groups. The y -axis indicates normalized miR-153 expression (normalized to U6) relative to control samples. Data were expressed as mean±SEM. n = 4 independent replicates. Asterisk indicates significant difference from control.

    Techniques Used: Expressing, Quantitative RT-PCR

    MiR-153 regulates Nfia and Nfib expression in fetal brains. (a) Photo-micrograph depicts ultrasound-guided trans-uterine insertion of a micro-capillary pipette (dashed green line) into the lateral ventricle in a GD13 fetal brain. Following in utero electroporation of control-GFP or miR-153/GFP vectors, fetuses were maintained for an additional period of 48 hours, before being analyzed at GD15.5. Double immunohistochemistry of anti-GFP with anti-Nfia or anti-Nfib in control-GFP (b) or Pre-miR-153-GFP (c–h) transfected GD15.5 mouse frozen sections. (b1,b2) Photomicrograph shows (b1) control-GFP (green) localizes to the cytoplasm of nuclear Nfib-labeled (red) neurons of the cortical plate and (b2) DAPI-counterstained nuclei. (c–h) Panels c1–h1, c2–h2 and c3–h3 show low magnification images of the same sections counterstained with DAPI (c1–h1) to visualize nuclei, or immuno-fluorescently labeled for GFP (c2–h2) as a marker for miR-153 over-expression, or Nfia (c3–e3) and Nfib (f3–h3). (c4,d4,f4,g4) High magnification photomicrographs showing that GFP expression from the pre-miR-153/GFP construct does not co-localize with nuclear immuno-labeling for Nfia (c4,d4) or Nfib (f4,g4). Residual cytoplasmic labeling in GFP/miR-153 over-expressing cells, represented by yellow immunofluorescence (e.g. c4), may represent incompletely suppressed translation or residual immuno-reactivity due to products of stalled translation. Dotted circles indicate regions depicted in high magnification images. Dotted squares depict regions of cortical plate with disrupted expression of Nfib overlying strong GFP expression in the ventricular/sub-ventricular zones. Pink arrows show nuclei immuno-stained for Nfia or Nfib, while green arrows indicate strong cytoplasmic miR-153-GFP immuno-staining. VZ: ventricular zone; SVZ: subventricular zone; CP: cortical plate. Scale bars: 25 µm.
    Figure Legend Snippet: MiR-153 regulates Nfia and Nfib expression in fetal brains. (a) Photo-micrograph depicts ultrasound-guided trans-uterine insertion of a micro-capillary pipette (dashed green line) into the lateral ventricle in a GD13 fetal brain. Following in utero electroporation of control-GFP or miR-153/GFP vectors, fetuses were maintained for an additional period of 48 hours, before being analyzed at GD15.5. Double immunohistochemistry of anti-GFP with anti-Nfia or anti-Nfib in control-GFP (b) or Pre-miR-153-GFP (c–h) transfected GD15.5 mouse frozen sections. (b1,b2) Photomicrograph shows (b1) control-GFP (green) localizes to the cytoplasm of nuclear Nfib-labeled (red) neurons of the cortical plate and (b2) DAPI-counterstained nuclei. (c–h) Panels c1–h1, c2–h2 and c3–h3 show low magnification images of the same sections counterstained with DAPI (c1–h1) to visualize nuclei, or immuno-fluorescently labeled for GFP (c2–h2) as a marker for miR-153 over-expression, or Nfia (c3–e3) and Nfib (f3–h3). (c4,d4,f4,g4) High magnification photomicrographs showing that GFP expression from the pre-miR-153/GFP construct does not co-localize with nuclear immuno-labeling for Nfia (c4,d4) or Nfib (f4,g4). Residual cytoplasmic labeling in GFP/miR-153 over-expressing cells, represented by yellow immunofluorescence (e.g. c4), may represent incompletely suppressed translation or residual immuno-reactivity due to products of stalled translation. Dotted circles indicate regions depicted in high magnification images. Dotted squares depict regions of cortical plate with disrupted expression of Nfib overlying strong GFP expression in the ventricular/sub-ventricular zones. Pink arrows show nuclei immuno-stained for Nfia or Nfib, while green arrows indicate strong cytoplasmic miR-153-GFP immuno-staining. VZ: ventricular zone; SVZ: subventricular zone; CP: cortical plate. Scale bars: 25 µm.

    Techniques Used: Expressing, Transferring, In Utero, Electroporation, Immunohistochemistry, Transfection, Labeling, Marker, Over Expression, Construct, Immunolabeling, Immunofluorescence, Staining, Immunostaining

    33) Product Images from "A screening cascade to identify ERβ ligands"

    Article Title: A screening cascade to identify ERβ ligands

    Journal: Nuclear Receptor Signaling

    doi: 10.1621/nrs.12003

    ÄKTA chromatogram and SDS-PAGE for the purification of ERβ LBD. Representative size-exclusion chromatogram from the ÄKTA purifier FPLC for the purification of ERβ LBD (A) and Coomassie stained SDS-PAGE gel after SEC of the pooled and concentrated eluted fractions at ~61 mL corresponding to ERβ LBD (B).
    Figure Legend Snippet: ÄKTA chromatogram and SDS-PAGE for the purification of ERβ LBD. Representative size-exclusion chromatogram from the ÄKTA purifier FPLC for the purification of ERβ LBD (A) and Coomassie stained SDS-PAGE gel after SEC of the pooled and concentrated eluted fractions at ~61 mL corresponding to ERβ LBD (B).

    Techniques Used: SDS Page, Purification, Fast Protein Liquid Chromatography, Staining, Size-exclusion Chromatography

    34) Product Images from "Bifidobacteria grown on human milk oligosaccharides downregulate the expression of inflammation-related genes in Caco-2 cells"

    Article Title: Bifidobacteria grown on human milk oligosaccharides downregulate the expression of inflammation-related genes in Caco-2 cells

    Journal: BMC Microbiology

    doi: 10.1186/s12866-015-0508-3

    Number of Caco-2 genes differentially expressed between B. infantis ATCC 15697 and B. breve SC95 in response to different substrates (GLU = glucose, LAC = lactose, HMO = human milk oligosaccharides). Venn diagram prepared using EulerAPE [ 62 ]
    Figure Legend Snippet: Number of Caco-2 genes differentially expressed between B. infantis ATCC 15697 and B. breve SC95 in response to different substrates (GLU = glucose, LAC = lactose, HMO = human milk oligosaccharides). Venn diagram prepared using EulerAPE [ 62 ]

    Techniques Used:

    Binding of B. breve SC95 and B. infantis ATCC 15697 to Caco-2 cell monolayers, expressed as percentage of initial bacterial input. Statistical analysis was performed by ANOVA, and binding percentages were compared to LAC. * P
    Figure Legend Snippet: Binding of B. breve SC95 and B. infantis ATCC 15697 to Caco-2 cell monolayers, expressed as percentage of initial bacterial input. Statistical analysis was performed by ANOVA, and binding percentages were compared to LAC. * P

    Techniques Used: Binding Assay

    35) Product Images from "Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay"

    Article Title: Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.01019

    Damaged leaf tissues in empty vector-treated or SCEI-silenced S. peruvianum plants at 20 dpi with Cmm .
    Figure Legend Snippet: Damaged leaf tissues in empty vector-treated or SCEI-silenced S. peruvianum plants at 20 dpi with Cmm .

    Techniques Used: Plasmid Preparation

    Scanning electron microscopy of longitudinal cut of a stem of non-treated, empty vector and SCE I-silenced Solanum plants with and without Cmm challenge . (A) S. peruvianum without inoculation of Cmm showing no bacteria or tissue damage (B) S. lycopersicum without inoculation of Cmm showing no bacteria or tissue damage (C) non-VIGS treated S. peruvianum at 10 dpi with Cmm showing some bacterial but limited parenchymal tissue damage. (D) S. lycopersicum at 10 dpi with Cmm revealing more bacterial structures with greater parenchymal tissue damage. (E) empty-vector treated S. peruvianum at 10 dpi with Cmm with some bacterial structures and limited parenchymal tissue damage. (F) SCE I-silenced S. peruvianum at 10 dpi with Cmm revealing considerable bacterial structures and parenchymal tissue damage. Magnification 6000x, scale bar ~10 μm.
    Figure Legend Snippet: Scanning electron microscopy of longitudinal cut of a stem of non-treated, empty vector and SCE I-silenced Solanum plants with and without Cmm challenge . (A) S. peruvianum without inoculation of Cmm showing no bacteria or tissue damage (B) S. lycopersicum without inoculation of Cmm showing no bacteria or tissue damage (C) non-VIGS treated S. peruvianum at 10 dpi with Cmm showing some bacterial but limited parenchymal tissue damage. (D) S. lycopersicum at 10 dpi with Cmm revealing more bacterial structures with greater parenchymal tissue damage. (E) empty-vector treated S. peruvianum at 10 dpi with Cmm with some bacterial structures and limited parenchymal tissue damage. (F) SCE I-silenced S. peruvianum at 10 dpi with Cmm revealing considerable bacterial structures and parenchymal tissue damage. Magnification 6000x, scale bar ~10 μm.

    Techniques Used: Electron Microscopy, Plasmid Preparation

    Phenotype of silencing of ChI I gene in S. peruvianum . (A) Phenotype of leaf bleaching at 10 dpi. (B) Close up of a bleached leaf at 10 dpi. (C) Close up of a bleached leaf 40 dpi. (D) Bleaching phenotype at 40 dpi.
    Figure Legend Snippet: Phenotype of silencing of ChI I gene in S. peruvianum . (A) Phenotype of leaf bleaching at 10 dpi. (B) Close up of a bleached leaf at 10 dpi. (C) Close up of a bleached leaf 40 dpi. (D) Bleaching phenotype at 40 dpi.

    Techniques Used:

    Cmm detection in S. peruvianum tissues by PCR . An amplicon of 233 bp was obtained using Cel- A primers por Cmm detection at 10 and 20 dpi. Lanes show PCR products shown with template of: (1) leaf tissue of SCE I-silenced S. peruvianum at 10 dpi, (2) leaf tissue of SCE I-silenced S. peruvianum at 20 dpi, (3) leaf tissue of empty vector treated S. peruvianum at 10 dpi, (4) leaf tissue of empty vector treated S. peruvianum at 20 dpi. Plasmid of pGEM T-Easy with Cel-A fragment. (-) Negative control. 100 ng of DNA were taken for each reaction.
    Figure Legend Snippet: Cmm detection in S. peruvianum tissues by PCR . An amplicon of 233 bp was obtained using Cel- A primers por Cmm detection at 10 and 20 dpi. Lanes show PCR products shown with template of: (1) leaf tissue of SCE I-silenced S. peruvianum at 10 dpi, (2) leaf tissue of SCE I-silenced S. peruvianum at 20 dpi, (3) leaf tissue of empty vector treated S. peruvianum at 10 dpi, (4) leaf tissue of empty vector treated S. peruvianum at 20 dpi. Plasmid of pGEM T-Easy with Cel-A fragment. (-) Negative control. 100 ng of DNA were taken for each reaction.

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Negative Control

    36) Product Images from "Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation"

    Article Title: Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005677

    rGal-9 is a potent mediator of HIV transcription ex vivo and synergizes with JQ1 in reactivating latent HIV . ( A ) Treatment of CD4+ T cells isolated from ART-suppressed HIV-infected individuals with DMSO 0.5% (negative control), PMA/ionomycin (2 nM / 500 nM), vorinostat (1μM), or varying concentrations of rGal-9 (500 nM and 1000 nM) for 24 hours. Fold increase in cell-associated HIV RNA was determined relative to the corresponding DMSO-treated control for each individual time point. Mean ± SEM is displayed, and statistical comparisons between rGal-9 and other treatments were performed using two-tailed paired Wilcoxon signed-rank tests. (B-C) CD4+ T cells were isolated from PBMCs of three HIV-infected ART-suppressed individuals using negative selection. Resting CD4+ T cells were further enriched through depletion of cells expressing CD69, CD25, or HLA-DR surface markers from half of the isolated CD4+ T cells. The remaining half was processed through the exact enrichment procedure, except PBS was added instead of the depleting antibodies. Both cell populations were treated with 0.5% DMSO (negative control), 500 nM rGal-9, 1000 nM rGal-9 or αCD3/αCD28-conjugated beads. Induction of cell-associated HIV RNA was measured 24 hours post-treatment using RT-qPCR. Each individual is represented with a different symbol. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t tests. Percentages reported reflect average values measured in the CD69- / CD25- / HLA-DR- CD4+ T cells with respect to values observed in total CD4+ T cells. (D) CD4+ T cells from HIV-infected ART-suppressed individuals were treated with 500 nM of rGal-9, 1 μM vorinostat, 40 nM romidepsin, 10 nM bryostatin, 300 nM prostratin, 1 μM JQ1, or 30 nM panobinostat alone or in combination with 500 nM of rGal-9 for 24 hours, and fold induction of cell-associated HIV RNA was determined using quantitative real-time PCR. * = p
    Figure Legend Snippet: rGal-9 is a potent mediator of HIV transcription ex vivo and synergizes with JQ1 in reactivating latent HIV . ( A ) Treatment of CD4+ T cells isolated from ART-suppressed HIV-infected individuals with DMSO 0.5% (negative control), PMA/ionomycin (2 nM / 500 nM), vorinostat (1μM), or varying concentrations of rGal-9 (500 nM and 1000 nM) for 24 hours. Fold increase in cell-associated HIV RNA was determined relative to the corresponding DMSO-treated control for each individual time point. Mean ± SEM is displayed, and statistical comparisons between rGal-9 and other treatments were performed using two-tailed paired Wilcoxon signed-rank tests. (B-C) CD4+ T cells were isolated from PBMCs of three HIV-infected ART-suppressed individuals using negative selection. Resting CD4+ T cells were further enriched through depletion of cells expressing CD69, CD25, or HLA-DR surface markers from half of the isolated CD4+ T cells. The remaining half was processed through the exact enrichment procedure, except PBS was added instead of the depleting antibodies. Both cell populations were treated with 0.5% DMSO (negative control), 500 nM rGal-9, 1000 nM rGal-9 or αCD3/αCD28-conjugated beads. Induction of cell-associated HIV RNA was measured 24 hours post-treatment using RT-qPCR. Each individual is represented with a different symbol. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t tests. Percentages reported reflect average values measured in the CD69- / CD25- / HLA-DR- CD4+ T cells with respect to values observed in total CD4+ T cells. (D) CD4+ T cells from HIV-infected ART-suppressed individuals were treated with 500 nM of rGal-9, 1 μM vorinostat, 40 nM romidepsin, 10 nM bryostatin, 300 nM prostratin, 1 μM JQ1, or 30 nM panobinostat alone or in combination with 500 nM of rGal-9 for 24 hours, and fold induction of cell-associated HIV RNA was determined using quantitative real-time PCR. * = p

    Techniques Used: Ex Vivo, Isolation, Infection, Negative Control, Two Tailed Test, Selection, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    37) Product Images from "Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation"

    Article Title: Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005677

    rGal-9 modulates the expression of genes involved in several signaling pathways associated with HIV latency. ( A ) Venn diagram showing the number of genes modulated by > 2 fold with FDR
    Figure Legend Snippet: rGal-9 modulates the expression of genes involved in several signaling pathways associated with HIV latency. ( A ) Venn diagram showing the number of genes modulated by > 2 fold with FDR

    Techniques Used: Expressing

    rGal-9 induces HIV transcription and reactivation in a glycan-dependent manner. ( A ) Effects of anti-Tim-3 antibody, anti-CD44 antibody, or anti-PDI antibody administration on rGal-9-mediated reactivation of HIV in J-Lat 5A8 cells. Antibodies were added 30 minutes prior to administration of 200 nM rGal-9. α-lactose (30 mM) was used as a positive control. ( B , C ) Treatment of J-Lat 5A8 cells with either 1 μg/ml tunicamycin, or with an enzymatic deglycosylation mix for 24 hours prior to rGal-9 stimulation. J-Lat cells were analyzed by flow cytometry to assess HIV-encoded GFP expression. Statistical comparisons were performed using two-tailed Mann-Whitney tests. ( D ) Effects of deglycosylation enzyme combinations on rGal-9-mediated HIV latency reversal in J-Lat 5A8 cells. N = PNGase F (Elizabethkingia miricola); O = O-Glycosidase (recombinant from Streptococcus pneumonia); S = α-(2→3,6,8,9)-Neuraminidase (recombinant from Arthrobacter ureafaciens); B = β(1→4)-Galactosidase (recombinant from Streptococcus pneumonia) + β-N-Acetylglucosaminidase (recombinant from Streptococcus pneumonia). Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t tests. * = p
    Figure Legend Snippet: rGal-9 induces HIV transcription and reactivation in a glycan-dependent manner. ( A ) Effects of anti-Tim-3 antibody, anti-CD44 antibody, or anti-PDI antibody administration on rGal-9-mediated reactivation of HIV in J-Lat 5A8 cells. Antibodies were added 30 minutes prior to administration of 200 nM rGal-9. α-lactose (30 mM) was used as a positive control. ( B , C ) Treatment of J-Lat 5A8 cells with either 1 μg/ml tunicamycin, or with an enzymatic deglycosylation mix for 24 hours prior to rGal-9 stimulation. J-Lat cells were analyzed by flow cytometry to assess HIV-encoded GFP expression. Statistical comparisons were performed using two-tailed Mann-Whitney tests. ( D ) Effects of deglycosylation enzyme combinations on rGal-9-mediated HIV latency reversal in J-Lat 5A8 cells. N = PNGase F (Elizabethkingia miricola); O = O-Glycosidase (recombinant from Streptococcus pneumonia); S = α-(2→3,6,8,9)-Neuraminidase (recombinant from Arthrobacter ureafaciens); B = β(1→4)-Galactosidase (recombinant from Streptococcus pneumonia) + β-N-Acetylglucosaminidase (recombinant from Streptococcus pneumonia). Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t tests. * = p

    Techniques Used: Positive Control, Flow Cytometry, Cytometry, Expressing, Two Tailed Test, MANN-WHITNEY, Recombinant

    rGal-9 is a potent mediator of HIV transcription in vitro . in vitro HIV reactivation in the J-Lat latency model (A) 5A8 clone, (B) 6.3 clone, and (C) 11.1 clone by varying doses of rGal-9 and other galectins (-1, -3, -4, -7, -8, and -9) after 24 hours of stimulation. αCD3 /αCD28 antibodies conjugated to beads, PMA/ionomycin (16 nM/500 nM), and TNFα (10 ng/ml) were used as positive controls. J-Lat cells were analyzed by flow cytometry to assess HIV-encoded GFP expression. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t tests. * = p
    Figure Legend Snippet: rGal-9 is a potent mediator of HIV transcription in vitro . in vitro HIV reactivation in the J-Lat latency model (A) 5A8 clone, (B) 6.3 clone, and (C) 11.1 clone by varying doses of rGal-9 and other galectins (-1, -3, -4, -7, -8, and -9) after 24 hours of stimulation. αCD3 /αCD28 antibodies conjugated to beads, PMA/ionomycin (16 nM/500 nM), and TNFα (10 ng/ml) were used as positive controls. J-Lat cells were analyzed by flow cytometry to assess HIV-encoded GFP expression. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t tests. * = p

    Techniques Used: In Vitro, Flow Cytometry, Cytometry, Expressing, Two Tailed Test

    rGal-9 induces the expression of the APOBEC3G anti-HIV host restriction factor in vitro . ( A–C ) Digital droplet PCR gene expression profiling quantifying HIV gag , host APOBEC3G, p21, and RNAseP (housekeeping control) mRNA in J-Lat 5A8 cells sorted into GFP-positive and GFP-negative populations after either rGal-9 treatment, αCD3/αCD28 stimulation, or a combination of both. Mean ± SEM is displayed, and statistical comparisons against the unstimulated control were performed using two-tailed unpaired t tests. * = p
    Figure Legend Snippet: rGal-9 induces the expression of the APOBEC3G anti-HIV host restriction factor in vitro . ( A–C ) Digital droplet PCR gene expression profiling quantifying HIV gag , host APOBEC3G, p21, and RNAseP (housekeeping control) mRNA in J-Lat 5A8 cells sorted into GFP-positive and GFP-negative populations after either rGal-9 treatment, αCD3/αCD28 stimulation, or a combination of both. Mean ± SEM is displayed, and statistical comparisons against the unstimulated control were performed using two-tailed unpaired t tests. * = p

    Techniques Used: Expressing, In Vitro, Polymerase Chain Reaction, Two Tailed Test

    rGal-9 treatment reduces viral infectivity. ( A ) Illustrative schematic of the viral infectivity experiment. ( B-D ) Effects of rGal-9 treatment of producer cells on HIV infectivity. The MOLT4-CCR5 cell line was infected for 6 hours, cells were washed and treated with either PBS, rGal-9 200nM, or interferon-α (5000 U/ml) for 24 hours, and cultures were incubated for 3 days. ( B ) HIV p24 levels produced by MOLT4-CCR5 cells were quantified after concentrating the culture supernatants. Concentrated culture supernatants were used to infect Jurkat cells by spinoculation. ( C ) Levels of integrated HIV DNA measured at days 3, 6, 9, and 12 post-infection of Jurkat cells. ( D ) Levels of integrated HIV DNA at days 3, 6, 9, and 12 post-infection of Jurkat cells, normalized to producer cell p24 supernatant levels. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t test. * = p
    Figure Legend Snippet: rGal-9 treatment reduces viral infectivity. ( A ) Illustrative schematic of the viral infectivity experiment. ( B-D ) Effects of rGal-9 treatment of producer cells on HIV infectivity. The MOLT4-CCR5 cell line was infected for 6 hours, cells were washed and treated with either PBS, rGal-9 200nM, or interferon-α (5000 U/ml) for 24 hours, and cultures were incubated for 3 days. ( B ) HIV p24 levels produced by MOLT4-CCR5 cells were quantified after concentrating the culture supernatants. Concentrated culture supernatants were used to infect Jurkat cells by spinoculation. ( C ) Levels of integrated HIV DNA measured at days 3, 6, 9, and 12 post-infection of Jurkat cells. ( D ) Levels of integrated HIV DNA at days 3, 6, 9, and 12 post-infection of Jurkat cells, normalized to producer cell p24 supernatant levels. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed unpaired t test. * = p

    Techniques Used: Infection, Incubation, Produced, Two Tailed Test

    rGal-9 is a potent mediator of HIV transcription ex vivo and synergizes with JQ1 in reactivating latent HIV . ( A ) Treatment of CD4+ T cells isolated from ART-suppressed HIV-infected individuals with DMSO 0.5% (negative control), PMA/ionomycin (2 nM / 500 nM), vorinostat (1μM), or varying concentrations of rGal-9 (500 nM and 1000 nM) for 24 hours. Fold increase in cell-associated HIV RNA was determined relative to the corresponding DMSO-treated control for each individual time point. Mean ± SEM is displayed, and statistical comparisons between rGal-9 and other treatments were performed using two-tailed paired Wilcoxon signed-rank tests. (B-C) CD4+ T cells were isolated from PBMCs of three HIV-infected ART-suppressed individuals using negative selection. Resting CD4+ T cells were further enriched through depletion of cells expressing CD69, CD25, or HLA-DR surface markers from half of the isolated CD4+ T cells. The remaining half was processed through the exact enrichment procedure, except PBS was added instead of the depleting antibodies. Both cell populations were treated with 0.5% DMSO (negative control), 500 nM rGal-9, 1000 nM rGal-9 or αCD3/αCD28-conjugated beads. Induction of cell-associated HIV RNA was measured 24 hours post-treatment using RT-qPCR. Each individual is represented with a different symbol. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t tests. Percentages reported reflect average values measured in the CD69- / CD25- / HLA-DR- CD4+ T cells with respect to values observed in total CD4+ T cells. (D) CD4+ T cells from HIV-infected ART-suppressed individuals were treated with 500 nM of rGal-9, 1 μM vorinostat, 40 nM romidepsin, 10 nM bryostatin, 300 nM prostratin, 1 μM JQ1, or 30 nM panobinostat alone or in combination with 500 nM of rGal-9 for 24 hours, and fold induction of cell-associated HIV RNA was determined using quantitative real-time PCR. * = p
    Figure Legend Snippet: rGal-9 is a potent mediator of HIV transcription ex vivo and synergizes with JQ1 in reactivating latent HIV . ( A ) Treatment of CD4+ T cells isolated from ART-suppressed HIV-infected individuals with DMSO 0.5% (negative control), PMA/ionomycin (2 nM / 500 nM), vorinostat (1μM), or varying concentrations of rGal-9 (500 nM and 1000 nM) for 24 hours. Fold increase in cell-associated HIV RNA was determined relative to the corresponding DMSO-treated control for each individual time point. Mean ± SEM is displayed, and statistical comparisons between rGal-9 and other treatments were performed using two-tailed paired Wilcoxon signed-rank tests. (B-C) CD4+ T cells were isolated from PBMCs of three HIV-infected ART-suppressed individuals using negative selection. Resting CD4+ T cells were further enriched through depletion of cells expressing CD69, CD25, or HLA-DR surface markers from half of the isolated CD4+ T cells. The remaining half was processed through the exact enrichment procedure, except PBS was added instead of the depleting antibodies. Both cell populations were treated with 0.5% DMSO (negative control), 500 nM rGal-9, 1000 nM rGal-9 or αCD3/αCD28-conjugated beads. Induction of cell-associated HIV RNA was measured 24 hours post-treatment using RT-qPCR. Each individual is represented with a different symbol. Mean ± SEM is displayed, and statistical comparisons were performed using two-tailed paired t tests. Percentages reported reflect average values measured in the CD69- / CD25- / HLA-DR- CD4+ T cells with respect to values observed in total CD4+ T cells. (D) CD4+ T cells from HIV-infected ART-suppressed individuals were treated with 500 nM of rGal-9, 1 μM vorinostat, 40 nM romidepsin, 10 nM bryostatin, 300 nM prostratin, 1 μM JQ1, or 30 nM panobinostat alone or in combination with 500 nM of rGal-9 for 24 hours, and fold induction of cell-associated HIV RNA was determined using quantitative real-time PCR. * = p

    Techniques Used: Ex Vivo, Isolation, Infection, Negative Control, Two Tailed Test, Selection, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    rGal-9 induces the expression of several anti-HIV host restriction factors including APOBEC3G ex vivo . ( A ) Heat map representing expression levels of host restriction factors in CD4+ T cells isolated from ART-suppressed individuals, after treatment with either 0.5% DMSO as negative control, 500 nM rGal-9, 1000 nM rGal-9, 1μM vorinostat, or a combination of PMA (2 nM) and Ionomycin (0.5 μM). Heat colors indicate fold modulation compared to the DMSO control. Red indicates induction of expression, and blue indicates reduction of expression. Statistical comparisons were performed using t tests, and p values were adjusted for multiple comparisons using false discovery rate. Asterisks indicate > 3-fold, statistically significant modulation of gene expression as compared to DMSO control, as follows: * = FDR
    Figure Legend Snippet: rGal-9 induces the expression of several anti-HIV host restriction factors including APOBEC3G ex vivo . ( A ) Heat map representing expression levels of host restriction factors in CD4+ T cells isolated from ART-suppressed individuals, after treatment with either 0.5% DMSO as negative control, 500 nM rGal-9, 1000 nM rGal-9, 1μM vorinostat, or a combination of PMA (2 nM) and Ionomycin (0.5 μM). Heat colors indicate fold modulation compared to the DMSO control. Red indicates induction of expression, and blue indicates reduction of expression. Statistical comparisons were performed using t tests, and p values were adjusted for multiple comparisons using false discovery rate. Asterisks indicate > 3-fold, statistically significant modulation of gene expression as compared to DMSO control, as follows: * = FDR

    Techniques Used: Expressing, Ex Vivo, Isolation, Negative Control

    rGal-9 partially activates primary CD4+ T cells and induces proliferation primarily in naïve CD4+ T cells. ( A , B ) Effects of rGal-9 stimulation on the cell surface expression of CD69 and CD25 activation markers on CD4+ T cells isolated from six ART-suppressed individuals. Mean ± SEM is displayed. Asterisks represent statistically significant differences as compared to DMSO control (p
    Figure Legend Snippet: rGal-9 partially activates primary CD4+ T cells and induces proliferation primarily in naïve CD4+ T cells. ( A , B ) Effects of rGal-9 stimulation on the cell surface expression of CD69 and CD25 activation markers on CD4+ T cells isolated from six ART-suppressed individuals. Mean ± SEM is displayed. Asterisks represent statistically significant differences as compared to DMSO control (p

    Techniques Used: Expressing, Activation Assay, Isolation

    38) Product Images from "A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins"

    Article Title: A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    Journal: Nature Communications

    doi: 10.1038/ncomms11685

    PsAvh262 is an essential virulence factor of Phytophthora sojae . ( a ) Expression profile of PsAvh262 during P. sojae strain P6497 infection of soybean hypocotyls. The susceptible soybean cultivar Williams was used as the host. Total RNA was extracted from mycelia (MY) or infected soybean leaves at 1.5, 3, 6, 12 and 24 h post inoculation (h.p.i.). Transcript levels of PsAvh262 were determined by qRT–PCR. The P. sojae actin gene (VMD GeneID: 108986) was used as the pathogen internal control gene, ( b ) Silencing of PsAvh262 in P. sojae greatly impaired the virulence in soybean hypocotyls. Relative transcript levels of PsAvh262 (upper panel) in the P. sojae transformants were determined by qRT–PCR. Disease symptoms (lower panel) in etiolated hypocotyls were observed. Pictures were taken at 7 days post inoculation (d.p.i.). S12 and S141 were non-silenced transformants carrying the same silencing construct. ( c ) Expression of PsAvh262 in soybean hairy roots enhanced P. sojae infection. Hairy roots expressing GFP-PsAvh262 or GFP were inoculated with mycelia plugs of RFP-labelled wild-type P. sojae strain P6497 (P6497-RFP). Oospore production in the infected hair roots was observed under a confocal microscope (left panel), and lesion length was determined (middle panel) at 48 h.p.i. Expression of GFP or GFP-PsAvh262 was confirmed by western blotting using an anti-GFP antibody (right panel). ( d ) Expression of PsAvh262 in N. benthamiana enhanced infection of Phytophthora capsici . Leaf regions transiently expressing PsAvh262 or GFP, were inoculated with mycelia plugs of P. capsici . Infected leaves were stained with Trypan blue at 36 h.p.i. to visualize disease lesions (left panel) and the sizes of the lesions were determined (middle panel). Expression of GFP or PsAvh262-GFP was confirmed by western blotting using an anti-GFP antibody (right panel). Δ, non-specific band when using anti-GFP, which are common contaminants of western blots present in many published articles detecting GFP-fused proteins expressed in the plant cells 13 64 ; #, PsAvh262 derived band; this may due to some unknown modification or degradation. Error bars represent the mean±s.d.( n =3) and asterisks ( ** or *** ) denote significant differences ( P
    Figure Legend Snippet: PsAvh262 is an essential virulence factor of Phytophthora sojae . ( a ) Expression profile of PsAvh262 during P. sojae strain P6497 infection of soybean hypocotyls. The susceptible soybean cultivar Williams was used as the host. Total RNA was extracted from mycelia (MY) or infected soybean leaves at 1.5, 3, 6, 12 and 24 h post inoculation (h.p.i.). Transcript levels of PsAvh262 were determined by qRT–PCR. The P. sojae actin gene (VMD GeneID: 108986) was used as the pathogen internal control gene, ( b ) Silencing of PsAvh262 in P. sojae greatly impaired the virulence in soybean hypocotyls. Relative transcript levels of PsAvh262 (upper panel) in the P. sojae transformants were determined by qRT–PCR. Disease symptoms (lower panel) in etiolated hypocotyls were observed. Pictures were taken at 7 days post inoculation (d.p.i.). S12 and S141 were non-silenced transformants carrying the same silencing construct. ( c ) Expression of PsAvh262 in soybean hairy roots enhanced P. sojae infection. Hairy roots expressing GFP-PsAvh262 or GFP were inoculated with mycelia plugs of RFP-labelled wild-type P. sojae strain P6497 (P6497-RFP). Oospore production in the infected hair roots was observed under a confocal microscope (left panel), and lesion length was determined (middle panel) at 48 h.p.i. Expression of GFP or GFP-PsAvh262 was confirmed by western blotting using an anti-GFP antibody (right panel). ( d ) Expression of PsAvh262 in N. benthamiana enhanced infection of Phytophthora capsici . Leaf regions transiently expressing PsAvh262 or GFP, were inoculated with mycelia plugs of P. capsici . Infected leaves were stained with Trypan blue at 36 h.p.i. to visualize disease lesions (left panel) and the sizes of the lesions were determined (middle panel). Expression of GFP or PsAvh262-GFP was confirmed by western blotting using an anti-GFP antibody (right panel). Δ, non-specific band when using anti-GFP, which are common contaminants of western blots present in many published articles detecting GFP-fused proteins expressed in the plant cells 13 64 ; #, PsAvh262 derived band; this may due to some unknown modification or degradation. Error bars represent the mean±s.d.( n =3) and asterisks ( ** or *** ) denote significant differences ( P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Construct, Microscopy, Western Blot, Staining, Derivative Assay, Modification

    39) Product Images from "Next generation deep sequencing identified a novel lncRNA n375709 associated with paclitaxel resistance in nasopharyngeal carcinoma"

    Article Title: Next generation deep sequencing identified a novel lncRNA n375709 associated with paclitaxel resistance in nasopharyngeal carcinoma

    Journal: Oncology Reports

    doi: 10.3892/or.2016.4981

    CCK-8 assays confirmed that CNE-2-Pr cells were significantly more resistant to paclitaxel than the CNE-2.
    Figure Legend Snippet: CCK-8 assays confirmed that CNE-2-Pr cells were significantly more resistant to paclitaxel than the CNE-2.

    Techniques Used: CCK-8 Assay

    40) Product Images from "β‐Estradiol results in a proprotein convertase subtilisin/kexin type 9‐dependent increase in low‐density lipoprotein receptor levels in human hepatic HuH7 cells"

    Article Title: β‐Estradiol results in a proprotein convertase subtilisin/kexin type 9‐dependent increase in low‐density lipoprotein receptor levels in human hepatic HuH7 cells

    Journal: The Febs Journal

    doi: 10.1111/febs.13309

    Assessment of sh RNA knockdown of PCSK 9 in HuH7 cells. PCSK 9 knockdown was evaluated in HuH7 cells that were transfected with nontarget scrambled sh RNA ( NT ) or with PCSK 9‐targeted sh RNA (shP). (A) The specificity and level of knockdown was assessed at the RNA level by qPCR , with primers for LDLR and PCSK 9, respectively. (B) The level of knockdown was assessed at the secreted protein level by PCSK 9 ELISA of cell‐conditioned media, relative to the nontarget conditioned media. (C) NT or shP cells were incubated in triplicate in serum‐free media for 24 h before the addition of vehicle or 10 μ m compactin for another 24 h. LDLR levels in total cell lysates were (C) evaluated by immunoblotting; the resultant densitometry results are shown relative to vehicle‐treated cells, and indicate that shP cells are as responsive to statins as their NT counterparts. n = 3 experiments. **** P
    Figure Legend Snippet: Assessment of sh RNA knockdown of PCSK 9 in HuH7 cells. PCSK 9 knockdown was evaluated in HuH7 cells that were transfected with nontarget scrambled sh RNA ( NT ) or with PCSK 9‐targeted sh RNA (shP). (A) The specificity and level of knockdown was assessed at the RNA level by qPCR , with primers for LDLR and PCSK 9, respectively. (B) The level of knockdown was assessed at the secreted protein level by PCSK 9 ELISA of cell‐conditioned media, relative to the nontarget conditioned media. (C) NT or shP cells were incubated in triplicate in serum‐free media for 24 h before the addition of vehicle or 10 μ m compactin for another 24 h. LDLR levels in total cell lysates were (C) evaluated by immunoblotting; the resultant densitometry results are shown relative to vehicle‐treated cells, and indicate that shP cells are as responsive to statins as their NT counterparts. n = 3 experiments. **** P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation

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    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae
    Article Snippet: .. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Genomic DNA was removed from total RNA by TURBO® DNase treatment (Ambion, New York, USA). .. Reverse transcription reactions were performed with 500 ng of RNA in 20 μl reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems, California, USA) by following the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics
    Article Snippet: .. RNA samples were DNase treated using TURBO DNase (Ambion) and purified using the RNeasy MinElute Cleanup Kit (Qiagen), to retain small RNAs according to manufacturer's instructions. cDNA was synthesized from 500 ng of extracted RNA using random hexamers at a concentration of 250 ng per 5 μg of RNA with the SuperScript III Reverse Transcriptase Kit (Invitrogen) as per manufacturer's instructions. .. The synthesized cDNA was diluted four times in RNase-free water and subsequently used for qRT-PCR to quantify mouse miR-29b target genes Col3a1 , Col1a1 , Col5a3 , Adam12, and Dnmt3a demonstrates primer binding sites for NIH3T3 qRT-PCR positive and negative control genes.

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  • 99
    Thermo Fisher nano drop 1000 spectrophotometer
    RNA extract from different numbers of worms. Results are presented as mean ± S.D. (n = 3). There is a linear relationship (y = 20.48x − 27.88, R 2 = 0.9976) between worm numbers and the yield of RNA extract. There is a correlation between the released RNA content after worm lysis measured by Qubit kit/fluorometer and RNA yield determined by <t>Nano-drop</t> 1000 Spectrophotometer after RNA extraction (Pearson correlation test: r = 0.9989, P
    Nano Drop 1000 Spectrophotometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano drop 1000 spectrophotometer/product/Thermo Fisher
    Average 99 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    nano drop 1000 spectrophotometer - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher nanodrop spectrophotometer nd 1000
    Scatter diagrams showing the quantity and quality of DNA in blood and milk. DNA samples were obtained from 2 BLV-negative cattle in Farm 3, 43 BLV-infected cattle without lymphoma in Farms 2–6, and 3 BLV-infected cattle with lymphoma in Farm 1. A The average quantities of genomic DNA in blood and milk samples were 7.9 µg and 6.7 µg, respectively, as determined with a <t>NanoDrop</t> spectrophotometer <t>ND-1000.</t> B The average A260/A280 ratios of genomic DNA in blood and milk were 1.88 and 1.87, respectively, as determined using a NanoDrop spectrophotometer ND-1000. C The threshold cycle values of the blood and milk samples were 22.12 and 22.37, respectively, as indicated with the red bold lines.
    Nanodrop Spectrophotometer Nd 1000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nanodrop spectrophotometer nd 1000/product/Thermo Fisher
    Average 94 stars, based on 251 article reviews
    Price from $9.99 to $1999.99
    nanodrop spectrophotometer nd 1000 - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    85
    Thermo Fisher nano drop kit
    Scatter diagrams showing the quantity and quality of DNA in blood and milk. DNA samples were obtained from 2 BLV-negative cattle in Farm 3, 43 BLV-infected cattle without lymphoma in Farms 2–6, and 3 BLV-infected cattle with lymphoma in Farm 1. A The average quantities of genomic DNA in blood and milk samples were 7.9 µg and 6.7 µg, respectively, as determined with a <t>NanoDrop</t> spectrophotometer <t>ND-1000.</t> B The average A260/A280 ratios of genomic DNA in blood and milk were 1.88 and 1.87, respectively, as determined using a NanoDrop spectrophotometer ND-1000. C The threshold cycle values of the blood and milk samples were 22.12 and 22.37, respectively, as indicated with the red bold lines.
    Nano Drop Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nano drop kit/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nano drop kit - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    RNA extract from different numbers of worms. Results are presented as mean ± S.D. (n = 3). There is a linear relationship (y = 20.48x − 27.88, R 2 = 0.9976) between worm numbers and the yield of RNA extract. There is a correlation between the released RNA content after worm lysis measured by Qubit kit/fluorometer and RNA yield determined by Nano-drop 1000 Spectrophotometer after RNA extraction (Pearson correlation test: r = 0.9989, P

    Journal: MethodsX

    Article Title: Inappropriateness of RNAlater to preserve Caenorhabditis elegans for RNA extraction

    doi: 10.1016/j.mex.2019.10.015

    Figure Lengend Snippet: RNA extract from different numbers of worms. Results are presented as mean ± S.D. (n = 3). There is a linear relationship (y = 20.48x − 27.88, R 2 = 0.9976) between worm numbers and the yield of RNA extract. There is a correlation between the released RNA content after worm lysis measured by Qubit kit/fluorometer and RNA yield determined by Nano-drop 1000 Spectrophotometer after RNA extraction (Pearson correlation test: r = 0.9989, P

    Article Snippet: The yield of total RNA was determined by Nano-drop 1000 Spectrophotometer (Thermo Scientific).

    Techniques: Lysis, Spectrophotometry, RNA Extraction

    Scatter diagrams showing the quantity and quality of DNA in blood and milk. DNA samples were obtained from 2 BLV-negative cattle in Farm 3, 43 BLV-infected cattle without lymphoma in Farms 2–6, and 3 BLV-infected cattle with lymphoma in Farm 1. A The average quantities of genomic DNA in blood and milk samples were 7.9 µg and 6.7 µg, respectively, as determined with a NanoDrop spectrophotometer ND-1000. B The average A260/A280 ratios of genomic DNA in blood and milk were 1.88 and 1.87, respectively, as determined using a NanoDrop spectrophotometer ND-1000. C The threshold cycle values of the blood and milk samples were 22.12 and 22.37, respectively, as indicated with the red bold lines.

    Journal: Veterinary Research

    Article Title: Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams

    doi: 10.1186/s13567-019-0724-1

    Figure Lengend Snippet: Scatter diagrams showing the quantity and quality of DNA in blood and milk. DNA samples were obtained from 2 BLV-negative cattle in Farm 3, 43 BLV-infected cattle without lymphoma in Farms 2–6, and 3 BLV-infected cattle with lymphoma in Farm 1. A The average quantities of genomic DNA in blood and milk samples were 7.9 µg and 6.7 µg, respectively, as determined with a NanoDrop spectrophotometer ND-1000. B The average A260/A280 ratios of genomic DNA in blood and milk were 1.88 and 1.87, respectively, as determined using a NanoDrop spectrophotometer ND-1000. C The threshold cycle values of the blood and milk samples were 22.12 and 22.37, respectively, as indicated with the red bold lines.

    Article Snippet: The quantity and quality of DNA samples extracted from milk sample was determined based on the A260/280 ratio using a Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific).

    Techniques: Infection, Spectrophotometry