Structured Review

Bio-Rad qrt pcr analysis
Expression analysis of five genes in cotton anthers by <t>qRT-PCR.</t> The housekeeping gene 18S was used as an internal control; H276A, CMS line; H276B, maintainer line; and H276A/H268, F1 plants from the descendant of restorer H268 hybridized with H276A. Error bars represent standard deviation (n = 3). Double asterisk represents significance at 1% probability
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1) Product Images from "RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A"

Article Title: RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A

Journal: Biological Research

doi: 10.1186/s40659-019-0212-0

Expression analysis of five genes in cotton anthers by qRT-PCR. The housekeeping gene 18S was used as an internal control; H276A, CMS line; H276B, maintainer line; and H276A/H268, F1 plants from the descendant of restorer H268 hybridized with H276A. Error bars represent standard deviation (n = 3). Double asterisk represents significance at 1% probability
Figure Legend Snippet: Expression analysis of five genes in cotton anthers by qRT-PCR. The housekeeping gene 18S was used as an internal control; H276A, CMS line; H276B, maintainer line; and H276A/H268, F1 plants from the descendant of restorer H268 hybridized with H276A. Error bars represent standard deviation (n = 3). Double asterisk represents significance at 1% probability

Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

2) Product Images from "Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease"

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.024745

Elevated IL10 expression and STAT3 activation in ADPKD kidney macrophages. (A) Elevated IL10 expression in human ADPKD kidneys. RNA was isolated from NHKs and non-cystic regions of ADPKD kidneys (three of each). IL10 expression was analyzed by performing qRT-PCR. The line indicates the mean relative value. (B) IL-10 is present in ADPKD cyst fluid. IL-10 in pooled cyst fluid collected from ADPKD kidneys from 12 different individuals was measured by using ELISA. Data are mean±s.e.m. (C-G) Phospho-STAT3 was present in macrophages of cystic ADPKD kidneys. Formalin-fixed, paraffin-embedded tissues from ADPKD kidneys were serially sectioned (C-E), and consecutive sections were stained by using immunohistochemistry with antibodies against the M2 macrophage marker CD163 (C), phospho-STAT3 (D) or secondary antibody only (E, No Ab) as a control. Arrows indicate phospho-STAT3-positive cells in macrophage-rich regions of interstitium. (F,G) Sections of ADPKD kidneys that had been fixed and prepared in a similar manner were co-stained with anti-CD163 (green) and anti-phospho-STAT3 (red) antibodies, followed by incubation with two distinct fluorescence-labeled secondary antibodies, each with the appropriate antibody specificity (F), and with DAPI (G). Arrowheads indicate co-staining cells. n =3 biological replicates. hIL-10, human IL-10.
Figure Legend Snippet: Elevated IL10 expression and STAT3 activation in ADPKD kidney macrophages. (A) Elevated IL10 expression in human ADPKD kidneys. RNA was isolated from NHKs and non-cystic regions of ADPKD kidneys (three of each). IL10 expression was analyzed by performing qRT-PCR. The line indicates the mean relative value. (B) IL-10 is present in ADPKD cyst fluid. IL-10 in pooled cyst fluid collected from ADPKD kidneys from 12 different individuals was measured by using ELISA. Data are mean±s.e.m. (C-G) Phospho-STAT3 was present in macrophages of cystic ADPKD kidneys. Formalin-fixed, paraffin-embedded tissues from ADPKD kidneys were serially sectioned (C-E), and consecutive sections were stained by using immunohistochemistry with antibodies against the M2 macrophage marker CD163 (C), phospho-STAT3 (D) or secondary antibody only (E, No Ab) as a control. Arrows indicate phospho-STAT3-positive cells in macrophage-rich regions of interstitium. (F,G) Sections of ADPKD kidneys that had been fixed and prepared in a similar manner were co-stained with anti-CD163 (green) and anti-phospho-STAT3 (red) antibodies, followed by incubation with two distinct fluorescence-labeled secondary antibodies, each with the appropriate antibody specificity (F), and with DAPI (G). Arrowheads indicate co-staining cells. n =3 biological replicates. hIL-10, human IL-10.

Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Marker, Incubation, Fluorescence, Labeling

3) Product Images from "Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo"

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo

Journal: Nutrition & Metabolism

doi: 10.1186/1743-7075-11-54

Relative mRNA expression of vimentin in VAA and VAD diet rat liver. Vimentin mRNA in VAA (black bars) or VAD (grey bars) rats, determined by qRT-PCR. Normalized values were expressed as the mean ± SEM of n = 5 ( n = 4 for VAD vehicle)/group, with the VAA control group set to 1.0. Diet was a significant main effect, P
Figure Legend Snippet: Relative mRNA expression of vimentin in VAA and VAD diet rat liver. Vimentin mRNA in VAA (black bars) or VAD (grey bars) rats, determined by qRT-PCR. Normalized values were expressed as the mean ± SEM of n = 5 ( n = 4 for VAD vehicle)/group, with the VAA control group set to 1.0. Diet was a significant main effect, P

Techniques Used: Expressing, Quantitative RT-PCR

4) Product Images from "CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression"

Article Title: CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression

Journal: Oncotarget

doi:

CBX7 and HMGA1b bind the SPP1 promoter in vivo HEK 293 cells were transiently transfected with a vector expressing HMGA1b-HA or CBX7-V5 or with the only empty vector. The chromatin was immunoprecipitated (IP) by using antibodies against HA ( A , left panel) or V5 ( B , left panel) tag. IgG were used as negative control. The IP chromatin was analyzed by qRT-PCR assay performed in triplicate with primers specific for the SPP1 promoter. GAPDH promoter primers were used as control of the binding specificity. Values are the mean of three (A) or two (B) independent experiments ± SEM. Mann Whitney test: ** p
Figure Legend Snippet: CBX7 and HMGA1b bind the SPP1 promoter in vivo HEK 293 cells were transiently transfected with a vector expressing HMGA1b-HA or CBX7-V5 or with the only empty vector. The chromatin was immunoprecipitated (IP) by using antibodies against HA ( A , left panel) or V5 ( B , left panel) tag. IgG were used as negative control. The IP chromatin was analyzed by qRT-PCR assay performed in triplicate with primers specific for the SPP1 promoter. GAPDH promoter primers were used as control of the binding specificity. Values are the mean of three (A) or two (B) independent experiments ± SEM. Mann Whitney test: ** p

Techniques Used: In Vivo, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Binding Assay, MANN-WHITNEY

HMGA1b induces the expression of the SPP1 gene (A) Left Panel. qRT-PCR analysis of SPP1 in B-CPAP cells transfected with increasing amount (1 μg, 3 μg and 5 μg) of HMGA1b-HA expressing vector. The total amount of the transfected DNA was balanced with the empty vector. Relative Expression represents values normalized to control cells transfected with the only empty vector, set equal to 1. Values are the mean of four independent experiments performed in triplicate ± SEM. Kruskal-Wallis test followed by Dunn's post test: * p
Figure Legend Snippet: HMGA1b induces the expression of the SPP1 gene (A) Left Panel. qRT-PCR analysis of SPP1 in B-CPAP cells transfected with increasing amount (1 μg, 3 μg and 5 μg) of HMGA1b-HA expressing vector. The total amount of the transfected DNA was balanced with the empty vector. Relative Expression represents values normalized to control cells transfected with the only empty vector, set equal to 1. Values are the mean of four independent experiments performed in triplicate ± SEM. Kruskal-Wallis test followed by Dunn's post test: * p

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation

5) Product Images from "Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress"

Article Title: Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19030915

Response of alternative oxidases (AOXs) in rice under salt stress. Fold change expression of AOX isoforms, AOX1a , AOX1c and AOX1d in seedling shoots ( A ) and roots ( B ) were analysed in response to 120 mM NaCl using qRT-PCR over a period of 12 days. Data are shown as the mean ± SE of three biological replicates relative to the control at each time point set as 1.0. Significant differences are indicated by asterisks (*) at p
Figure Legend Snippet: Response of alternative oxidases (AOXs) in rice under salt stress. Fold change expression of AOX isoforms, AOX1a , AOX1c and AOX1d in seedling shoots ( A ) and roots ( B ) were analysed in response to 120 mM NaCl using qRT-PCR over a period of 12 days. Data are shown as the mean ± SE of three biological replicates relative to the control at each time point set as 1.0. Significant differences are indicated by asterisks (*) at p

Techniques Used: Expressing, Quantitative RT-PCR

Expression of OsNDB2 in rice under salt stress. Expression was analysed in seedling roots ( A ) and shoots ( B ) exposed to 120 mM NaCl over the period of 15 days using qRT-PCR. Data are shown as the mean relative gene expression ± SE of three biological replicates; ( C ). An immunoblot of the OsNDB2 protein present in mitochondria isolated from salt-treated and control shoots harvested 9 days after the start of salt application. CR-control roots; TR-treated roots; CS-control shoots; TS-treated shoots.
Figure Legend Snippet: Expression of OsNDB2 in rice under salt stress. Expression was analysed in seedling roots ( A ) and shoots ( B ) exposed to 120 mM NaCl over the period of 15 days using qRT-PCR. Data are shown as the mean relative gene expression ± SE of three biological replicates; ( C ). An immunoblot of the OsNDB2 protein present in mitochondria isolated from salt-treated and control shoots harvested 9 days after the start of salt application. CR-control roots; TR-treated roots; CS-control shoots; TS-treated shoots.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation

Expression of alternative dehydrogenases (NDs) in rice under salt stress. Fold change expression of ND isoforms, OsNDA1 , OsNDA2 , OsNDB1 , OsNDB2 , OsNDB3 and OsNDC1 , in seedling shoots ( A ) and roots ( B ) were analysed in response to 120 mM NaCl using qRT-PCR over a period of 12 days. Data are shown as the mean ± SE of three biological replicates relative to the control at each time point set as 1.00. Significant differences are indicated by asterisks (*) at p
Figure Legend Snippet: Expression of alternative dehydrogenases (NDs) in rice under salt stress. Fold change expression of ND isoforms, OsNDA1 , OsNDA2 , OsNDB1 , OsNDB2 , OsNDB3 and OsNDC1 , in seedling shoots ( A ) and roots ( B ) were analysed in response to 120 mM NaCl using qRT-PCR over a period of 12 days. Data are shown as the mean ± SE of three biological replicates relative to the control at each time point set as 1.00. Significant differences are indicated by asterisks (*) at p

Techniques Used: Expressing, Quantitative RT-PCR

6) Product Images from "The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis"

Article Title: The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis

Journal: Journal of Cell Science

doi: 10.1242/jcs.165233

miR-199a/b regulates LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells. (A,B) qRT-PCR analysis of LDLR , CLTC , Rab5A and Rab21 expression in Huh7 cells transfected with non-targeting control mimic (CM), miR-199a-5p mimic, or control inhibitor (CI) and miR-199a-5p inhibitor. (C) Western blot analysis of LDLR, CLTC, Rab5A and Rab21 in Huh7 cells transfected with control mimic or miR-199a-5p mimic (upper panels) and control inhibitor or inh-199a-5p (lower panels). A densitometry analysis is shown in the corresponding histograms; Hsp90 was used as a loading control. (D) Human LDLR , CLTC , Rab21 and Rab5a 3′UTR containing the indicated point mutations (PM) in the miR-199a/b-5p target sites. (E) Luciferase reporter activity in COS7 cells transfected with control mimic or miR-199a-5p mimic and the indicated human 3′UTR containing or not (wild-type, WT) the indicated point mutation in the target miR-199a-5p-binding sites. In A and B, data are expressed as mean±s.e.m. and representative of ≥3 experiments in triplicate. In A–C, data are expressed as mean±s.e.m. and representative of ≥3 experiments performed in duplicate. In E, data are expressed as a percentage of 3′UTR activity of control mimic (±s.e.m.) and are representative of ≥3 experiments performed in triplicate. * P ≤0.05.
Figure Legend Snippet: miR-199a/b regulates LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells. (A,B) qRT-PCR analysis of LDLR , CLTC , Rab5A and Rab21 expression in Huh7 cells transfected with non-targeting control mimic (CM), miR-199a-5p mimic, or control inhibitor (CI) and miR-199a-5p inhibitor. (C) Western blot analysis of LDLR, CLTC, Rab5A and Rab21 in Huh7 cells transfected with control mimic or miR-199a-5p mimic (upper panels) and control inhibitor or inh-199a-5p (lower panels). A densitometry analysis is shown in the corresponding histograms; Hsp90 was used as a loading control. (D) Human LDLR , CLTC , Rab21 and Rab5a 3′UTR containing the indicated point mutations (PM) in the miR-199a/b-5p target sites. (E) Luciferase reporter activity in COS7 cells transfected with control mimic or miR-199a-5p mimic and the indicated human 3′UTR containing or not (wild-type, WT) the indicated point mutation in the target miR-199a-5p-binding sites. In A and B, data are expressed as mean±s.e.m. and representative of ≥3 experiments in triplicate. In A–C, data are expressed as mean±s.e.m. and representative of ≥3 experiments performed in duplicate. In E, data are expressed as a percentage of 3′UTR activity of control mimic (±s.e.m.) and are representative of ≥3 experiments performed in triplicate. * P ≤0.05.

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Luciferase, Activity Assay, Mutagenesis, Binding Assay

7) Product Images from "Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation"

Article Title: Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation

Journal: Biological Research

doi: 10.1186/s40659-018-0189-0

SETDB1 was regulated by miR-381-3p in a direct interaction in breast cancer cell lines. a Schematic of potential binding sites for miR-381-3p in the SETDB1 3′-UTR, the seed and the mutated sequences of potential binding sites. Luciferase reporter assays were performed to verify the interaction between miR-381-3p and SETDB1 by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p mimic into MDA-MB-231 cells ( b ) and MCF-7 cells ( c ), or by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p inhibitor into MDA-MB-231 cells ( d ) and MCF-7 cells ( e ). f RIP analysis was used to evaluate the binding between miR-381-3p and SETDB1 using anti-Ago1 in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic, followed by measurement of SETDB1 mRNA by qRT-PCR assay. g Western blot analysis of SETDB1 protein expression in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic or miR-381-3p inhibitor. * P
Figure Legend Snippet: SETDB1 was regulated by miR-381-3p in a direct interaction in breast cancer cell lines. a Schematic of potential binding sites for miR-381-3p in the SETDB1 3′-UTR, the seed and the mutated sequences of potential binding sites. Luciferase reporter assays were performed to verify the interaction between miR-381-3p and SETDB1 by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p mimic into MDA-MB-231 cells ( b ) and MCF-7 cells ( c ), or by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p inhibitor into MDA-MB-231 cells ( d ) and MCF-7 cells ( e ). f RIP analysis was used to evaluate the binding between miR-381-3p and SETDB1 using anti-Ago1 in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic, followed by measurement of SETDB1 mRNA by qRT-PCR assay. g Western blot analysis of SETDB1 protein expression in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic or miR-381-3p inhibitor. * P

Techniques Used: Binding Assay, Luciferase, Cotransfection, Construct, Multiple Displacement Amplification, Transfection, Quantitative RT-PCR, Western Blot, Expressing

SETDB1 depletion repressed tumor growth in vivo. Nude mice were subcutaneously injected with 1.0 × 10 6 MCF-7 cells transfected with siNC or siSETDB1. 50 days later, mice were killed and tumor masses were removed. a Tumor volume was mearsured by a caliper every 10 days. b Bright-field imaging of the xenograft tumors. c The average weight of the xenograft tumors. d qRT-PCR assay of SETDB1 mRNA expression in removed tumor tissues. * P
Figure Legend Snippet: SETDB1 depletion repressed tumor growth in vivo. Nude mice were subcutaneously injected with 1.0 × 10 6 MCF-7 cells transfected with siNC or siSETDB1. 50 days later, mice were killed and tumor masses were removed. a Tumor volume was mearsured by a caliper every 10 days. b Bright-field imaging of the xenograft tumors. c The average weight of the xenograft tumors. d qRT-PCR assay of SETDB1 mRNA expression in removed tumor tissues. * P

Techniques Used: In Vivo, Mouse Assay, Injection, Transfection, Imaging, Quantitative RT-PCR, Expressing

SETDB1 mRNA expression was upregulated in breast cancer tissues and cell lines. a SETDB1 mRNA expression was measured in 45 pairs breast cancer tissues and adjacent normal breast tissues by qRT-PCR assay. b qRT-PCR assay of SETDB1 mRNA level in breast cancer cell lines (MCF-7, MDA-MB-231) and human mammary epithelial cell line (MCF-10A). c Kaplan–Meier survival assay and log-rank test were used to evaluate the correlation between SETDB1 mRNA level and breast cancer patient prognosis in 45 breast cancer patients in low- and high-risk groups based on SETDB1 mRNA expression. * P
Figure Legend Snippet: SETDB1 mRNA expression was upregulated in breast cancer tissues and cell lines. a SETDB1 mRNA expression was measured in 45 pairs breast cancer tissues and adjacent normal breast tissues by qRT-PCR assay. b qRT-PCR assay of SETDB1 mRNA level in breast cancer cell lines (MCF-7, MDA-MB-231) and human mammary epithelial cell line (MCF-10A). c Kaplan–Meier survival assay and log-rank test were used to evaluate the correlation between SETDB1 mRNA level and breast cancer patient prognosis in 45 breast cancer patients in low- and high-risk groups based on SETDB1 mRNA expression. * P

Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Clonogenic Cell Survival Assay

8) Product Images from "EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways"

Article Title: EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002798

Noncanonical signaling contributes to lifting hif-1 -mediated repression of the host defense response. A. hif-1 animals overexpressing wild type HIF-1 ( hif-1;[hif-1] ) or non-hydroxylatable HIF-1 ( hif-1;[hif-1 P621G ] ) were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test); †, p ≤0.05 (compared hif-1;[hif-1] with hif-1;[hif-1 P621G ] by two-sample t test). B. swan-1(ok267) mutants were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. egl-9(sa307) data from Figure 3 are included for comparison. Results are means of 3–5 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test). C. Genes whose expression levels were intermediate in swan-1; [hif-1 P621G ] animals compared with swan-1 and egl-9 animals. swan-1 animals overexpressing non-hydroxylatable HIF-1 ( swan-1;hif-1 P621G ) were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test). Data for swan-1 and egl-9 mutants from Figure 5A and 3 are included for comparison. D. Genes whose expression levels did not appear intermediate in swan-1;hif-1 P621G animals compared with egl-9 and swan-1 animals. Data for swan-1 and egl-9 mutants from Figure 5A and 3 are included for comparison. E. swan-1(ok267) mutants exhibit enhanced susceptibility to S. aureus . Survival analysis: wild type MS = 65 h, N = 110/4; swan-1 MS = 48 h, N = 108/1, p = 0.0036 (compared with wild type); egl-9 MS = 40 h, N = 87/2, p
Figure Legend Snippet: Noncanonical signaling contributes to lifting hif-1 -mediated repression of the host defense response. A. hif-1 animals overexpressing wild type HIF-1 ( hif-1;[hif-1] ) or non-hydroxylatable HIF-1 ( hif-1;[hif-1 P621G ] ) were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test); †, p ≤0.05 (compared hif-1;[hif-1] with hif-1;[hif-1 P621G ] by two-sample t test). B. swan-1(ok267) mutants were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. egl-9(sa307) data from Figure 3 are included for comparison. Results are means of 3–5 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test). C. Genes whose expression levels were intermediate in swan-1; [hif-1 P621G ] animals compared with swan-1 and egl-9 animals. swan-1 animals overexpressing non-hydroxylatable HIF-1 ( swan-1;hif-1 P621G ) were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test). Data for swan-1 and egl-9 mutants from Figure 5A and 3 are included for comparison. D. Genes whose expression levels did not appear intermediate in swan-1;hif-1 P621G animals compared with egl-9 and swan-1 animals. Data for swan-1 and egl-9 mutants from Figure 5A and 3 are included for comparison. E. swan-1(ok267) mutants exhibit enhanced susceptibility to S. aureus . Survival analysis: wild type MS = 65 h, N = 110/4; swan-1 MS = 48 h, N = 108/1, p = 0.0036 (compared with wild type); egl-9 MS = 40 h, N = 87/2, p

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Mass Spectrometry

egl-9 is required to lift repression of host defense genes by hif-1 . A, B, C. egl-9(sa307) and egl-9(sa307);hif-1(ia4) animals were fed heat-killed non-pathogenic E. coli for 8 h and gene expression, measured by qRT-PCR, was normalized to parallel wild type controls. Genes were divided into three groups, according to their expression in infected egl-9 animals (see G, H, I): A. egl-9- repressed genes, B. egl-9- independent genes, and C. egl-9- induced genes. D, E, F. vhl-1(ok161) and vhl-1(ok161);hif-1(ia4) animals were fed heat-killed non-pathogenic E. coli and gene expression was normalized to wild type. Genes were grouped as in A, B, C. G, H, I. egl-9(sa307) and egl-9(sa307);hif-1(ia4) animals were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Genes are divided into three groups: G. egl-9- repressed genes, H. egl-9- independent genes, and I. egl-9- induced genes. J, K, L. vhl-1(ok161) and vhl-1(ok161);hif-1(ia4) animals were infected and gene expression was normalized to wild type. Genes are grouped as in G, H, I. Data are means of 2–5 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test).
Figure Legend Snippet: egl-9 is required to lift repression of host defense genes by hif-1 . A, B, C. egl-9(sa307) and egl-9(sa307);hif-1(ia4) animals were fed heat-killed non-pathogenic E. coli for 8 h and gene expression, measured by qRT-PCR, was normalized to parallel wild type controls. Genes were divided into three groups, according to their expression in infected egl-9 animals (see G, H, I): A. egl-9- repressed genes, B. egl-9- independent genes, and C. egl-9- induced genes. D, E, F. vhl-1(ok161) and vhl-1(ok161);hif-1(ia4) animals were fed heat-killed non-pathogenic E. coli and gene expression was normalized to wild type. Genes were grouped as in A, B, C. G, H, I. egl-9(sa307) and egl-9(sa307);hif-1(ia4) animals were infected with S. aureus for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Genes are divided into three groups: G. egl-9- repressed genes, H. egl-9- independent genes, and I. egl-9- induced genes. J, K, L. vhl-1(ok161) and vhl-1(ok161);hif-1(ia4) animals were infected and gene expression was normalized to wild type. Genes are grouped as in G, H, I. Data are means of 2–5 independent biological replicates, error bars are SEM. *, p ≤0.05 (compared with wild type by two-sample t test).

Techniques Used: Expressing, Quantitative RT-PCR, Infection

9) Product Images from "Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors"

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq248

The effects of in vivo depletion of splicing factors on the splicing patterns of endogenous and synthetic genes containing conserved intronic hexamers. ( A ) qRT-PCR analysis of the siRNA treated GFP-SMN1 control cell lines with primer sets specific for exon included (primer set 4, Figure 1 D) and excluded (primer set 5) products of 10 endogenous genes. The splicing patterns of each gene are diagrammed where black bars represent exons and red bars represent the location of conserved ISRE hexamer motifs. Data is reported as the ratio of the mean expression of the exon excluded isoform to the exon included isoform normalized to the ratio for the GFP-SMN1 control ± the average error. P -values derived from the Student's t-test are as follows: * P
Figure Legend Snippet: The effects of in vivo depletion of splicing factors on the splicing patterns of endogenous and synthetic genes containing conserved intronic hexamers. ( A ) qRT-PCR analysis of the siRNA treated GFP-SMN1 control cell lines with primer sets specific for exon included (primer set 4, Figure 1 D) and excluded (primer set 5) products of 10 endogenous genes. The splicing patterns of each gene are diagrammed where black bars represent exons and red bars represent the location of conserved ISRE hexamer motifs. Data is reported as the ratio of the mean expression of the exon excluded isoform to the exon included isoform normalized to the ratio for the GFP-SMN1 control ± the average error. P -values derived from the Student's t-test are as follows: * P

Techniques Used: In Vivo, Quantitative RT-PCR, Expressing, Derivative Assay

A Screening PLatform for Intronic Control Elements (SPLICE) provides a generalizable in vivo screening strategy for ISREs. ( A ) SPLICE couples an exon inclusion event in a mini-gene (SMN1) to the expression level of a fluorescent protein (GFP) through a NMD-based reporter system. A random nucleotide library cloned upstream of the 3′ ss is screened for ISREs by sorting cells based on fluorescence levels. The enriched cells are sorted into sections (A, B, C) in a second screening round. ( B ) Microscope images of stable cell lines expressing the negative (NMD) and positive (GFP-SMN1) control constructs. (Upper panels) GFP fluorescence, lower panels: phase contrast images. ( C ) Flow cytometry histograms of stable cell lines expressing the control constructs. An untransfected HEK-293 FLP-In cell population (Untrans.) was also analyzed for reference. ( D ) Schematic representing the relative locations of primer set binding for transcript isoform analysis by qRT-PCR. Primer sets were used to quantify levels of total transcript (set 1), intron 6 retention (set 2), intron 7 retention (set 3), exon 7 included isoform (set 4) and exon excluded isoform (set 5). Primer binding locations within the SMN1 mini-gene are presented in Supplementary Figure S3 . ( E ) qRT-PCR analysis of the NMD and GFP-SMN1 control cell lines supports decay of the PTC harboring isoform. Expression levels were normalized to the levels of HPRT (hypoxanthine-guanine phosphoribosyltransferase). Data presented is the mean expression of duplicate PCR samples ± the average error. ( F ) DNA sequencing analysis of purified genomic DNA from HEK-293 cell lines harboring the library constructs. ( G ) The enriched cell populations maintain the fluorescence levels of the sorted sections (A, B, C). Following the second round of sorting, the fluorescence levels of expanded populations were re-analyzed through flow cytometry to confirm maintenance of expression levels.
Figure Legend Snippet: A Screening PLatform for Intronic Control Elements (SPLICE) provides a generalizable in vivo screening strategy for ISREs. ( A ) SPLICE couples an exon inclusion event in a mini-gene (SMN1) to the expression level of a fluorescent protein (GFP) through a NMD-based reporter system. A random nucleotide library cloned upstream of the 3′ ss is screened for ISREs by sorting cells based on fluorescence levels. The enriched cells are sorted into sections (A, B, C) in a second screening round. ( B ) Microscope images of stable cell lines expressing the negative (NMD) and positive (GFP-SMN1) control constructs. (Upper panels) GFP fluorescence, lower panels: phase contrast images. ( C ) Flow cytometry histograms of stable cell lines expressing the control constructs. An untransfected HEK-293 FLP-In cell population (Untrans.) was also analyzed for reference. ( D ) Schematic representing the relative locations of primer set binding for transcript isoform analysis by qRT-PCR. Primer sets were used to quantify levels of total transcript (set 1), intron 6 retention (set 2), intron 7 retention (set 3), exon 7 included isoform (set 4) and exon excluded isoform (set 5). Primer binding locations within the SMN1 mini-gene are presented in Supplementary Figure S3 . ( E ) qRT-PCR analysis of the NMD and GFP-SMN1 control cell lines supports decay of the PTC harboring isoform. Expression levels were normalized to the levels of HPRT (hypoxanthine-guanine phosphoribosyltransferase). Data presented is the mean expression of duplicate PCR samples ± the average error. ( F ) DNA sequencing analysis of purified genomic DNA from HEK-293 cell lines harboring the library constructs. ( G ) The enriched cell populations maintain the fluorescence levels of the sorted sections (A, B, C). Following the second round of sorting, the fluorescence levels of expanded populations were re-analyzed through flow cytometry to confirm maintenance of expression levels.

Techniques Used: In Vivo, Expressing, Clone Assay, Fluorescence, Microscopy, Stable Transfection, Construct, Flow Cytometry, Cytometry, Binding Assay, Quantitative RT-PCR, Polymerase Chain Reaction, DNA Sequencing, Purification

10) Product Images from "In Vivo Determination of Direct Targets of the Nonsense-Mediated Decay Pathway in Drosophila"

Article Title: In Vivo Determination of Direct Targets of the Nonsense-Mediated Decay Pathway in Drosophila

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.113.009357

Copia RNA levels are indirectly regulated by the NMD pathway. (A) Copia genomic (boxes) and transcript structures. Also indicated is the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplicon. (B) qRT-PCR analysis of Copia of the same time course described in Figure 2 . (C) Copia levels in S2 cells, measured by qRT-PCR, using the assay described Figure S2 (fold change after Upf1 depletion indicated in parentheses). Error bars represent ± 1 SD.
Figure Legend Snippet: Copia RNA levels are indirectly regulated by the NMD pathway. (A) Copia genomic (boxes) and transcript structures. Also indicated is the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplicon. (B) qRT-PCR analysis of Copia of the same time course described in Figure 2 . (C) Copia levels in S2 cells, measured by qRT-PCR, using the assay described Figure S2 (fold change after Upf1 depletion indicated in parentheses). Error bars represent ± 1 SD.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification

11) Product Images from "A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice"

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060913

Changes in gene expression in the hypomorphic hearts. Expression of gene products, examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. Data represents mean +/− SD from 3 to 5 animals of each genotype, and was normalized such that the expression of the mRNA in wild-type animals was equal to 1 (*: p
Figure Legend Snippet: Changes in gene expression in the hypomorphic hearts. Expression of gene products, examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. Data represents mean +/− SD from 3 to 5 animals of each genotype, and was normalized such that the expression of the mRNA in wild-type animals was equal to 1 (*: p

Techniques Used: Expressing, Quantitative RT-PCR, Isolation

12) Product Images from "High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma"

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma

Journal: Journal of Cancer Research and Clinical Oncology

doi: 10.1007/s00432-018-2625-5

MLL2 expression in ESCC. a MLL2 mRNA expression was examined in 42 ESCC (tumor) and paired adjacent normal tissues (non-tumor) by qRT-PCR and MLL2 expression was up-regulated in ESCC ( P
Figure Legend Snippet: MLL2 expression in ESCC. a MLL2 mRNA expression was examined in 42 ESCC (tumor) and paired adjacent normal tissues (non-tumor) by qRT-PCR and MLL2 expression was up-regulated in ESCC ( P

Techniques Used: Expressing, Quantitative RT-PCR

13) Product Images from "RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)"

Article Title: RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)

Journal: Genes

doi: 10.3390/genes8110291

qRT-PCR analysis of the gene expression of 12 differentially expressed genes selected at the stem elongation stage between the early and late maturing lines of crested wheatgrass. Each panel shows gene ID and RNA-Seq readings of fragments per kilobase of transcript per million mapped reads (FPKM) for both lines. REL: relative expression level.
Figure Legend Snippet: qRT-PCR analysis of the gene expression of 12 differentially expressed genes selected at the stem elongation stage between the early and late maturing lines of crested wheatgrass. Each panel shows gene ID and RNA-Seq readings of fragments per kilobase of transcript per million mapped reads (FPKM) for both lines. REL: relative expression level.

Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay

14) Product Images from "p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans"

Article Title: p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0020183

PMK-1 Regulates Basal and Inducible Expression of P. aeruginosa –Induced Genes (A) Venn diagram of overlap between genes regulated by PMK-1 and P. aeruginosa. (B) (C) qRT-PCR analysis of PA14-induced gene expression in wild-type animals and in pmk-1 mutants. Results are the average of two biological replicates, each replicate measured in duplicate and normalized to a control gene. Error bars are SEM. (D) Diagram of different gene classes regulated by PMK-1 and/or P. aeruginosa. PMK-1 is required for basal and inducible regulations of class A genes. PMK-1 is required for basal, but not inducible expression of class B genes. PMK-1 is required for inducible but not basal expression of class C genes. PMK-1 is required for neither basal nor inducible expression of class D genes. PMK-1 regulates basal expression of class E genes, but these genes are not induced by P. aeruginosa.
Figure Legend Snippet: PMK-1 Regulates Basal and Inducible Expression of P. aeruginosa –Induced Genes (A) Venn diagram of overlap between genes regulated by PMK-1 and P. aeruginosa. (B) (C) qRT-PCR analysis of PA14-induced gene expression in wild-type animals and in pmk-1 mutants. Results are the average of two biological replicates, each replicate measured in duplicate and normalized to a control gene. Error bars are SEM. (D) Diagram of different gene classes regulated by PMK-1 and/or P. aeruginosa. PMK-1 is required for basal and inducible regulations of class A genes. PMK-1 is required for basal, but not inducible expression of class B genes. PMK-1 is required for inducible but not basal expression of class C genes. PMK-1 is required for neither basal nor inducible expression of class D genes. PMK-1 regulates basal expression of class E genes, but these genes are not induced by P. aeruginosa.

Techniques Used: Expressing, Quantitative RT-PCR

15) Product Images from "Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression"

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117758

GPR158 effects on AR expression in the LNCaP/C4-2B model. LNCaP (A, B, C) and C4-2B (B and C) were transiently transfected using Lipofectamine LTX reagent with either GPR158 expression plasmid or vector (A and B) OR either GPR158 siRNA or control scrambled siRNA (A and C) . After 3 days of transfection, total isolated RNA and protein cell lysates were used for qRT-PCR (A) and western blotting (B and C) , respectively for the expression analysis of GPR158, AR, PSA and β-actin mRNA and protein. The data represent two independent experiments performed in duplicate.
Figure Legend Snippet: GPR158 effects on AR expression in the LNCaP/C4-2B model. LNCaP (A, B, C) and C4-2B (B and C) were transiently transfected using Lipofectamine LTX reagent with either GPR158 expression plasmid or vector (A and B) OR either GPR158 siRNA or control scrambled siRNA (A and C) . After 3 days of transfection, total isolated RNA and protein cell lysates were used for qRT-PCR (A) and western blotting (B and C) , respectively for the expression analysis of GPR158, AR, PSA and β-actin mRNA and protein. The data represent two independent experiments performed in duplicate.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Western Blot

Effects of an androgen on GPR158 expression. (A-C and E-G) Cells of the indicated lines were incubated in media containing 10% CSS for androgen starvation overnight (0 time point), followed by treatment with DHT (10 nM) for the indicated time periods. Total RNA (A-C) or cell lysates (E-G) were isolated and subjected to qRT-PCR and western blotting, respectively to estimate AR, PSA, GPR158 and beta-actin expression. (D and H) PHPEC were grown in media containing 10% FCS or 10% CSS, treated with DHT (10nM) alone for 24 hrs or pre-incubated with bicalutamide (10μM) for 1-hr prior to DHT treatment in media containing 10% CSS. Total RNA or the whole cell lysates were isolated and qRT-PCR and western blotting was performed, respectively to detect GPR158, AR, PSA and beta-actin levels (D) The mRNA level of the above indicated genes was considered 1 in cells grown in 10% FCS for calculating the relative fold difference of these genes in other experimental conditions. (A-H) The data represent three independent experiments.
Figure Legend Snippet: Effects of an androgen on GPR158 expression. (A-C and E-G) Cells of the indicated lines were incubated in media containing 10% CSS for androgen starvation overnight (0 time point), followed by treatment with DHT (10 nM) for the indicated time periods. Total RNA (A-C) or cell lysates (E-G) were isolated and subjected to qRT-PCR and western blotting, respectively to estimate AR, PSA, GPR158 and beta-actin expression. (D and H) PHPEC were grown in media containing 10% FCS or 10% CSS, treated with DHT (10nM) alone for 24 hrs or pre-incubated with bicalutamide (10μM) for 1-hr prior to DHT treatment in media containing 10% CSS. Total RNA or the whole cell lysates were isolated and qRT-PCR and western blotting was performed, respectively to detect GPR158, AR, PSA and beta-actin levels (D) The mRNA level of the above indicated genes was considered 1 in cells grown in 10% FCS for calculating the relative fold difference of these genes in other experimental conditions. (A-H) The data represent three independent experiments.

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

GPR158 expression in NED. (A, B, C, D) LNCaP cells were androgen-starved for a short (A, B, C) and long (D) duration as indicated. Following incubation, total RNA was subjected to qRT-PCR analysis (A and B) and whole cell lysates were processed for western blotting (C and D) for estimation of GPR158, AR, PSA and NSE mRNA and protein expression levels. The results are representative of two independent experiments performed in duplicate. (E) Representative microscopic image of LNCaP cell grown in complete media or in CSS for androgen-starvation for 4-wks is shown. (F) LNCaP cells were grown in 10% CSS for 4-wks, leading to NED. (G) Lenti-GPR158-LNCaP cells were induced with Dox at 100ng/mL or 500ng/mL for 3 days. (F and G) The cells were then subjected to subcellular fractionation as described in Materials and Methods. The loaded amount of protein per fraction is shown. The western blotting was performed using anti-ICD GPR158 antibody. Specific protein markers were used to validate and confirm the purity of the five subcellular fractions examined: cytoplasmic extract (CE) = alpha-tubulin, membrane extract (ME) = EGFR, soluble nuclear extract (NE) = Sp1, chromatin-bound nuclear extract (CBE) = histone H3 and insoluble cytoskeletal extract (CSE) lamin A/C. Image J analysis of protein band intensities was used to estimate the percentage of GPR158 protein present in each fraction in relation to the total amount of protein in all fractions.
Figure Legend Snippet: GPR158 expression in NED. (A, B, C, D) LNCaP cells were androgen-starved for a short (A, B, C) and long (D) duration as indicated. Following incubation, total RNA was subjected to qRT-PCR analysis (A and B) and whole cell lysates were processed for western blotting (C and D) for estimation of GPR158, AR, PSA and NSE mRNA and protein expression levels. The results are representative of two independent experiments performed in duplicate. (E) Representative microscopic image of LNCaP cell grown in complete media or in CSS for androgen-starvation for 4-wks is shown. (F) LNCaP cells were grown in 10% CSS for 4-wks, leading to NED. (G) Lenti-GPR158-LNCaP cells were induced with Dox at 100ng/mL or 500ng/mL for 3 days. (F and G) The cells were then subjected to subcellular fractionation as described in Materials and Methods. The loaded amount of protein per fraction is shown. The western blotting was performed using anti-ICD GPR158 antibody. Specific protein markers were used to validate and confirm the purity of the five subcellular fractions examined: cytoplasmic extract (CE) = alpha-tubulin, membrane extract (ME) = EGFR, soluble nuclear extract (NE) = Sp1, chromatin-bound nuclear extract (CBE) = histone H3 and insoluble cytoskeletal extract (CSE) lamin A/C. Image J analysis of protein band intensities was used to estimate the percentage of GPR158 protein present in each fraction in relation to the total amount of protein in all fractions.

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Fractionation

GPR158 effects on cell proliferation in PCa cell lines. (A) RT-PCR analysis of GPR158 mRNA expression in indicated PCa cell lines (B) Western blot analysis for the detection of GPR158 protein using anti-ICD GPR158 antibody in whole cell lysates of indicated PCa cell lines. The same membrane was probed for beta-actin, which served as a loading control. The vertical line indicates repositioned gel lanes from the same membrane to match the sample order of Fig. 1A . (C, D, E) All four indicated PCa cell lines were transfected at 70% confluence with either GPR158 expression plasmid or empty pcDNA3.1 (+) vector (C and D) OR either GPR158 siRNA or control scrambled siRNA (E) as indicated using Lipofectamine LTX reagent and incubated in growth medium for 3 days in a 6-well culture plates. (C and E) After 3 days, the trypsinized cells were counted using trypan blue dye in a hemocytometer chamber. (D) Western blotting for the detection of GPR158 in whole cell lysates isolated from cells transfected with indicated plasmids using anti-ICD GPR158 antibody. (F) Total RNA was isolated from DU145 cells that were transfected with the indicated concentration of either GPR158 siRNA or control scrambled siRNA for analyzing the levels of GPR158 mRNA by qRT-PCR. GAPDH was internal reference control. (C, E, F) The data shows mean ± SE of 3 independent experiments, each performed in duplicate. ***p
Figure Legend Snippet: GPR158 effects on cell proliferation in PCa cell lines. (A) RT-PCR analysis of GPR158 mRNA expression in indicated PCa cell lines (B) Western blot analysis for the detection of GPR158 protein using anti-ICD GPR158 antibody in whole cell lysates of indicated PCa cell lines. The same membrane was probed for beta-actin, which served as a loading control. The vertical line indicates repositioned gel lanes from the same membrane to match the sample order of Fig. 1A . (C, D, E) All four indicated PCa cell lines were transfected at 70% confluence with either GPR158 expression plasmid or empty pcDNA3.1 (+) vector (C and D) OR either GPR158 siRNA or control scrambled siRNA (E) as indicated using Lipofectamine LTX reagent and incubated in growth medium for 3 days in a 6-well culture plates. (C and E) After 3 days, the trypsinized cells were counted using trypan blue dye in a hemocytometer chamber. (D) Western blotting for the detection of GPR158 in whole cell lysates isolated from cells transfected with indicated plasmids using anti-ICD GPR158 antibody. (F) Total RNA was isolated from DU145 cells that were transfected with the indicated concentration of either GPR158 siRNA or control scrambled siRNA for analyzing the levels of GPR158 mRNA by qRT-PCR. GAPDH was internal reference control. (C, E, F) The data shows mean ± SE of 3 independent experiments, each performed in duplicate. ***p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Plasmid Preparation, Incubation, Isolation, Concentration Assay, Quantitative RT-PCR

16) Product Images from "GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role"

Article Title: GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057843

GPR158 regulates the proliferation of TBM-1 cells. TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either GPR158 expression plasmids or vector alone ( A ) OR either GPR158 siRNA or control scrambled siRNA as indicated ( B and C ) and incubated in growth medium for 3 days in a 6-well culture dishes. ( A and C ) After 3 days of transfection, the cells were trypsinized and counted using trypan blue dye in a hemocytometer chamber for the cells transfected with indicated plasmids. ( B ) Total RNA was isolated for analyzing the levels of GPR158 mRNA by qRT-PCR. β-actin was used as a reference gene. ( A, B and C ) The data represent mean ± SEM of three independent experiments. ***P
Figure Legend Snippet: GPR158 regulates the proliferation of TBM-1 cells. TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either GPR158 expression plasmids or vector alone ( A ) OR either GPR158 siRNA or control scrambled siRNA as indicated ( B and C ) and incubated in growth medium for 3 days in a 6-well culture dishes. ( A and C ) After 3 days of transfection, the cells were trypsinized and counted using trypan blue dye in a hemocytometer chamber for the cells transfected with indicated plasmids. ( B ) Total RNA was isolated for analyzing the levels of GPR158 mRNA by qRT-PCR. β-actin was used as a reference gene. ( A, B and C ) The data represent mean ± SEM of three independent experiments. ***P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Incubation, Isolation, Quantitative RT-PCR

Expression and GC-mediated induction of GPR158 in trabecular meshwork cells. ( A ) RT-PCR analysis of GPR158, aquaporin-1, myocilin and β-actin mRNA expression in TBM-1 cells. ( B ) Western blotting for GPR158 in cellular extracts from untreated and Dex treated (250 nM) for 6 days using anti-C-terminal GPR158 antibodies (1∶1000 dilution). The data are representative of three independent experiments. The vertical line indicates repositioned gel lanes. ( C and D ) TBM-1 cells were stimulated with either Dex ( C ) or TA ( D ) for the indicated time periods. Total RNA was isolated for quantitation of GPR158 mRNA by qRT-PCR. GAPDH was used as a reference gene. The cells treated with ethanol (0.1%) were used as a negative control. The data are representative of three independent experiments. ***P
Figure Legend Snippet: Expression and GC-mediated induction of GPR158 in trabecular meshwork cells. ( A ) RT-PCR analysis of GPR158, aquaporin-1, myocilin and β-actin mRNA expression in TBM-1 cells. ( B ) Western blotting for GPR158 in cellular extracts from untreated and Dex treated (250 nM) for 6 days using anti-C-terminal GPR158 antibodies (1∶1000 dilution). The data are representative of three independent experiments. The vertical line indicates repositioned gel lanes. ( C and D ) TBM-1 cells were stimulated with either Dex ( C ) or TA ( D ) for the indicated time periods. Total RNA was isolated for quantitation of GPR158 mRNA by qRT-PCR. GAPDH was used as a reference gene. The cells treated with ethanol (0.1%) were used as a negative control. The data are representative of three independent experiments. ***P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Quantitation Assay, Quantitative RT-PCR, Negative Control

17) Product Images from "A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3"

Article Title: A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.02.007

qRT-PCR analysis of recombinant ALKBH3 demethylation activity against 3-meC ssDNA. Demethylation activity of recombinant ALKBH3 was analyzed by qRT-PCR. (A) The amount of demethylated product was estimated from a standard curve using ssDNA oligonucleotides without 3-meC. (B) The demethylation of 3-meC ssDNA by FLAG-His-ALKBH3 was measured in the presence or absence of 2-OG and/or Fe (II). Heat denatured: recombinant FLAG-His-ALKBH3 was heat-inactivated at 95 °C for 10 min. (C) The level of demethylated 3-meC ssDNA converted by recombinant ALKBH3 was determined in a time-dependent manner. All data shown are representative of at least three independent experiments. *p
Figure Legend Snippet: qRT-PCR analysis of recombinant ALKBH3 demethylation activity against 3-meC ssDNA. Demethylation activity of recombinant ALKBH3 was analyzed by qRT-PCR. (A) The amount of demethylated product was estimated from a standard curve using ssDNA oligonucleotides without 3-meC. (B) The demethylation of 3-meC ssDNA by FLAG-His-ALKBH3 was measured in the presence or absence of 2-OG and/or Fe (II). Heat denatured: recombinant FLAG-His-ALKBH3 was heat-inactivated at 95 °C for 10 min. (C) The level of demethylated 3-meC ssDNA converted by recombinant ALKBH3 was determined in a time-dependent manner. All data shown are representative of at least three independent experiments. *p

Techniques Used: Quantitative RT-PCR, Recombinant, Activity Assay

Comparison of 3-meC ssDNA demethylase activity of ALKBH2 and ALKBH3 from E. coli and FLAG-His-ALKBH3 from silkworms. Concentration-dependent demethylase activity of ALKBH2 and ALKBH3 from E. coli were determined in our qRT-PCR assay and compared to the activity of 4 ng of recombinant FLAG-His-ALKBH3 from silkworms (open circle). Data shown are representative of three independent experiments.
Figure Legend Snippet: Comparison of 3-meC ssDNA demethylase activity of ALKBH2 and ALKBH3 from E. coli and FLAG-His-ALKBH3 from silkworms. Concentration-dependent demethylase activity of ALKBH2 and ALKBH3 from E. coli were determined in our qRT-PCR assay and compared to the activity of 4 ng of recombinant FLAG-His-ALKBH3 from silkworms (open circle). Data shown are representative of three independent experiments.

Techniques Used: Activity Assay, Concentration Assay, Quantitative RT-PCR, Recombinant

18) Product Images from "Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus"

Article Title: Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000982

The C. elegans host response is comprised of pathogen-specific and -shared components. A . Comparison of genes induced 2-fold or higher ( p ≤0.01) upon infection with S. aureus for 8 h, P. aeruginosa for 8 h, and M. nematophilum for 6 h. Gene identities are presented in Table 3 . B . Quadrant analysis of genes with expression changes both during S. aureus and P. aeruginosa infection. X axis, Fold Change for each gene during P. aeruginosa infection (log 10 values). Y axis, Fold Change for each gene during S. aureus infection (log 10 values). C . qRT-PCR analysis of P. aeruginosa –induced genes during S. aureus infection. D . qRT-PCR analysis of S. aureus –induced genes during P. aeruginosa infection. Transcript levels were measured in synchronized young adult wild-type animals feeding on non-pathogenic E. coli OP50 or infected with pathogen for 8 h ( S. aureus NCTC8325) or 4 h ( P. aeruginosa PA14). Results are the average of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding P. aeruginosa - induced levels and the basal E. coli levels. E . qRT-PCR analysis of overlap genes during S. aureus or P. aeruginosa infection. Transcript levels were measured in synchronized young adult wild-type animals feeding on non-pathogenic E. coli or infected for 8 h or 4 h, respectively (on P. aeruginosa , these genes were predicted to change by 4 h as well as 8 h). Results are the average of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding pathogen - induced levels and the basal E. coli levels. Previous microarray analysis wrongly predicted acs-2 to be induced by P. aeruginosa , the only example of this kind that we have found.
Figure Legend Snippet: The C. elegans host response is comprised of pathogen-specific and -shared components. A . Comparison of genes induced 2-fold or higher ( p ≤0.01) upon infection with S. aureus for 8 h, P. aeruginosa for 8 h, and M. nematophilum for 6 h. Gene identities are presented in Table 3 . B . Quadrant analysis of genes with expression changes both during S. aureus and P. aeruginosa infection. X axis, Fold Change for each gene during P. aeruginosa infection (log 10 values). Y axis, Fold Change for each gene during S. aureus infection (log 10 values). C . qRT-PCR analysis of P. aeruginosa –induced genes during S. aureus infection. D . qRT-PCR analysis of S. aureus –induced genes during P. aeruginosa infection. Transcript levels were measured in synchronized young adult wild-type animals feeding on non-pathogenic E. coli OP50 or infected with pathogen for 8 h ( S. aureus NCTC8325) or 4 h ( P. aeruginosa PA14). Results are the average of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding P. aeruginosa - induced levels and the basal E. coli levels. E . qRT-PCR analysis of overlap genes during S. aureus or P. aeruginosa infection. Transcript levels were measured in synchronized young adult wild-type animals feeding on non-pathogenic E. coli or infected for 8 h or 4 h, respectively (on P. aeruginosa , these genes were predicted to change by 4 h as well as 8 h). Results are the average of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding pathogen - induced levels and the basal E. coli levels. Previous microarray analysis wrongly predicted acs-2 to be induced by P. aeruginosa , the only example of this kind that we have found.

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Microarray

The C. elegans host response to S. aureus infection is comprised of two kinetic groups. A, B . qRT-PCR analysis of genes predicted to be up-regulated by microarray analysis. Transcript levels were measured in synchronized young adult wild-type animals feeding on heat-killed E. coli OP50 or infected with S. aureus NCTC8325 for 4, 8, and 12 h. Data are the means of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding S. aureus- induced levels and the basal E. coli levels. A . Immediate-early-induced genes were highly induced by 4 h of infection. B . Later induction of early response genes. C . qRT-PCR analysis of genes predicted to be repressed by microarray analysis. Transcript levels were measured as in A , after 8 h infection with S. aureus . Data are the means of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding S. aureus- induced levels and the basal E. coli levels. Error bars are SEM.
Figure Legend Snippet: The C. elegans host response to S. aureus infection is comprised of two kinetic groups. A, B . qRT-PCR analysis of genes predicted to be up-regulated by microarray analysis. Transcript levels were measured in synchronized young adult wild-type animals feeding on heat-killed E. coli OP50 or infected with S. aureus NCTC8325 for 4, 8, and 12 h. Data are the means of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding S. aureus- induced levels and the basal E. coli levels. A . Immediate-early-induced genes were highly induced by 4 h of infection. B . Later induction of early response genes. C . qRT-PCR analysis of genes predicted to be repressed by microarray analysis. Transcript levels were measured as in A , after 8 h infection with S. aureus . Data are the means of three biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding S. aureus- induced levels and the basal E. coli levels. Error bars are SEM.

Techniques Used: Infection, Quantitative RT-PCR, Microarray

The C. elegans host response to S. aureus is induced in the intestinal epithelial cells. Transcriptional reporters using upstream sequences to fmo-2, ilys-3 , F53A9.8, clec-52, clec-60 , and clec-70 fused to GFP were induced in the intestinal epithelial cells after 24 h of S. aureus NCTC8325 infection (left panels) compared to parallel E. coli OP50 - fed controls (right panels). Despite strong induction of fmo-2 and ilys-3 as measured by qRT-PCR, the corresponding GFP reporters exhibited low levels of expression; this could be due to their low basal expression on E. coli (not shown). Red, myo-2::NLS::cherry coinjection marker expressed in the pharynx in the head. A fold induction of 1 indicates no induction. a, anterior end; p, posterior end. Arrows indicate pharyngeal expression. ilys-3 was also expressed in the pharynx, in an unidentified cell superimposed on the pharynx, and in unidentified cells, possibly epithelial cells, in the vulva ( Fig. S6A,B ). clec-70 was also expressed in unknown cells in the pharynx ( Fig. S6C ) and in the uterine muscle ( Fig. S6D ), but only in one transgenic line of three. F53A9.8 was also expressed in a group of cells surrounding the rectum, possibly the rectal gland cells that secrete molecules into the rectal lumen ( Fig. S6G, H ).
Figure Legend Snippet: The C. elegans host response to S. aureus is induced in the intestinal epithelial cells. Transcriptional reporters using upstream sequences to fmo-2, ilys-3 , F53A9.8, clec-52, clec-60 , and clec-70 fused to GFP were induced in the intestinal epithelial cells after 24 h of S. aureus NCTC8325 infection (left panels) compared to parallel E. coli OP50 - fed controls (right panels). Despite strong induction of fmo-2 and ilys-3 as measured by qRT-PCR, the corresponding GFP reporters exhibited low levels of expression; this could be due to their low basal expression on E. coli (not shown). Red, myo-2::NLS::cherry coinjection marker expressed in the pharynx in the head. A fold induction of 1 indicates no induction. a, anterior end; p, posterior end. Arrows indicate pharyngeal expression. ilys-3 was also expressed in the pharynx, in an unidentified cell superimposed on the pharynx, and in unidentified cells, possibly epithelial cells, in the vulva ( Fig. S6A,B ). clec-70 was also expressed in unknown cells in the pharynx ( Fig. S6C ) and in the uterine muscle ( Fig. S6D ), but only in one transgenic line of three. F53A9.8 was also expressed in a group of cells surrounding the rectum, possibly the rectal gland cells that secrete molecules into the rectal lumen ( Fig. S6G, H ).

Techniques Used: Infection, Quantitative RT-PCR, Expressing, Marker, Transgenic Assay

Potential PAMP-mediated, TLR-independent sensing of S. aureus . A . qRT-PCR analysis of induced genes in animals feeding on heat-killed S. aureus . Transcript levels were measured in synchronized young adult wild-type animals infected with live S. aureus NCTC8325 or feeding on dead S. aureus or E. coli OP50 for 8 h. B . qRT-PCR analysis of induced genes in animals feeding on B. subtilis . Transcript levels were measured in synchronized young adult wild-type animals feeding on B. subtilis PY79 or heat-killed E. coli for 8 h. C . qRT-PCR analysis of induced genes in tol-1 mutant animals. Transcript levels were measured in synchronized young adult animals feeding on heat-killed E. coli or infected with S. aureus for 8 h. D . qRT-PCR analysis of genes induced by live or heat-killed P. aeruginosa . Transcript levels were measured in synchronized young adult animals feeding on heat-killed E. coli or P. aeruginosa , or infected with live P. aeruginosa PA14 for 4 h. In all cases, data are the means of two biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding bacteria - induced levels and the basal E. coli levels. Error bars are SEM.
Figure Legend Snippet: Potential PAMP-mediated, TLR-independent sensing of S. aureus . A . qRT-PCR analysis of induced genes in animals feeding on heat-killed S. aureus . Transcript levels were measured in synchronized young adult wild-type animals infected with live S. aureus NCTC8325 or feeding on dead S. aureus or E. coli OP50 for 8 h. B . qRT-PCR analysis of induced genes in animals feeding on B. subtilis . Transcript levels were measured in synchronized young adult wild-type animals feeding on B. subtilis PY79 or heat-killed E. coli for 8 h. C . qRT-PCR analysis of induced genes in tol-1 mutant animals. Transcript levels were measured in synchronized young adult animals feeding on heat-killed E. coli or infected with S. aureus for 8 h. D . qRT-PCR analysis of genes induced by live or heat-killed P. aeruginosa . Transcript levels were measured in synchronized young adult animals feeding on heat-killed E. coli or P. aeruginosa , or infected with live P. aeruginosa PA14 for 4 h. In all cases, data are the means of two biological replicates, each replicate measured in duplicate and normalized to a control gene, expressed as the ratio of the corresponding bacteria - induced levels and the basal E. coli levels. Error bars are SEM.

Techniques Used: Quantitative RT-PCR, Infection, Mutagenesis

19) Product Images from "miR-150 Modulates Cisplatin Chemosensitivity and Invasiveness of Muscle-Invasive Bladder Cancer Cells via Targeting PDCD4 In Vitro"

Article Title: miR-150 Modulates Cisplatin Chemosensitivity and Invasiveness of Muscle-Invasive Bladder Cancer Cells via Targeting PDCD4 In Vitro

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.891340

miR-150 inhibition enhances chemosensitivity to Cisplatin and suppresses invasiveness of MIBC cells. ( A ) miR-150 expression in MIBC cell lines tested by qRT-PCR. ( B ) Dose-inhibition rate curves plotted from three independent MTS assays showing the effect of miR-150 inhibition on MIBC cells’ chemosensitivity to cisplatin. ( C ) IC50 values affected by miR-150 inhibition. ( D ) Transwell assay performed on the control cell lines and miR-150-inhibited cell lines. * p
Figure Legend Snippet: miR-150 inhibition enhances chemosensitivity to Cisplatin and suppresses invasiveness of MIBC cells. ( A ) miR-150 expression in MIBC cell lines tested by qRT-PCR. ( B ) Dose-inhibition rate curves plotted from three independent MTS assays showing the effect of miR-150 inhibition on MIBC cells’ chemosensitivity to cisplatin. ( C ) IC50 values affected by miR-150 inhibition. ( D ) Transwell assay performed on the control cell lines and miR-150-inhibited cell lines. * p

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Transwell Assay

20) Product Images from "OsFH15, a class I formin, interacts with microfilaments and microtubules to regulate grain size via affecting cell expansion in rice"

Article Title: OsFH15, a class I formin, interacts with microfilaments and microtubules to regulate grain size via affecting cell expansion in rice

Journal: Scientific Reports

doi: 10.1038/s41598-017-06431-5

Phenotype with Overexpression and Repression of OsFH15 . ( A ) qRT-PCR analysis of OsFH15 expression pattern in various tissues. ( B ) SANGER sequencing chromatography showing the mutations in Osfh15 mutant. ( C ) qRT-PCR analysis of OsFH15 expression from 14 d-old seedling in wild-type and transgenic plants, UBQ5 as control. ( D ) Spikelets of WT, Cas9 #13, RNAi #4 and OE #11 before heading. Bar = 5 mm. ( E ) Grains of WT, Cas9 #13, RNAi #4 and OE #11. Bar = 1 cm. ( F ) to ( I ) Grain length ( F ), Grain width ( G ) and Grain thickness ( H ) were determined by vernier depth. 1,000-grain weight ( I ) was weighed. Data are represented as mean ± SEM (n ≥ 100 in F-H, n = 15 in I). *P
Figure Legend Snippet: Phenotype with Overexpression and Repression of OsFH15 . ( A ) qRT-PCR analysis of OsFH15 expression pattern in various tissues. ( B ) SANGER sequencing chromatography showing the mutations in Osfh15 mutant. ( C ) qRT-PCR analysis of OsFH15 expression from 14 d-old seedling in wild-type and transgenic plants, UBQ5 as control. ( D ) Spikelets of WT, Cas9 #13, RNAi #4 and OE #11 before heading. Bar = 5 mm. ( E ) Grains of WT, Cas9 #13, RNAi #4 and OE #11. Bar = 1 cm. ( F ) to ( I ) Grain length ( F ), Grain width ( G ) and Grain thickness ( H ) were determined by vernier depth. 1,000-grain weight ( I ) was weighed. Data are represented as mean ± SEM (n ≥ 100 in F-H, n = 15 in I). *P

Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Sequencing, Chromatography, Mutagenesis, Transgenic Assay

21) Product Images from "N6-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential"

Article Title: N6-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky130

The let-7/Lin28 pathway restores the impaired capacity of mESCs in the absence of m 6 A - linc1281 . ( A ) qRT-PCR analysis of cardiomyocyte markers on day 10 of EB differentiation in shControl and shlinc1281 mESCs in the presence of FlucC or the let-7 miRNA sponge. Sponge, the construct expresses a sponge RNA that contains three tandemly repeated complementary binding sites to each of the 5 miRNAs. ( B ) Percentage of beating EBs. ( C ) Representative images of bodies stained for cTnT and Ho.33342 on day 12 of EB formation. ( D – F ) FACS isolation of Sox1 + cells ( D ), qRT-PCR analysis of neural markers ( E ) and representative images of cells stained for Sox1-GFP, N-cad and Ho.33342 on day 5 of neural differentiation ( F ). ( G – I ) The spontaneous differentiation capacity of shControl and shlinc1281 mESCs overexpressing FlucC, Lin28a or Lin28b, based on EB formation. ( J – L ) Examination of the neural lineage differentiation capacity of the indicated mESC lines. Scale bar represents 100 μm. Error bars show ± SEM ( n ≥ 3). The asterisk (*) denotes a significant difference from ‘shCtrl+FlucC’, and the hash mark (#) denotes a significant difference from ‘sh1+FlucC’ or ‘sh2+FlucC’. *** , ### P
Figure Legend Snippet: The let-7/Lin28 pathway restores the impaired capacity of mESCs in the absence of m 6 A - linc1281 . ( A ) qRT-PCR analysis of cardiomyocyte markers on day 10 of EB differentiation in shControl and shlinc1281 mESCs in the presence of FlucC or the let-7 miRNA sponge. Sponge, the construct expresses a sponge RNA that contains three tandemly repeated complementary binding sites to each of the 5 miRNAs. ( B ) Percentage of beating EBs. ( C ) Representative images of bodies stained for cTnT and Ho.33342 on day 12 of EB formation. ( D – F ) FACS isolation of Sox1 + cells ( D ), qRT-PCR analysis of neural markers ( E ) and representative images of cells stained for Sox1-GFP, N-cad and Ho.33342 on day 5 of neural differentiation ( F ). ( G – I ) The spontaneous differentiation capacity of shControl and shlinc1281 mESCs overexpressing FlucC, Lin28a or Lin28b, based on EB formation. ( J – L ) Examination of the neural lineage differentiation capacity of the indicated mESC lines. Scale bar represents 100 μm. Error bars show ± SEM ( n ≥ 3). The asterisk (*) denotes a significant difference from ‘shCtrl+FlucC’, and the hash mark (#) denotes a significant difference from ‘sh1+FlucC’ or ‘sh2+FlucC’. *** , ### P

Techniques Used: Quantitative RT-PCR, Construct, Binding Assay, Staining, FACS, Isolation

Linc1281 is required for proper mESC differentiation. ( A ) Expression levels of linc1281 in mESCs in the presence or absence LIF for 4 days. ( B ) RNA FISH for linc1281 in mESCs. Scale bar represents 100 or 20 μm. ( C ) qRT-PCR analysis of transcripts derived from the cytoplasmic or nuclear fractions of mESCs. The log 10 ratio of cytoplasmic to nuclear transcripts is presented. ( D ) qRT-PCR analysis of linc1281 knockdown efficiency. pLKO.1-scramble shRNA was used as a negative control. ( E ) mRNA levels of Oct4, Sox2 and Nanog upon linc1281 depletion determined via qRT-PCR. ( F ) Weight differences between teratomas generated from shControl and shlinc1281 cells. ( G ) Representative sections of teratomas stained with H E. Scale bar represents 25 μm. ( H ) Lineage-specific marker expression in teratomas. ( I ) qRT-PCR analysis of mesoderm (left), cardiac progenitor (middle) and cardiomyocyte markers (right) during EB formation. EB, embryoid bodies. ( J ) Percentage of EBs with beating activity. ( K ) Representative images of bodies stained for cTnT and Hoechst 33342 (Ho.33342) on day 12 of EB formation. Scale bar represents 100 μm. ( L ) Percentage of Sox1 + cells analyzed via FACS during neural lineage differentiation. ( M ) qRT-PCR analysis of neural markers on day 5 of neural differentiation. ( N ) Representative images of cells stained for Sox1-GFP, N-cad and Ho.33342 on day 5 of neural differentiation. Scale bar represents 100 μm. Error bars show ± SEM ( n ≥ 3). *** P
Figure Legend Snippet: Linc1281 is required for proper mESC differentiation. ( A ) Expression levels of linc1281 in mESCs in the presence or absence LIF for 4 days. ( B ) RNA FISH for linc1281 in mESCs. Scale bar represents 100 or 20 μm. ( C ) qRT-PCR analysis of transcripts derived from the cytoplasmic or nuclear fractions of mESCs. The log 10 ratio of cytoplasmic to nuclear transcripts is presented. ( D ) qRT-PCR analysis of linc1281 knockdown efficiency. pLKO.1-scramble shRNA was used as a negative control. ( E ) mRNA levels of Oct4, Sox2 and Nanog upon linc1281 depletion determined via qRT-PCR. ( F ) Weight differences between teratomas generated from shControl and shlinc1281 cells. ( G ) Representative sections of teratomas stained with H E. Scale bar represents 25 μm. ( H ) Lineage-specific marker expression in teratomas. ( I ) qRT-PCR analysis of mesoderm (left), cardiac progenitor (middle) and cardiomyocyte markers (right) during EB formation. EB, embryoid bodies. ( J ) Percentage of EBs with beating activity. ( K ) Representative images of bodies stained for cTnT and Hoechst 33342 (Ho.33342) on day 12 of EB formation. Scale bar represents 100 μm. ( L ) Percentage of Sox1 + cells analyzed via FACS during neural lineage differentiation. ( M ) qRT-PCR analysis of neural markers on day 5 of neural differentiation. ( N ) Representative images of cells stained for Sox1-GFP, N-cad and Ho.33342 on day 5 of neural differentiation. Scale bar represents 100 μm. Error bars show ± SEM ( n ≥ 3). *** P

Techniques Used: Expressing, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Derivative Assay, shRNA, Negative Control, Generated, Staining, Marker, Activity Assay, FACS

22) Product Images from "PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells"

Article Title: PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells

Journal: Scientific Reports

doi: 10.1038/srep25268

miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P
Figure Legend Snippet: miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P

Techniques Used: Western Blot, Transfection, Software, Expressing, Quantitative RT-PCR

Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P
Figure Legend Snippet: Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P

Techniques Used: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

23) Product Images from "Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes"

Article Title: Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

Journal: Scientific Reports

doi: 10.1038/srep25723

Involvement of histone deacetylation at the miR-193b-3p promoter regions in HepG2 cells exposed to 0.01 Gy irradiation. ( A ) Alterations of miR-193b-3p expression were examined in normal mouse cells (NCTC), mouse hepatoma cells, and human hepatoma cells (HepG2) by qRT-PCR. The cells were irradiated with 0.01 Gy (6.5 mGy/h). Six hours post-irradiation, miRNA expression was evaluated and reported as fold-change differences relative to the sham-irradiated controls. ( B ) When the cells were pretreated with NaB (a histone deacetylase inhibitor) for 24 h, alterations in miR-193b-3p expression were observed in response to irradiation in HepG2 cells. The data were normalized using a mammalian U6 gene and are expressed as the mean ± S.D. ( C ) Chromatin immunoprecipitation (ChIP) assays were performed to evaluate the histone deacetylation of the miR-193b-3p promoter regions in HepG2 cells with or without NAC pretreatment (20 mM) 3 h after exposure to 0.01 Gy irradiation. Chromatin was immunoprecipitated with the anti-H3K9ac, anti-H4K16ac, or anti-IgG antibodies, and DNA was analyzed by qRT-PCR using miR-193b-3p-specific primers. The results are shown as a percentage of the chromatin input. The ChIP samples were quantified by qRT-PCR. The input was used as an internal control, and IgG was used as the antibody control. The statistically significant differences between the non-irradiated and irradiated samples are indicated (* p
Figure Legend Snippet: Involvement of histone deacetylation at the miR-193b-3p promoter regions in HepG2 cells exposed to 0.01 Gy irradiation. ( A ) Alterations of miR-193b-3p expression were examined in normal mouse cells (NCTC), mouse hepatoma cells, and human hepatoma cells (HepG2) by qRT-PCR. The cells were irradiated with 0.01 Gy (6.5 mGy/h). Six hours post-irradiation, miRNA expression was evaluated and reported as fold-change differences relative to the sham-irradiated controls. ( B ) When the cells were pretreated with NaB (a histone deacetylase inhibitor) for 24 h, alterations in miR-193b-3p expression were observed in response to irradiation in HepG2 cells. The data were normalized using a mammalian U6 gene and are expressed as the mean ± S.D. ( C ) Chromatin immunoprecipitation (ChIP) assays were performed to evaluate the histone deacetylation of the miR-193b-3p promoter regions in HepG2 cells with or without NAC pretreatment (20 mM) 3 h after exposure to 0.01 Gy irradiation. Chromatin was immunoprecipitated with the anti-H3K9ac, anti-H4K16ac, or anti-IgG antibodies, and DNA was analyzed by qRT-PCR using miR-193b-3p-specific primers. The results are shown as a percentage of the chromatin input. The ChIP samples were quantified by qRT-PCR. The input was used as an internal control, and IgG was used as the antibody control. The statistically significant differences between the non-irradiated and irradiated samples are indicated (* p

Techniques Used: Irradiation, Expressing, Quantitative RT-PCR, Histone Deacetylase Assay, Chromatin Immunoprecipitation, Immunoprecipitation

Validation of miRNA expression in mouse tissues in response to irradiation. A qRT-PCR validation of differentially expressed miRNAs (listed in Table 1) was performed. Mice were irradiated with 0.01 Gy (6.5 mGy/h). Six hours post-irradiation, miRNA expression was evaluated in the spleens ( A ), livers ( B ), kidneys ( C ), and lungs ( D ) and reported as fold-change differences relative to compared with the sham-irradiated controls. The mouse U6 and 5S genes were used as the normalization factors. The data shown are the mean ± S.D. (n = 5). Statistically significant differences between the non-irradiated and irradiated samples are indicated (* p
Figure Legend Snippet: Validation of miRNA expression in mouse tissues in response to irradiation. A qRT-PCR validation of differentially expressed miRNAs (listed in Table 1) was performed. Mice were irradiated with 0.01 Gy (6.5 mGy/h). Six hours post-irradiation, miRNA expression was evaluated in the spleens ( A ), livers ( B ), kidneys ( C ), and lungs ( D ) and reported as fold-change differences relative to compared with the sham-irradiated controls. The mouse U6 and 5S genes were used as the normalization factors. The data shown are the mean ± S.D. (n = 5). Statistically significant differences between the non-irradiated and irradiated samples are indicated (* p

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

24) Product Images from "Diminished PRRX1 Expression is Associated with Increased Risk of Atrial Fibrillation and Shortening of the Cardiac Action Potential"

Article Title: Diminished PRRX1 Expression is Associated with Increased Risk of Atrial Fibrillation and Shortening of the Cardiac Action Potential

Journal: Circulation. Cardiovascular genetics

doi: 10.1161/CIRCGENETICS.117.001902

Identification of the PRRX1 enhancer underlying the 1q24 AF-association locus A. The representative zebrafish for the initial enhancer (E–K) / promoter(Pr) screen displaying the tissue distribution and intensity of the enhancer and promoter activity. Arrows indicate the cardiac region. Scale bar indicates 1mm. B. qRT-PCR results measuring relative eGFP expression levels in zebrafish enhancer/promoter screen (n=3). C. Hi-C data showing organization of topologically associated domains around E and F enhancers in fibroblast cell lines (IMR90) as compared to human embryonic stem cells (hESC). D. Results of chromatin conformation capture (3C) experiment displaying increased frequency of interaction between the E and F enhancers and PRRX1 promoter in human cardiac fibroblasts (HCF)(n=5) as compared to hESC (n=4). *Represents p
Figure Legend Snippet: Identification of the PRRX1 enhancer underlying the 1q24 AF-association locus A. The representative zebrafish for the initial enhancer (E–K) / promoter(Pr) screen displaying the tissue distribution and intensity of the enhancer and promoter activity. Arrows indicate the cardiac region. Scale bar indicates 1mm. B. qRT-PCR results measuring relative eGFP expression levels in zebrafish enhancer/promoter screen (n=3). C. Hi-C data showing organization of topologically associated domains around E and F enhancers in fibroblast cell lines (IMR90) as compared to human embryonic stem cells (hESC). D. Results of chromatin conformation capture (3C) experiment displaying increased frequency of interaction between the E and F enhancers and PRRX1 promoter in human cardiac fibroblasts (HCF)(n=5) as compared to hESC (n=4). *Represents p

Techniques Used: Activity Assay, Quantitative RT-PCR, Expressing, Hi-C

25) Product Images from "Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza"

Article Title: Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

Journal: PeerJ

doi: 10.7717/peerj.4461

Transcript abundance of SmMET1 (A), SmCMT1 (B), SmCMT2a (C), SmCMT2b (D), SmCMT3 (E), SmDRM1 (F), SmDRM2 (G), and SmDNMT2 (H) in roots (Rt), Stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza . Transcript level was analyzed using quantitative real time RT-PCR. SmUBQ10 was used as a reference. qRT-PCR was performed in triplicates for each independent biological replicate. Transcript levels in roots were arbitrarily set to 1 and the levels in other tissues were given relative to this. One-way ANOVA was performed using IBM SPSS 20 software. P
Figure Legend Snippet: Transcript abundance of SmMET1 (A), SmCMT1 (B), SmCMT2a (C), SmCMT2b (D), SmCMT3 (E), SmDRM1 (F), SmDRM2 (G), and SmDNMT2 (H) in roots (Rt), Stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza . Transcript level was analyzed using quantitative real time RT-PCR. SmUBQ10 was used as a reference. qRT-PCR was performed in triplicates for each independent biological replicate. Transcript levels in roots were arbitrarily set to 1 and the levels in other tissues were given relative to this. One-way ANOVA was performed using IBM SPSS 20 software. P

Techniques Used: Quantitative RT-PCR, Software

26) Product Images from "Sonic Hedgehog-Signalling Patterns the Developing Chicken Comb as Revealed by Exploration of the Pea-comb Mutation"

Article Title: Sonic Hedgehog-Signalling Patterns the Developing Chicken Comb as Revealed by Exploration of the Pea-comb Mutation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050890

Candidate gene expression analysis in Pea- and single-comb tissue. Bar graphs with qRT-PCR analysis data for (A) RUNX2, (B) ETS1, (C) PAX3, (D) COL1A2, (E) ITGB3, (F) IHH, (G) SHH, (H) PTCH1, (I) SMO, (J) GLI1 and (K) GLI3. Bar graph data are normalized to ß-actin mRNA levels and is relative to the ß-actin mRNA level in Pea- and single-comb tissue respectively. Bar graphs are mean±s.e.m., ANOVA, n = 6 (single-comb) n = 5 (Pea-comb). * p
Figure Legend Snippet: Candidate gene expression analysis in Pea- and single-comb tissue. Bar graphs with qRT-PCR analysis data for (A) RUNX2, (B) ETS1, (C) PAX3, (D) COL1A2, (E) ITGB3, (F) IHH, (G) SHH, (H) PTCH1, (I) SMO, (J) GLI1 and (K) GLI3. Bar graph data are normalized to ß-actin mRNA levels and is relative to the ß-actin mRNA level in Pea- and single-comb tissue respectively. Bar graphs are mean±s.e.m., ANOVA, n = 6 (single-comb) n = 5 (Pea-comb). * p

Techniques Used: Expressing, Quantitative RT-PCR

Comparison of Pea- and single-comb with respect to comb morphology, Alcian blue cartilage staining and SOX5 expression. (A–D) Morphology of E12 and E18, Pea- and single-combs. (E–H) Alcian blue stained cross section of E12 and E18 Pea- and single-combs to visualize cartilaginous structures. Note that in spite of the differences in Pea- and single-comb morphology the underlying cartilage structures are normal. The sections are not exactly on the same level. (I) Bar graph with qRT-PCR results and fluorescence micrographs of immunohistological analysis of SOX5 mRNA levels. Bar graph data are normalized to the ß-actin mRNA levels and is related to the ß-actin mRNA level. Bars±s.e.m., ANOVA, n > 4 combs per sample * p
Figure Legend Snippet: Comparison of Pea- and single-comb with respect to comb morphology, Alcian blue cartilage staining and SOX5 expression. (A–D) Morphology of E12 and E18, Pea- and single-combs. (E–H) Alcian blue stained cross section of E12 and E18 Pea- and single-combs to visualize cartilaginous structures. Note that in spite of the differences in Pea- and single-comb morphology the underlying cartilage structures are normal. The sections are not exactly on the same level. (I) Bar graph with qRT-PCR results and fluorescence micrographs of immunohistological analysis of SOX5 mRNA levels. Bar graph data are normalized to the ß-actin mRNA levels and is related to the ß-actin mRNA level. Bars±s.e.m., ANOVA, n > 4 combs per sample * p

Techniques Used: Staining, Expressing, Quantitative RT-PCR, Fluorescence

27) Product Images from "The Expression of CARK1 or RCAR11 Driven by Synthetic Promoters Increases Drought Tolerance in Arabidopsis thaliana"

Article Title: The Expression of CARK1 or RCAR11 Driven by Synthetic Promoters Increases Drought Tolerance in Arabidopsis thaliana

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19071945

Identification of transgenic lines and analysis of drought-related genes by qRT-PCR. ( A ) ABA. Relative expressional levels of cytosolic ABA receptor kinase 1 ( CARK1 ), RD29A , and RD29B in transgenic plants. Twelve-day-old seedlings were incubated in MS liquid medium with or without 50 μM ABA for 3 h; ( B ) d -Mannitol. Relative expressional levels of CARK1 , regulatory components of ABA receptor 11 ( RCAR11 ), RD29A , and RD29B in transgenic plants. Twelve-day-old seedlings were incubated in MS liquid medium with or without 200 mM d -mannitol for 2 h. Values are means ± SD ( n = 3). ACTIN2/8 was used as an internal control. The change ratio of gene expression in wild type (WT) is 1. The experiments were repeated three times. (* p
Figure Legend Snippet: Identification of transgenic lines and analysis of drought-related genes by qRT-PCR. ( A ) ABA. Relative expressional levels of cytosolic ABA receptor kinase 1 ( CARK1 ), RD29A , and RD29B in transgenic plants. Twelve-day-old seedlings were incubated in MS liquid medium with or without 50 μM ABA for 3 h; ( B ) d -Mannitol. Relative expressional levels of CARK1 , regulatory components of ABA receptor 11 ( RCAR11 ), RD29A , and RD29B in transgenic plants. Twelve-day-old seedlings were incubated in MS liquid medium with or without 200 mM d -mannitol for 2 h. Values are means ± SD ( n = 3). ACTIN2/8 was used as an internal control. The change ratio of gene expression in wild type (WT) is 1. The experiments were repeated three times. (* p

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Incubation, Mass Spectrometry, Expressing

28) Product Images from "Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis"

Article Title: Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis

Journal: Oncotarget

doi: 10.18632/oncotarget.23858

( A ) Representative qRT-PCR analysis of BAG3 , CAV1, EZR, SERPINB2, BCLAF1 and HMGA2 mRNA expression in 8505C ATC cells transfected with scrambled (si SCR #1) or BAG3 -specific siRNA (si BAG3 #1) (grown in Light SILAC Media) compared to non-transfected 8505C cells (grown in Heavy SILAC media). Data were represented as relative expression on GAPDH . ( B ) Representative Western Blot analysis of BAG3, SERPINB2 and CAV1 protein expression in 8505C ATC cells transfected with scrambled (si SCR #1) or BAG3 -specific siRNA (si BAG3 #1) (grown in Light SILAC Media) compared to non-transfected 8505C cells (grown in Heavy SILAC media). α-TUBULIN and GAPDH were used as loading control. These transfected cells were used for Mass Spectrometry analysis.
Figure Legend Snippet: ( A ) Representative qRT-PCR analysis of BAG3 , CAV1, EZR, SERPINB2, BCLAF1 and HMGA2 mRNA expression in 8505C ATC cells transfected with scrambled (si SCR #1) or BAG3 -specific siRNA (si BAG3 #1) (grown in Light SILAC Media) compared to non-transfected 8505C cells (grown in Heavy SILAC media). Data were represented as relative expression on GAPDH . ( B ) Representative Western Blot analysis of BAG3, SERPINB2 and CAV1 protein expression in 8505C ATC cells transfected with scrambled (si SCR #1) or BAG3 -specific siRNA (si BAG3 #1) (grown in Light SILAC Media) compared to non-transfected 8505C cells (grown in Heavy SILAC media). α-TUBULIN and GAPDH were used as loading control. These transfected cells were used for Mass Spectrometry analysis.

Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Mass Spectrometry

29) Product Images from "C/EBP? Gene Targets in Human Keratinocytes"

Article Title: C/EBP? Gene Targets in Human Keratinocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013789

Identification of C/EBPδ-regulated genes in human keratinocytes. A . Left Panels, RT-PCR analysis of human primary keratinocytes after C/EBPδ RNAi inactivation at 48 hours post-transfection. cDNA normalization was performed with GAPDH. Right Panels, Western Blot analysis of the same C/EBPδ-inactivated human primary keratinocytes with the C/EBPδ antibody. Vinculin was used as a loading control. B . Heat map showing the mRNA expression levels of several classes of genes after C/EBPδ inactivation. C . qRT-PCR analysis of C/EBPδ regulated genes that emerged from the expression profiling. D . ChIP analysis of promoter regions of C/EBPδ-regulated genes with chromatin from human primary keratinocytes, with α-C/EBPδ and control antibodies. In the Left Panel, adult and neonatal keratinocytes were analyzed in semi-quantitative PCR. In the Right Panel, the targets were validated by qPCR in adult keratinocytes.
Figure Legend Snippet: Identification of C/EBPδ-regulated genes in human keratinocytes. A . Left Panels, RT-PCR analysis of human primary keratinocytes after C/EBPδ RNAi inactivation at 48 hours post-transfection. cDNA normalization was performed with GAPDH. Right Panels, Western Blot analysis of the same C/EBPδ-inactivated human primary keratinocytes with the C/EBPδ antibody. Vinculin was used as a loading control. B . Heat map showing the mRNA expression levels of several classes of genes after C/EBPδ inactivation. C . qRT-PCR analysis of C/EBPδ regulated genes that emerged from the expression profiling. D . ChIP analysis of promoter regions of C/EBPδ-regulated genes with chromatin from human primary keratinocytes, with α-C/EBPδ and control antibodies. In the Left Panel, adult and neonatal keratinocytes were analyzed in semi-quantitative PCR. In the Right Panel, the targets were validated by qPCR in adult keratinocytes.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Role of C/EBPδ target genes in keratinocyte differentiation. A . Analysis of differentiation markers by qRT-PCR of primary keratinocytes induced to differentiate after transient transfection of MafB expression plasmid and empty vector as control. After 24 h, half of the cells were induced to differentiate by addition of calcium, as in Fig. 5 . B . Same as A, except that Sox2 was overexpressed.
Figure Legend Snippet: Role of C/EBPδ target genes in keratinocyte differentiation. A . Analysis of differentiation markers by qRT-PCR of primary keratinocytes induced to differentiate after transient transfection of MafB expression plasmid and empty vector as control. After 24 h, half of the cells were induced to differentiate by addition of calcium, as in Fig. 5 . B . Same as A, except that Sox2 was overexpressed.

Techniques Used: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

C/EBPδ is important for keratinocyte differentiation. Analysis of differentiation markers by qRT-PCR of C/EBPδ-inactivated cells induced to differentiate. Primary keratinocytes were transfected with scamble and C/EBPδ siRNAs. After 24 h, half of the cells were induced to differentiate by addition of calcium. Samples were harvested after 3 days, RNAs prepared and qPCR performed on the indicated genes. Values are normalized to GAPDH, used as an internal control.
Figure Legend Snippet: C/EBPδ is important for keratinocyte differentiation. Analysis of differentiation markers by qRT-PCR of C/EBPδ-inactivated cells induced to differentiate. Primary keratinocytes were transfected with scamble and C/EBPδ siRNAs. After 24 h, half of the cells were induced to differentiate by addition of calcium. Samples were harvested after 3 days, RNAs prepared and qPCR performed on the indicated genes. Values are normalized to GAPDH, used as an internal control.

Techniques Used: Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction

30) Product Images from "Diminished PRRX1 Expression is Associated with Increased Risk of Atrial Fibrillation and Shortening of the Cardiac Action Potential"

Article Title: Diminished PRRX1 Expression is Associated with Increased Risk of Atrial Fibrillation and Shortening of the Cardiac Action Potential

Journal: Circulation. Cardiovascular genetics

doi: 10.1161/CIRCGENETICS.117.001902

Identification of the PRRX1 enhancer underlying the 1q24 AF-association locus A. The representative zebrafish for the initial enhancer (E–K) / promoter(Pr) screen displaying the tissue distribution and intensity of the enhancer and promoter activity. Arrows indicate the cardiac region. Scale bar indicates 1mm. B. qRT-PCR results measuring relative eGFP expression levels in zebrafish enhancer/promoter screen (n=3). C. Hi-C data showing organization of topologically associated domains around E and F enhancers in fibroblast cell lines (IMR90) as compared to human embryonic stem cells (hESC). D. Results of chromatin conformation capture (3C) experiment displaying increased frequency of interaction between the E and F enhancers and PRRX1 promoter in human cardiac fibroblasts (HCF)(n=5) as compared to hESC (n=4). *Represents p
Figure Legend Snippet: Identification of the PRRX1 enhancer underlying the 1q24 AF-association locus A. The representative zebrafish for the initial enhancer (E–K) / promoter(Pr) screen displaying the tissue distribution and intensity of the enhancer and promoter activity. Arrows indicate the cardiac region. Scale bar indicates 1mm. B. qRT-PCR results measuring relative eGFP expression levels in zebrafish enhancer/promoter screen (n=3). C. Hi-C data showing organization of topologically associated domains around E and F enhancers in fibroblast cell lines (IMR90) as compared to human embryonic stem cells (hESC). D. Results of chromatin conformation capture (3C) experiment displaying increased frequency of interaction between the E and F enhancers and PRRX1 promoter in human cardiac fibroblasts (HCF)(n=5) as compared to hESC (n=4). *Represents p

Techniques Used: Activity Assay, Quantitative RT-PCR, Expressing, Hi-C

31) Product Images from "ACVR1 R206H cooperates with H3.1K27M in promoting diffuse intrinsic pontine glioma pathogenesis"

Article Title: ACVR1 R206H cooperates with H3.1K27M in promoting diffuse intrinsic pontine glioma pathogenesis

Journal: Nature Communications

doi: 10.1038/s41467-019-08823-9

ACVR1 mutations have differential effects on proliferation and survival. a – d Brainstem progenitor cells isolated from p3 Ntv-a;p53 fl/fl mice were cultured in vitro as neurospheres and infected with ACVR1 WT, ACVR1 R206H, ACVR1 G328V, or ACVR1 G328E virus. a Proliferation of infected neurospheres ( n = 5). b Cell viability of infected neurospheres ( n = 4). c , d pSMAD1/5/8 ( c ) ( n = 4) and Id1 ( d ) ( n = 6) protein expression in infected neurospheres. Also see Supplementary Figure 1 . e – h Brainstem progenitor cells isolated from p3 Ntv-a;p53 fl/fl mice were cultured in vitro as neurospheres and infected with ACVR1 WT or R206H virus in the presence of H3.1K27M virus. e Heat map of differentially expressed genes between ACVR1 WT and H3.1K27M infected neurospheres compared to ACVR1 R206H and H3.1K27M infected neurospheres based on p value identified by RNA-Seq analysis. Also see Supplementary Data 1 . f GSEA enrichment plot of epithelial to mesenchymal transition genes identified by RNA-Seq analysis from ( e ). Also see Supplementary Data 2 . g GSEA enrichment plot of IL-6_JAK_STAT3 signaling genes identified by RNA-Seq analysis from ( e ). Also see Supplementary Data 2 . h qRT-PCR validation of select genes from neurospheres infected with ACVR1 WT, ACVR1 R206H, or ACVR1 G328V and H3.1K27M virus ( n = 3). i pSTAT3 Y705 protein expression in infected neurospheres as described in ( a ) ( n = 8). Also see Supplementary Figure 1 . All data are represented as mean with SEM, * p
Figure Legend Snippet: ACVR1 mutations have differential effects on proliferation and survival. a – d Brainstem progenitor cells isolated from p3 Ntv-a;p53 fl/fl mice were cultured in vitro as neurospheres and infected with ACVR1 WT, ACVR1 R206H, ACVR1 G328V, or ACVR1 G328E virus. a Proliferation of infected neurospheres ( n = 5). b Cell viability of infected neurospheres ( n = 4). c , d pSMAD1/5/8 ( c ) ( n = 4) and Id1 ( d ) ( n = 6) protein expression in infected neurospheres. Also see Supplementary Figure 1 . e – h Brainstem progenitor cells isolated from p3 Ntv-a;p53 fl/fl mice were cultured in vitro as neurospheres and infected with ACVR1 WT or R206H virus in the presence of H3.1K27M virus. e Heat map of differentially expressed genes between ACVR1 WT and H3.1K27M infected neurospheres compared to ACVR1 R206H and H3.1K27M infected neurospheres based on p value identified by RNA-Seq analysis. Also see Supplementary Data 1 . f GSEA enrichment plot of epithelial to mesenchymal transition genes identified by RNA-Seq analysis from ( e ). Also see Supplementary Data 2 . g GSEA enrichment plot of IL-6_JAK_STAT3 signaling genes identified by RNA-Seq analysis from ( e ). Also see Supplementary Data 2 . h qRT-PCR validation of select genes from neurospheres infected with ACVR1 WT, ACVR1 R206H, or ACVR1 G328V and H3.1K27M virus ( n = 3). i pSTAT3 Y705 protein expression in infected neurospheres as described in ( a ) ( n = 8). Also see Supplementary Figure 1 . All data are represented as mean with SEM, * p

Techniques Used: Isolation, Mouse Assay, Cell Culture, In Vitro, Infection, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

32) Product Images from "Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer"

Article Title: Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4260

Gene expression in genetically modified adipose-derived stem cells aggregate cultures . Adipose-derived stem cells ( ASCs) were transduced with 100 multiplicity of infections of respective adenoviral vectors as indicated, cultured into aggregates, and maintained in a defined serum-free medium for 3, 7, 14, 21 or 28 days. For each treatment group and time point indicated, RNA was extracted from three aggregates, and both expression of (A) tranduced genes (3, 7, 14, 21 and 28 days) and (B,C,D,E,F,G,H) the cartilage-specific marker genes aggrecan (AGC), biglycan (BGC), cartilage matrix (CM), and proteoglycan (PGC), collagen (COL) I, COL II, COL X, (3, 14, and 28 days) were determined by quantitative real time (qRT)-PCR. RNA isolated from ASCs differentiated by a commercial established medium and RNA extracted immediately from ASCs newly transduced (time 0) were used as comparative controls. The primer sequences, product sizes, and annealing temperatures for qRT-PCR are listed in Additional file 1 . The expression level of each targeted gene was normalized to the housekeeping gene GAPDH. Values are expressed as the fold induction of means ± standard deviations of normalized expression levels. Statistical differences between groups and positive control were analyzed using a t test; *differences were considered significant when P
Figure Legend Snippet: Gene expression in genetically modified adipose-derived stem cells aggregate cultures . Adipose-derived stem cells ( ASCs) were transduced with 100 multiplicity of infections of respective adenoviral vectors as indicated, cultured into aggregates, and maintained in a defined serum-free medium for 3, 7, 14, 21 or 28 days. For each treatment group and time point indicated, RNA was extracted from three aggregates, and both expression of (A) tranduced genes (3, 7, 14, 21 and 28 days) and (B,C,D,E,F,G,H) the cartilage-specific marker genes aggrecan (AGC), biglycan (BGC), cartilage matrix (CM), and proteoglycan (PGC), collagen (COL) I, COL II, COL X, (3, 14, and 28 days) were determined by quantitative real time (qRT)-PCR. RNA isolated from ASCs differentiated by a commercial established medium and RNA extracted immediately from ASCs newly transduced (time 0) were used as comparative controls. The primer sequences, product sizes, and annealing temperatures for qRT-PCR are listed in Additional file 1 . The expression level of each targeted gene was normalized to the housekeeping gene GAPDH. Values are expressed as the fold induction of means ± standard deviations of normalized expression levels. Statistical differences between groups and positive control were analyzed using a t test; *differences were considered significant when P

Techniques Used: Expressing, Genetically Modified, Derivative Assay, Transduction, Cell Culture, Marker, Pyrolysis Gas Chromatography, Quantitative RT-PCR, Isolation, Positive Control

33) Product Images from "Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1"

Article Title: Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1

Journal: Viruses

doi: 10.3390/v7031492

Detection of RSV accumulation levels in SBPHs after feeding-based RNA interference (RNAi). ( A ) The levels of HiPV VP1 and RSV RNP transcripts in insects after feeding them artificial diets without dsRNA (CK), dsGFP and dsVP1 (with 150 ng/μl dsRNA concentration for 6 days). After qRT-PCR, the levels of VP1 and RNP transcripts were normalized relative to the β -actin transcript according to the ΔC T algorithm, and the resulting 2 −ΔCt values were used to plot with different feeding treatments as the abscissa. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP . Each histogram bar represents the mean (±SE) from four repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p
Figure Legend Snippet: Detection of RSV accumulation levels in SBPHs after feeding-based RNA interference (RNAi). ( A ) The levels of HiPV VP1 and RSV RNP transcripts in insects after feeding them artificial diets without dsRNA (CK), dsGFP and dsVP1 (with 150 ng/μl dsRNA concentration for 6 days). After qRT-PCR, the levels of VP1 and RNP transcripts were normalized relative to the β -actin transcript according to the ΔC T algorithm, and the resulting 2 −ΔCt values were used to plot with different feeding treatments as the abscissa. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP . Each histogram bar represents the mean (±SE) from four repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p

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

RSV-acquisition ability of SBPH after feeding-based RNAi. After qRT-PCR, the levels of VP1 transcripts in SBPH via different feeding treatments (CK, dsGFP and dsVP1; 150 ng/μL dsRNA concentration for 6 days) were normalized relative to the β -actin transcript according to the ΔC T algorithm. The virus-acquisition rate of SBPH was detected using the DIBA method after a 10-day latent period. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates RSV-acquisition rate of SBPH. Each histogram bar represents the mean (±SE) from three (CK and dsGFP) or five (dsVP1) repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p
Figure Legend Snippet: RSV-acquisition ability of SBPH after feeding-based RNAi. After qRT-PCR, the levels of VP1 transcripts in SBPH via different feeding treatments (CK, dsGFP and dsVP1; 150 ng/μL dsRNA concentration for 6 days) were normalized relative to the β -actin transcript according to the ΔC T algorithm. The virus-acquisition rate of SBPH was detected using the DIBA method after a 10-day latent period. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates RSV-acquisition rate of SBPH. Each histogram bar represents the mean (±SE) from three (CK and dsGFP) or five (dsVP1) repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p

Techniques Used: Quantitative RT-PCR, Concentration Assay, Dot Immunobinding, Expressing

Titers of HiPV and RSV in single SBPH. After qRT-PCR, the levels of HiPV VP1 and RSV RNP transcripts in single SBPH were normalized relative to the β -actin transcript according to the ΔC T algorithm. The 118 individual SBPHs are arranged according to descending VP1 expression quantity as the abscissa; the left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP .
Figure Legend Snippet: Titers of HiPV and RSV in single SBPH. After qRT-PCR, the levels of HiPV VP1 and RSV RNP transcripts in single SBPH were normalized relative to the β -actin transcript according to the ΔC T algorithm. The 118 individual SBPHs are arranged according to descending VP1 expression quantity as the abscissa; the left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP .

Techniques Used: Quantitative RT-PCR, Expressing

34) Product Images from "EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression"

Article Title: EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

Journal: Blood

doi: 10.1182/blood-2015-06-649087

EHMT1 or EHMT2 knockdown increases γ-globin gene expression and HbF synthesis in primary adult human erythroid cells. (A) Validation of shRNA-mediated knockdown of EHMT1 or EHMT2 in primary adult human erythroid cells by qRT-PCR at day 14 of erythroid
Figure Legend Snippet: EHMT1 or EHMT2 knockdown increases γ-globin gene expression and HbF synthesis in primary adult human erythroid cells. (A) Validation of shRNA-mediated knockdown of EHMT1 or EHMT2 in primary adult human erythroid cells by qRT-PCR at day 14 of erythroid

Techniques Used: Expressing, shRNA, Quantitative RT-PCR

35) Product Images from "bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2+"

Article Title: bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2+

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01490

The mco , mntABC , sod , and bifA mRNA levels in biofilm cells as well as during biofilm development with exogenous Mn 2+ . mco (A) , mntABC (B) , sod (C) , and bifA (D) mRNA levels in colonized cells incubated with 200 μM Mn 2+ for 2 h after 6, 12, and 24 h cultivation for adhesion, were detected by qRT-PCR, respectively. The cellular mRNA levels of mntABC (E) , sod (F) , and bifA (G) at different stages of biofilm formation were detected by qRT-PCR, respectively. Relative gene expression levels of mco, mntABC , sod and bifA were normalized to 16s rRNA ( n = 4), respectively. Data were expressed as mean ± SEM. Significance, ∗ P
Figure Legend Snippet: The mco , mntABC , sod , and bifA mRNA levels in biofilm cells as well as during biofilm development with exogenous Mn 2+ . mco (A) , mntABC (B) , sod (C) , and bifA (D) mRNA levels in colonized cells incubated with 200 μM Mn 2+ for 2 h after 6, 12, and 24 h cultivation for adhesion, were detected by qRT-PCR, respectively. The cellular mRNA levels of mntABC (E) , sod (F) , and bifA (G) at different stages of biofilm formation were detected by qRT-PCR, respectively. Relative gene expression levels of mco, mntABC , sod and bifA were normalized to 16s rRNA ( n = 4), respectively. Data were expressed as mean ± SEM. Significance, ∗ P

Techniques Used: Incubation, Quantitative RT-PCR, Expressing

Expression of mco, mntABC , sod , and bifA gene between the planktonic and biofilm lifestyles as well as during biofilm development. mco (A) , mntABC (B) , sod (C) , and bifA (D) mRNA levels in planktonic cells and colonized cells at 6, 12, and 24 h incubation were determined by qRT-PCR, respectively. (E) The cellular mRNA levels of mco, mntABC , sod and bifA during biofilm development in the colonized cells were determined by qRT-PCR. These expression levels were normalized to 16s rRNA ( n = 4). Data were expressed as mean ± SEM. Significance, ∗ P
Figure Legend Snippet: Expression of mco, mntABC , sod , and bifA gene between the planktonic and biofilm lifestyles as well as during biofilm development. mco (A) , mntABC (B) , sod (C) , and bifA (D) mRNA levels in planktonic cells and colonized cells at 6, 12, and 24 h incubation were determined by qRT-PCR, respectively. (E) The cellular mRNA levels of mco, mntABC , sod and bifA during biofilm development in the colonized cells were determined by qRT-PCR. These expression levels were normalized to 16s rRNA ( n = 4). Data were expressed as mean ± SEM. Significance, ∗ P

Techniques Used: Expressing, Incubation, Quantitative RT-PCR

The sod and bifA mRNA levels in biofilm cells as well as during biofilm development with exogenous H 2 O 2 . sod (A) and bifA (C) mRNA levels were detected by qRT-PCR analysis in colonized cells incubated with 200 μM H 2 O 2 for 2 h after 3 h, 6, 12, and 24 h cultivation for adhesion, respectively. Relative gene expression levels of sod and bifA were normalized to 16s rRNA ( n = 4), respectively. The cellular mRNA levels of sod (B) and bifA (D) during different stages of biofilm formation. Data were expressed as mean ± SEM. Significance, ∗ P
Figure Legend Snippet: The sod and bifA mRNA levels in biofilm cells as well as during biofilm development with exogenous H 2 O 2 . sod (A) and bifA (C) mRNA levels were detected by qRT-PCR analysis in colonized cells incubated with 200 μM H 2 O 2 for 2 h after 3 h, 6, 12, and 24 h cultivation for adhesion, respectively. Relative gene expression levels of sod and bifA were normalized to 16s rRNA ( n = 4), respectively. The cellular mRNA levels of sod (B) and bifA (D) during different stages of biofilm formation. Data were expressed as mean ± SEM. Significance, ∗ P

Techniques Used: Quantitative RT-PCR, Incubation, Expressing

36) Product Images from "Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae"

Article Title: Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae

Journal: BMC Biology

doi: 10.1186/s12915-016-0325-7

Deletion of SUT457 accelerates senescence in telomerase-deficient cells. a Schematic representation of the SUT457 locus on Chromosome III. Arrows indicate direction of transcription. b Expression levels of SYP1 , SUT457 , SNR65 and RPS14A determined by qRT-PCR using total RNA extracted from isogenic wild-type and sut457Δ strains. The expression levels of each gene were normalised to the expression of ACT1 . Error bars represent standard error of the mean, resulting from three independent replicates. *** P
Figure Legend Snippet: Deletion of SUT457 accelerates senescence in telomerase-deficient cells. a Schematic representation of the SUT457 locus on Chromosome III. Arrows indicate direction of transcription. b Expression levels of SYP1 , SUT457 , SNR65 and RPS14A determined by qRT-PCR using total RNA extracted from isogenic wild-type and sut457Δ strains. The expression levels of each gene were normalised to the expression of ACT1 . Error bars represent standard error of the mean, resulting from three independent replicates. *** P

Techniques Used: Expressing, Quantitative RT-PCR

37) Product Images from "Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3"

Article Title: Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3

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

doi: 10.1073/pnas.1710866114

Mutants of sh3p2 and sh3p1sh3p3 do not show apparent phenotypes. ( A ) Schematic presentation of the T-DNA insertion sites in sh3p1 and sh3p3 and position of the amiRNA target sequence of amish3p2 . Gray lines indicate untranslated regions, and introns and black boxes indicate exons. Positions of start and stop codons are indicated in the figure. Binding sites of the forward and reverse primers and the T-DNA left border primer used for genotyping are indicated by arrows. ( B ) PCR analysis of cDNA generated from total RNA of sh3p1 and sh3p3 homozygous mutants. Expression of SH3P1 and SH3P3 in wild-type (WT), sh3p1 , and sh3p3 seedlings was analyzed using SH3P1 - , SH3P3 -, and ACTIN2 -specific primers. ( C ) qRT-PCR analysis of amish3p2 . Expression of SH3P2 in amish3p2 -, WT, and sh3p1sh3p3 double-homozygous seedlings was analyzed using gene-specific primers of SH3P2 and normalized against transcript levels of ACT8 . ( D ) Photographs of 9-d-old WT, ami sh3p2 , and sh3p1sh3p3 seedlings. Seedlings were grown on GM under short-day conditions. (Scale bars: 2 cm.) ( E ) Measurement of the root length of 6-d-old WT and mutant seedlings grown on GM or GM supplemented with 3 μM BFA. ( F ) Measurement of FM4-64 uptake at the plasma membrane in root epidermis cells of 7-d-old WT (blue line) and amish3p2 (red line) seedlings. Seedlings were incubated for 5 min in liquid GM with 2 μM FM4-64 and washed twice in liquid GM before the analysis under a confocal microscope. FM4-64 signals were imaged using the same settings over a period of 15 min. Signal intensities were analyzed using Olympus Fluoview software. The signal intensity at 0 min was set to 100%. ( G , Left ) Photographs of WT, ami sh3p2 , and atg7 seedlings after a 6-d dark treatment. (Scale bars: 1 cm.) ( G , Right ) Total chlorophyll content in the seedlings was analyzed. Seedlings were grown for 7 d on half-strength MS under long-day conditions before being transferred into the dark. Note that starvation-induced chlorosis is not enhanced in amish3p2 compared with the autophagy mutant atg7 . The value of the WT was set to 100%.
Figure Legend Snippet: Mutants of sh3p2 and sh3p1sh3p3 do not show apparent phenotypes. ( A ) Schematic presentation of the T-DNA insertion sites in sh3p1 and sh3p3 and position of the amiRNA target sequence of amish3p2 . Gray lines indicate untranslated regions, and introns and black boxes indicate exons. Positions of start and stop codons are indicated in the figure. Binding sites of the forward and reverse primers and the T-DNA left border primer used for genotyping are indicated by arrows. ( B ) PCR analysis of cDNA generated from total RNA of sh3p1 and sh3p3 homozygous mutants. Expression of SH3P1 and SH3P3 in wild-type (WT), sh3p1 , and sh3p3 seedlings was analyzed using SH3P1 - , SH3P3 -, and ACTIN2 -specific primers. ( C ) qRT-PCR analysis of amish3p2 . Expression of SH3P2 in amish3p2 -, WT, and sh3p1sh3p3 double-homozygous seedlings was analyzed using gene-specific primers of SH3P2 and normalized against transcript levels of ACT8 . ( D ) Photographs of 9-d-old WT, ami sh3p2 , and sh3p1sh3p3 seedlings. Seedlings were grown on GM under short-day conditions. (Scale bars: 2 cm.) ( E ) Measurement of the root length of 6-d-old WT and mutant seedlings grown on GM or GM supplemented with 3 μM BFA. ( F ) Measurement of FM4-64 uptake at the plasma membrane in root epidermis cells of 7-d-old WT (blue line) and amish3p2 (red line) seedlings. Seedlings were incubated for 5 min in liquid GM with 2 μM FM4-64 and washed twice in liquid GM before the analysis under a confocal microscope. FM4-64 signals were imaged using the same settings over a period of 15 min. Signal intensities were analyzed using Olympus Fluoview software. The signal intensity at 0 min was set to 100%. ( G , Left ) Photographs of WT, ami sh3p2 , and atg7 seedlings after a 6-d dark treatment. (Scale bars: 1 cm.) ( G , Right ) Total chlorophyll content in the seedlings was analyzed. Seedlings were grown for 7 d on half-strength MS under long-day conditions before being transferred into the dark. Note that starvation-induced chlorosis is not enhanced in amish3p2 compared with the autophagy mutant atg7 . The value of the WT was set to 100%.

Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Generated, Expressing, Quantitative RT-PCR, Mutagenesis, Incubation, Microscopy, Software, Mass Spectrometry

38) Product Images from "Functional Characterization of TaFUSCA3, a B3-Superfamily Transcription Factor Gene in the Wheat"

Article Title: Functional Characterization of TaFUSCA3, a B3-Superfamily Transcription Factor Gene in the Wheat

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.01133

Organ-specific expression assay of TaFUSCA3 in wheat by real-time qRT-PCR. The organs include roots, young stems, young leaves, endosperms, mature stems, mature leaves, flag leaves, stamens, and pistils. Data are means ± SD ( n = 3) in the organ-specific expression assays. Three biological experiments showed the same results.
Figure Legend Snippet: Organ-specific expression assay of TaFUSCA3 in wheat by real-time qRT-PCR. The organs include roots, young stems, young leaves, endosperms, mature stems, mature leaves, flag leaves, stamens, and pistils. Data are means ± SD ( n = 3) in the organ-specific expression assays. Three biological experiments showed the same results.

Techniques Used: Expressing, Quantitative RT-PCR

Expression analyses of the TaFUSCA3 gene by real-time qRT-PCR. (A) mRNA relative expression index of TaFUSCA3 and TaSPA TFs. (B) Expression levels of the Ta1Bx7 gene encoding the SSP in developing endosperms from 4 to 32 DAP are shown, respectively. The Taβ-actin gene was used as an internal control for standardization. Error bars represent the SD for three technical replicates. Three biological experiments gave the same results. The Pearson correlation coefficient between the expression levels of TaFUSCA3 and TaSPA was 0.8670 and that between TaFUSCA3 and Ta1Bx7 was 0.8038.
Figure Legend Snippet: Expression analyses of the TaFUSCA3 gene by real-time qRT-PCR. (A) mRNA relative expression index of TaFUSCA3 and TaSPA TFs. (B) Expression levels of the Ta1Bx7 gene encoding the SSP in developing endosperms from 4 to 32 DAP are shown, respectively. The Taβ-actin gene was used as an internal control for standardization. Error bars represent the SD for three technical replicates. Three biological experiments gave the same results. The Pearson correlation coefficient between the expression levels of TaFUSCA3 and TaSPA was 0.8670 and that between TaFUSCA3 and Ta1Bx7 was 0.8038.

Techniques Used: Expressing, Quantitative RT-PCR

39) Product Images from "The Effect of Oxandrolone Treatment on Human Osteoblastic Cells"

Article Title: The Effect of Oxandrolone Treatment on Human Osteoblastic Cells

Journal: Journal of Burns and Wounds

doi:

Consequence of oxandrolone treatment on gene expression in normal human osteocytes: time course analysis. Normal human osteoblastic cells were cultured for 10 days to confluence, then switched to differentiation medium containing ascorbic acid and β-glycerol phosphate. After 7 days, osteocytic cells were treated with 15 μg/mL oxandrolone. Total RNA was isolated after 24 hours or 5 days treatment, and gene expression characterized by qRT-PCR analysis using human primers specific for AR, type I collagen (col), alkaline phosphatase (ALP), and osteocalcin (OC) and osteoprotegerin (OPG). n = 3 to 4. ** P
Figure Legend Snippet: Consequence of oxandrolone treatment on gene expression in normal human osteocytes: time course analysis. Normal human osteoblastic cells were cultured for 10 days to confluence, then switched to differentiation medium containing ascorbic acid and β-glycerol phosphate. After 7 days, osteocytic cells were treated with 15 μg/mL oxandrolone. Total RNA was isolated after 24 hours or 5 days treatment, and gene expression characterized by qRT-PCR analysis using human primers specific for AR, type I collagen (col), alkaline phosphatase (ALP), and osteocalcin (OC) and osteoprotegerin (OPG). n = 3 to 4. ** P

Techniques Used: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, ALP Assay

Consequence of oxandrolone treatment on gene expression in normal human osteocytes: dose-response analysis. Normal human osteoblastic cells were cultured as described in Figure 5 . Osteocytic cultures were treated for 5 days with 0, 1, 5, 10, and 15 μg/mL oxandrolone. Total RNA was isolated and gene expression characterized by qRT-PCR analysis using human primers specific for AR, type I collagen (col), alkaline phosphatase (ALP), osteocalcin (OC) and osteoprotegerin (OPG). n = 2 to 4. * P
Figure Legend Snippet: Consequence of oxandrolone treatment on gene expression in normal human osteocytes: dose-response analysis. Normal human osteoblastic cells were cultured as described in Figure 5 . Osteocytic cultures were treated for 5 days with 0, 1, 5, 10, and 15 μg/mL oxandrolone. Total RNA was isolated and gene expression characterized by qRT-PCR analysis using human primers specific for AR, type I collagen (col), alkaline phosphatase (ALP), osteocalcin (OC) and osteoprotegerin (OPG). n = 2 to 4. * P

Techniques Used: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, ALP Assay

40) Product Images from "Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells"

Article Title: Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0563-9

Extracellular matrix expression of degenerate NPCs after NPCs were stimulated by BM-MSC exosomes. a qRT-PCR analysis showed an increase in ACAN ( a ), COL2A1 ( b ), SOX-9 ( c ) and TIMP-1 ( f ) and a decrease in MMP-1 ( d ) and MMP-3 ( e ) mRNA expression with the time of stimulation by BM-MSC exosomes. * P
Figure Legend Snippet: Extracellular matrix expression of degenerate NPCs after NPCs were stimulated by BM-MSC exosomes. a qRT-PCR analysis showed an increase in ACAN ( a ), COL2A1 ( b ), SOX-9 ( c ) and TIMP-1 ( f ) and a decrease in MMP-1 ( d ) and MMP-3 ( e ) mRNA expression with the time of stimulation by BM-MSC exosomes. * P

Techniques Used: Expressing, Quantitative RT-PCR

Comparison of gene expression in BM-MSCs after coculture with NPCs and after stimulation by NPC exosomes. a Schematic diagram of experimental groups. NPCs and BM-MSCs were cocultured by an indirect method using a transwell system. NPCs were cultured in the upper chamber and BM-MSCs in the lower chamber to coculture for 14 days. NPC exosomes alone were added to BM-MSCs in the Exo group. BM-MSCs without treatment were taken as the control group. b After 14 days of treatment, qRT-PCR detected relative mRNA expression in BM-MSCs. Expression levels of ACAN ( P
Figure Legend Snippet: Comparison of gene expression in BM-MSCs after coculture with NPCs and after stimulation by NPC exosomes. a Schematic diagram of experimental groups. NPCs and BM-MSCs were cocultured by an indirect method using a transwell system. NPCs were cultured in the upper chamber and BM-MSCs in the lower chamber to coculture for 14 days. NPC exosomes alone were added to BM-MSCs in the Exo group. BM-MSCs without treatment were taken as the control group. b After 14 days of treatment, qRT-PCR detected relative mRNA expression in BM-MSCs. Expression levels of ACAN ( P

Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR

41) Product Images from "SF3B1 is a stress-sensitive splicing factor that regulates both HSF1 concentration and activity"

Article Title: SF3B1 is a stress-sensitive splicing factor that regulates both HSF1 concentration and activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176382

SF3B1 activity is sensitive to temperature. qRT-PCR analysis of (A) an alternatively spliced variant of the RBM5 transcript that is known to be regulated by SF3B1 and (B) the normal splice variant of ACT1B in cells incubated with varying levels of PB for 16 hours +/- heat shock at 42° for 1 hour before harvest. Data is normalized to the no drug, no HS control. * indicates p-value
Figure Legend Snippet: SF3B1 activity is sensitive to temperature. qRT-PCR analysis of (A) an alternatively spliced variant of the RBM5 transcript that is known to be regulated by SF3B1 and (B) the normal splice variant of ACT1B in cells incubated with varying levels of PB for 16 hours +/- heat shock at 42° for 1 hour before harvest. Data is normalized to the no drug, no HS control. * indicates p-value

Techniques Used: Activity Assay, Quantitative RT-PCR, Variant Assay, Incubation

The SF3B1 inhibitor Pladienolide B inhibits the HSR in a dose-dependent manner. (A) qRT-PCR analysis of HSPA6 mRNA levels in cells treated with varying levels of Pladienolide B (PB) and normalized to the no drug control. Cells were incubated with PB for 16 hours and then subjected to a 1 hour heat shock at 42° before harvest. (B) qRT-PCR analysis of an alternatively spliced variant of the RBM5 transcript that is known to be sensitive to SF3B1 [ 22 ]. Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (C) qRT-PCR analysis of the normal spliced transcript for ACT1B and RBM5 . Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (D) Diagram indicating primer locations for qPCR. * indicates p-value
Figure Legend Snippet: The SF3B1 inhibitor Pladienolide B inhibits the HSR in a dose-dependent manner. (A) qRT-PCR analysis of HSPA6 mRNA levels in cells treated with varying levels of Pladienolide B (PB) and normalized to the no drug control. Cells were incubated with PB for 16 hours and then subjected to a 1 hour heat shock at 42° before harvest. (B) qRT-PCR analysis of an alternatively spliced variant of the RBM5 transcript that is known to be sensitive to SF3B1 [ 22 ]. Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (C) qRT-PCR analysis of the normal spliced transcript for ACT1B and RBM5 . Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (D) Diagram indicating primer locations for qPCR. * indicates p-value

Techniques Used: Quantitative RT-PCR, Incubation, Variant Assay, Real-time Polymerase Chain Reaction

SF3B1 knockdown and high but not low levels of an SF3B1 inhibitor decreases HSF1 mRNA and protein levels. (A) qRT-PCR analysis of HSF1 mRNA levels in cells that have been treated with either HSF1 siRNA or SF3B1 siRNA relative to nonsilencing siRNA control cells. (B) Western blot analysis of cells that have been treated with non-silencing control siRNA (NSC), HSF1 siRNA, or SF3B1 siRNA and probed with either anti-HSF1 or anti-ACTIN antibodies. (C) qRT-PCR analysis of HSF1 mRNA levels in cells that have been treated with varying amounts of Pladienolide B relative to control cells. (D) Western blot analysis of cells that have been treated with varying amounts of Pladienolide B, with and without heat shock at 42° for 1 hour and probed with either anti-HSF1 or anti-ACTIN antibodies.
Figure Legend Snippet: SF3B1 knockdown and high but not low levels of an SF3B1 inhibitor decreases HSF1 mRNA and protein levels. (A) qRT-PCR analysis of HSF1 mRNA levels in cells that have been treated with either HSF1 siRNA or SF3B1 siRNA relative to nonsilencing siRNA control cells. (B) Western blot analysis of cells that have been treated with non-silencing control siRNA (NSC), HSF1 siRNA, or SF3B1 siRNA and probed with either anti-HSF1 or anti-ACTIN antibodies. (C) qRT-PCR analysis of HSF1 mRNA levels in cells that have been treated with varying amounts of Pladienolide B relative to control cells. (D) Western blot analysis of cells that have been treated with varying amounts of Pladienolide B, with and without heat shock at 42° for 1 hour and probed with either anti-HSF1 or anti-ACTIN antibodies.

Techniques Used: Quantitative RT-PCR, Western Blot

Depletion of SF3B1 inhibits the HSR. (A) qRT-PCR analysis of mRNA levels of two heat shock genes, HSPA6 and DNAJB1 , in cells that have been treated with either HSF1 siRNA or SF3B1 siRNA and subjected to a 1 hour heat shock at 42°. Data is shown relative to mRNA levels in heat shocked cells treated with a nonsilencing control siRNA. (B) Western blot analysis of cells treated with SF3B1 siRNA or a nonsilencing control siRNA probed with both anti-SF3B1 and anti-ACTIN antibodies. Two biologically independent replicates for each sample are shown. (C) Western blot analysis of cells treated with SF3B1 siRNA or a nonsilencing control siRNA and probed with anti-HSP70 or anti-ACTIN antibodies. Heat shocked cells were subjected to a 1 hour at 42° and then allowed to recover for 4 hours at 37° before harvesting. Induction levels of HSP70 were quantitated by densitometry from the blots and normalized to ACTIN. Fold induction upon heat shock is shown. (D) Diagram showing the qPCR primer locations for HSPA6 and DNAJB1 .
Figure Legend Snippet: Depletion of SF3B1 inhibits the HSR. (A) qRT-PCR analysis of mRNA levels of two heat shock genes, HSPA6 and DNAJB1 , in cells that have been treated with either HSF1 siRNA or SF3B1 siRNA and subjected to a 1 hour heat shock at 42°. Data is shown relative to mRNA levels in heat shocked cells treated with a nonsilencing control siRNA. (B) Western blot analysis of cells treated with SF3B1 siRNA or a nonsilencing control siRNA probed with both anti-SF3B1 and anti-ACTIN antibodies. Two biologically independent replicates for each sample are shown. (C) Western blot analysis of cells treated with SF3B1 siRNA or a nonsilencing control siRNA and probed with anti-HSP70 or anti-ACTIN antibodies. Heat shocked cells were subjected to a 1 hour at 42° and then allowed to recover for 4 hours at 37° before harvesting. Induction levels of HSP70 were quantitated by densitometry from the blots and normalized to ACTIN. Fold induction upon heat shock is shown. (D) Diagram showing the qPCR primer locations for HSPA6 and DNAJB1 .

Techniques Used: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

42) Product Images from "Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes †"

Article Title: Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes †

Journal:

doi: 10.1128/MCB.02317-05

qRT-PCR validation of GLI1/EGF-regulated gene expression in primary human keratinocytes. Note that the lower induction values compared to N/TERT keratinocytes (see Table ) are likely to be due to lower expression levels of EGFR.
Figure Legend Snippet: qRT-PCR validation of GLI1/EGF-regulated gene expression in primary human keratinocytes. Note that the lower induction values compared to N/TERT keratinocytes (see Table ) are likely to be due to lower expression levels of EGFR.

Techniques Used: Quantitative RT-PCR, Expressing

Inhibition of EGFR and MEK/ERK signaling abrogates synergistic activation of GLI/EGF target genes. (A through D) qRT-PCR analysis of GLI/EGF (IL1R2, S100A9, and ARC [and EGR3 in panel A]) and EGF-insensitive GLI target genes (PTCH and BCL2 genes) in HaCaT
Figure Legend Snippet: Inhibition of EGFR and MEK/ERK signaling abrogates synergistic activation of GLI/EGF target genes. (A through D) qRT-PCR analysis of GLI/EGF (IL1R2, S100A9, and ARC [and EGR3 in panel A]) and EGF-insensitive GLI target genes (PTCH and BCL2 genes) in HaCaT

Techniques Used: Inhibition, Activation Assay, Quantitative RT-PCR

43) Product Images from "Flowering time in banana (Musa spp.), a day neutral plant, is controlled by at least three FLOWERING LOCUS T homologues"

Article Title: Flowering time in banana (Musa spp.), a day neutral plant, is controlled by at least three FLOWERING LOCUS T homologues

Journal: Scientific Reports

doi: 10.1038/s41598-017-06118-x

Detection of Arabidopsis AP1 expression by qRT-PCR in transgenic Arabidopsis lines ectopically expressing banana MaFT / TSF genes. The SAND (At2g28390) gene of Arabidopsis was used as reference gene.
Figure Legend Snippet: Detection of Arabidopsis AP1 expression by qRT-PCR in transgenic Arabidopsis lines ectopically expressing banana MaFT / TSF genes. The SAND (At2g28390) gene of Arabidopsis was used as reference gene.

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

Diurnal expression patterns of FT / TSF -like genes of banana by qRT-PCR. Black and white bars at the bottom represent day and night periods. The relative expression was normalized against RPS2 . The values are means of three biological and three technical replicates (±SE).
Figure Legend Snippet: Diurnal expression patterns of FT / TSF -like genes of banana by qRT-PCR. Black and white bars at the bottom represent day and night periods. The relative expression was normalized against RPS2 . The values are means of three biological and three technical replicates (±SE).

Techniques Used: Expressing, Quantitative RT-PCR

Time-course expression patterns of banana FT / TSF genes during development in Grand Nain by qRT-PCR. The RPS2 gene of each is used as reference gene. The transition from vegetative to reproductive stage is shown by an asterisk. The values are means of two biological (each containing a pool of leaves of five plants) and three technical replicates (±SE). Analysis was performed using the Tukey-Kramer multiple comparison test by GraphPad software. The asterisks (*) indicate significant differences (P
Figure Legend Snippet: Time-course expression patterns of banana FT / TSF genes during development in Grand Nain by qRT-PCR. The RPS2 gene of each is used as reference gene. The transition from vegetative to reproductive stage is shown by an asterisk. The values are means of two biological (each containing a pool of leaves of five plants) and three technical replicates (±SE). Analysis was performed using the Tukey-Kramer multiple comparison test by GraphPad software. The asterisks (*) indicate significant differences (P

Techniques Used: Expressing, Quantitative RT-PCR, Software

Time-course expression patterns of banana FT / TSF genes during development in Hill banana by qRT-PCR. The RPS2 gene of each is used as reference gene. The transition from vegetative to reproductive stage is shown by a black circle. The values are means of two biological (each containing a pool of leaves of five plants) and three technical replicates (±SE). Analysis was performed using the Tukey-Kramer multiple comparison test by GraphPad software. The asterisks (*) indicate significant differences (P
Figure Legend Snippet: Time-course expression patterns of banana FT / TSF genes during development in Hill banana by qRT-PCR. The RPS2 gene of each is used as reference gene. The transition from vegetative to reproductive stage is shown by a black circle. The values are means of two biological (each containing a pool of leaves of five plants) and three technical replicates (±SE). Analysis was performed using the Tukey-Kramer multiple comparison test by GraphPad software. The asterisks (*) indicate significant differences (P

Techniques Used: Expressing, Quantitative RT-PCR, Software

44) Product Images from "Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus"

Article Title: Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus

Journal: Nature Communications

doi: 10.1038/s41467-018-04390-7

Gene knockdown in vitro and in vivo cytotoxicity of fusogenic porous Si nanoparticle constructs. a siRNA knockdown results (via qRT-PCR) from Raw 264.7 macrophage cells incubated with nanoparticles for 24 h. Error bars indicate SD ( n = 6). The fusogenic formulations show substantial knockdown, comparable to standard lipofectamine transfection agent. No significant difference in knockdown efficiency is observed between the two fusogenic formulations (F-siIRF5-mPEG, F-siIRF5-CRV) and lipofectamine. *Significant difference (one-way ANOVA with Tukey’s HSD post hoc test, p -level
Figure Legend Snippet: Gene knockdown in vitro and in vivo cytotoxicity of fusogenic porous Si nanoparticle constructs. a siRNA knockdown results (via qRT-PCR) from Raw 264.7 macrophage cells incubated with nanoparticles for 24 h. Error bars indicate SD ( n = 6). The fusogenic formulations show substantial knockdown, comparable to standard lipofectamine transfection agent. No significant difference in knockdown efficiency is observed between the two fusogenic formulations (F-siIRF5-mPEG, F-siIRF5-CRV) and lipofectamine. *Significant difference (one-way ANOVA with Tukey’s HSD post hoc test, p -level

Techniques Used: In Vitro, In Vivo, Construct, Quantitative RT-PCR, Incubation, Transfection

45) Product Images from "A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells"

Article Title: A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-018-4711-0

Silencing of GNB4 in 182 R -6 and TAM R -1 cells via DNMT3B. a , Total RNA isolated from S05, 182 R -6, and TAM R -1 cells was subjected to qRT-PCR using a primer set specific to GNB4. Whole cellular lysate was prepared from S05, 182 R -6, and TAM R -1 cells, and Western blot analysis was performed using an antibody against GNB4. b , Whole cellular lysate was prepared from S05, 182 R -6, and TAM R -1 cells, and Western blot analysis was performed using antibodies against DNMT1, DNMT3A, DNMT3B, and MeCP2. c , 182 R -6 and TAM R -1 cells were transiently transfected with either 200 nM DNMT3B siRNA or 200 nM negative control siRNA; 72 h after transfection, total RNA isolated from these cells was subjected to qRT-PCR using a primer set specific to GNB4. d , Seventy-two hours after transfection, whole cellular lysate prepared from 182 R -6 and TAM R -1 cells was transfected with either 200 nM DNMT3B siRNA or 200 nM negative control siRNA, and was subjected to Western blot analysis using antibodies against DNMT1, DNMT3A, DNMT3B and GNB4. Asterisk indicates p
Figure Legend Snippet: Silencing of GNB4 in 182 R -6 and TAM R -1 cells via DNMT3B. a , Total RNA isolated from S05, 182 R -6, and TAM R -1 cells was subjected to qRT-PCR using a primer set specific to GNB4. Whole cellular lysate was prepared from S05, 182 R -6, and TAM R -1 cells, and Western blot analysis was performed using an antibody against GNB4. b , Whole cellular lysate was prepared from S05, 182 R -6, and TAM R -1 cells, and Western blot analysis was performed using antibodies against DNMT1, DNMT3A, DNMT3B, and MeCP2. c , 182 R -6 and TAM R -1 cells were transiently transfected with either 200 nM DNMT3B siRNA or 200 nM negative control siRNA; 72 h after transfection, total RNA isolated from these cells was subjected to qRT-PCR using a primer set specific to GNB4. d , Seventy-two hours after transfection, whole cellular lysate prepared from 182 R -6 and TAM R -1 cells was transfected with either 200 nM DNMT3B siRNA or 200 nM negative control siRNA, and was subjected to Western blot analysis using antibodies against DNMT1, DNMT3A, DNMT3B and GNB4. Asterisk indicates p

Techniques Used: Isolation, Quantitative RT-PCR, Western Blot, Transfection, Negative Control

46) Product Images from "A Small Molecule Polyamine Oxidase Inhibitor Blocks Androgen-Induced Oxidative Stress and Delays Prostate Cancer Progression in the TRAMP Mouse Model"

Article Title: A Small Molecule Polyamine Oxidase Inhibitor Blocks Androgen-Induced Oxidative Stress and Delays Prostate Cancer Progression in the TRAMP Mouse Model

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-08-2472

(a) qRT-PCR amplification of SSAT mRNA from LNCaP cells stably transfected with siSSAT vector and scrambled vector control (see Methods for detail). Cells are either grown in the presence of 0 nM ( ) or 1 nM R1881 ( ). All data are normalized to corresponding
Figure Legend Snippet: (a) qRT-PCR amplification of SSAT mRNA from LNCaP cells stably transfected with siSSAT vector and scrambled vector control (see Methods for detail). Cells are either grown in the presence of 0 nM ( ) or 1 nM R1881 ( ). All data are normalized to corresponding

Techniques Used: Quantitative RT-PCR, Amplification, Stable Transfection, Transfection, Plasmid Preparation

qRT-PCR amplification of SSAT mRNA from LNCaP cells (a) either control untreated ( ) or treated with 0.05 nM ( ) and 1 nM R1881 ( ) for 96 h; (b) treated with 1 nM R1881 for 24 h ( ), 48 h ( ), 72 h ( ) and 96 h ( ); (c) ROS levels in LNCaP cells determined
Figure Legend Snippet: qRT-PCR amplification of SSAT mRNA from LNCaP cells (a) either control untreated ( ) or treated with 0.05 nM ( ) and 1 nM R1881 ( ) for 96 h; (b) treated with 1 nM R1881 for 24 h ( ), 48 h ( ), 72 h ( ) and 96 h ( ); (c) ROS levels in LNCaP cells determined

Techniques Used: Quantitative RT-PCR, Amplification

47) Product Images from "LncRNA RP11-79H23.3 Functions as a Competing Endogenous RNA to Regulate PTEN Expression through Sponging hsa-miR-107 in the Development of Bladder Cancer"

Article Title: LncRNA RP11-79H23.3 Functions as a Competing Endogenous RNA to Regulate PTEN Expression through Sponging hsa-miR-107 in the Development of Bladder Cancer

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19092531

RP11-79H23.3 and miR-107 impact the expressions of the target gene PTEN and PI3K/AKT signaling pathway molecules in vitro. ( A , B ) Immunofluorescent assays were used to detect the expressions of PTEN in EJ and T24 cells transfected with pIRES2-RP11-79H23.3 or siRP11-79H23.3 or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107 (scale bar, 100 μm). ( C ) The mRNA levels of PTEN were determined by qRT-PCR in EJ and T24 cells after being transfected with miR-107 mimics or inhibitors. ( D ) The protein levels of PTEN were determined in EJ and T24 cells transfected with miR-107 mimics or inhibitors by Western blotting. ( E ) The expressions of RP11-79H23.3 and PTEN were detected in cells transfected with pIRES2-RP11-79H23.3 and si RP11-79H23.3 by qRT-PCR. ( F ) qRT-PCR assays were performed to detect the mRNA levels of PTEN in EJ and T24 cells transfected with pIRES2-RP11-79H23.3 or siRP11-79H23.3 or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107. ( G , H ) The expressions of PTEN and PI3K/AKT signaling pathway molecules were assayed in BC cells transfected with the indicated plasmids or siRNAs or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107 by Western blotting. Each experiment was independently repeated at least three times. Data are shown as mean ± SD, * p
Figure Legend Snippet: RP11-79H23.3 and miR-107 impact the expressions of the target gene PTEN and PI3K/AKT signaling pathway molecules in vitro. ( A , B ) Immunofluorescent assays were used to detect the expressions of PTEN in EJ and T24 cells transfected with pIRES2-RP11-79H23.3 or siRP11-79H23.3 or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107 (scale bar, 100 μm). ( C ) The mRNA levels of PTEN were determined by qRT-PCR in EJ and T24 cells after being transfected with miR-107 mimics or inhibitors. ( D ) The protein levels of PTEN were determined in EJ and T24 cells transfected with miR-107 mimics or inhibitors by Western blotting. ( E ) The expressions of RP11-79H23.3 and PTEN were detected in cells transfected with pIRES2-RP11-79H23.3 and si RP11-79H23.3 by qRT-PCR. ( F ) qRT-PCR assays were performed to detect the mRNA levels of PTEN in EJ and T24 cells transfected with pIRES2-RP11-79H23.3 or siRP11-79H23.3 or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107. ( G , H ) The expressions of PTEN and PI3K/AKT signaling pathway molecules were assayed in BC cells transfected with the indicated plasmids or siRNAs or pIRES2-RP11-79H23.3 and miR-107 mimics or siRP11-79H23.3 and inh-107 by Western blotting. Each experiment was independently repeated at least three times. Data are shown as mean ± SD, * p

Techniques Used: In Vitro, Transfection, Quantitative RT-PCR, Western Blot

RP11-79H23.3 influences BC cell proliferation, migration, and invasion in vitro. ( A , B ) The expressions of RP11-79H23.3 was detected in BC cells transfected with pIRES2-RP11-79H23.3 or si-RNA fragments (si-RP11-79H23.3I, si-RP11-79H23.3II) by qRT-PCR. ( C – E ) The cell proliferation ability was assessed by CCK-8, EdU, and colony formation assays after upregulation and downregulation of RP11-79H23.3 in EJ and T24 cells. CCK-8 cell growth curve, relative quantification of EdU-incorporating cells, and soft agar colony formation numbers are shown, respectively (scale bar, 100 μm). ( F , G ) The cell migration and invasion capacities were detected in upregulating or downregulating RP11-79H23.3 cells by wound healing and transwell assays (scale bar, 200 μm; scale bar, 100 μm. ( H , I ) Quantitative analyses of wound healing and transwell assay are shown. Each experiment was independently repeated at least three times. Data is shown as mean ± SD. ( J ) Cytoskeleton was surveyed by phalloidine-FITC staining of microfilament and immunofluorescence staining for paxillion in BC cells under confocal laser scanning microscopy when RP11-79H23.3 was upregulated or downregulated (scale bar, 20 μm). * p
Figure Legend Snippet: RP11-79H23.3 influences BC cell proliferation, migration, and invasion in vitro. ( A , B ) The expressions of RP11-79H23.3 was detected in BC cells transfected with pIRES2-RP11-79H23.3 or si-RNA fragments (si-RP11-79H23.3I, si-RP11-79H23.3II) by qRT-PCR. ( C – E ) The cell proliferation ability was assessed by CCK-8, EdU, and colony formation assays after upregulation and downregulation of RP11-79H23.3 in EJ and T24 cells. CCK-8 cell growth curve, relative quantification of EdU-incorporating cells, and soft agar colony formation numbers are shown, respectively (scale bar, 100 μm). ( F , G ) The cell migration and invasion capacities were detected in upregulating or downregulating RP11-79H23.3 cells by wound healing and transwell assays (scale bar, 200 μm; scale bar, 100 μm. ( H , I ) Quantitative analyses of wound healing and transwell assay are shown. Each experiment was independently repeated at least three times. Data is shown as mean ± SD. ( J ) Cytoskeleton was surveyed by phalloidine-FITC staining of microfilament and immunofluorescence staining for paxillion in BC cells under confocal laser scanning microscopy when RP11-79H23.3 was upregulated or downregulated (scale bar, 20 μm). * p

Techniques Used: Migration, In Vitro, Transfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Staining, Immunofluorescence, Confocal Laser Scanning Microscopy

RP11-79H23.3 and PTEN could competitively bind to miR-107. ( A ) The miR-107 binding sites on RP11-79H23.3 and PTEN were predicted by miRcode and TargetScan, respectively. ( B ) Schematic of RP11-79H23.3 wild-type (wt) and mutant (mut) luciferase reporter vector is shown. ( C ) Schematic of miR107 and the putative binding sequence in the 3′-UTR sequence of PTEN as well as the mutant sequence of PTEN 3′-UTR for luciferase assays is shown. ( D ) The relative luciferase activities of EJ cells were tested after co-transfection with pmirGLO-RP11-79H23.3-wt or pmirGLO-RP11-79H23.3-mut and miR-107 mimics or miR-NC. ( E ) The relative luciferase activities were analyzed in EJ cells co-transfected with inh-107 or inh-NC and luciferase reporter vectors pmirGLO-RP11-79H23.3-wt or pmirGLO-RP11-79H23.3-mut. ( F ) The relative luciferase activities were analyzed in EJ cells co-transfected with miR-107 mimics or miR-NC and luciferase reporter vectors pmirGLO-PTEN 3′UTR-wt or pmirGLO- PTEN3′UTR-mut. ( G ) The subcellular localization for RP11-79H23.3 and miR-107 was detected in BC cells with FISH (scale bar, 20 μm). ( H ) The schematic diagram of GFP-MS2-RIP is shown. ( I ) GFP-MS2-RIP followed by qRT-PCR to detect miR-107 and endogenously associated RP11-79H23.3 in EJ cells. ( J ) Anti-AGO2 RIP was executed in EJ cells transiently transfected with miR-107 mimics, followed by RT-PCR and qRT-PCR to detect miR-107 and RP11-79H23.3 associated with AGO2. ( K ) The relative luciferase activities were analyzed in EJ cells co-transfected with luciferase reporter vectors pmirGLO-PTEN 3′UTR-wt and pIRES2-RP11-79H23.3 or vector and/or miR-107 mimics or miR-NC. ( L , N ) EdU assay showed that ectopic expression of miR-107 abolished the decreased proliferation by RP11-79H23.3 overexpression in EJ cells, whereas miR-107 inhibitor reversed the increased proliferation by RP11-79H23.3 knockdown in T24 cells (scale bar, 100 μm). ( M , O ) Transwell assay indicated that overexpression of miR-107 relieved the reduced invasion by upregulation of RP11-79H23.3 in EJ cells, whereas co-transfected miR-107 inhibitor abated the increased invasion by siRP11-79H23.3 in T24 cells (scale bar, 100 μm). Each experiment was independently repeated at least three times. Data is shown as mean ± SD, * p
Figure Legend Snippet: RP11-79H23.3 and PTEN could competitively bind to miR-107. ( A ) The miR-107 binding sites on RP11-79H23.3 and PTEN were predicted by miRcode and TargetScan, respectively. ( B ) Schematic of RP11-79H23.3 wild-type (wt) and mutant (mut) luciferase reporter vector is shown. ( C ) Schematic of miR107 and the putative binding sequence in the 3′-UTR sequence of PTEN as well as the mutant sequence of PTEN 3′-UTR for luciferase assays is shown. ( D ) The relative luciferase activities of EJ cells were tested after co-transfection with pmirGLO-RP11-79H23.3-wt or pmirGLO-RP11-79H23.3-mut and miR-107 mimics or miR-NC. ( E ) The relative luciferase activities were analyzed in EJ cells co-transfected with inh-107 or inh-NC and luciferase reporter vectors pmirGLO-RP11-79H23.3-wt or pmirGLO-RP11-79H23.3-mut. ( F ) The relative luciferase activities were analyzed in EJ cells co-transfected with miR-107 mimics or miR-NC and luciferase reporter vectors pmirGLO-PTEN 3′UTR-wt or pmirGLO- PTEN3′UTR-mut. ( G ) The subcellular localization for RP11-79H23.3 and miR-107 was detected in BC cells with FISH (scale bar, 20 μm). ( H ) The schematic diagram of GFP-MS2-RIP is shown. ( I ) GFP-MS2-RIP followed by qRT-PCR to detect miR-107 and endogenously associated RP11-79H23.3 in EJ cells. ( J ) Anti-AGO2 RIP was executed in EJ cells transiently transfected with miR-107 mimics, followed by RT-PCR and qRT-PCR to detect miR-107 and RP11-79H23.3 associated with AGO2. ( K ) The relative luciferase activities were analyzed in EJ cells co-transfected with luciferase reporter vectors pmirGLO-PTEN 3′UTR-wt and pIRES2-RP11-79H23.3 or vector and/or miR-107 mimics or miR-NC. ( L , N ) EdU assay showed that ectopic expression of miR-107 abolished the decreased proliferation by RP11-79H23.3 overexpression in EJ cells, whereas miR-107 inhibitor reversed the increased proliferation by RP11-79H23.3 knockdown in T24 cells (scale bar, 100 μm). ( M , O ) Transwell assay indicated that overexpression of miR-107 relieved the reduced invasion by upregulation of RP11-79H23.3 in EJ cells, whereas co-transfected miR-107 inhibitor abated the increased invasion by siRP11-79H23.3 in T24 cells (scale bar, 100 μm). Each experiment was independently repeated at least three times. Data is shown as mean ± SD, * p

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation, Sequencing, Cotransfection, Transfection, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, EdU Assay, Expressing, Over Expression, Transwell Assay

48) Product Images from "Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway"

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

Journal: Cancer Cell International

doi: 10.1186/s12935-019-1050-4

Uev1 depletion inactivates AKT pathway signaling and reduces cell survival under serum starvation conditions in breast cancer cells. MDA-MB-231 or MCF7 cells were transfected with shRNA lentiviral particles against UEV1 (sh UEV1 ) or non-specific target (shCK). shUEV1-1 and shUEV1-2 represent two independent stable shUEV1 MDA-MB-231 cell lines. shUEV1-8 represents a stable shUEV1 MCF7 cell line. a UEV1A transcript levels in shCK and shUEV1 lines were determined by qRT-PCR in MDA-MB-231 cells. b UEV1A transcript levels in shCK and shUEV1 line were determined by qRT-PCR in MCF7 cells. c AKT pathway proteins in whole-cell extracts (WCE) or nuclear fractions (N) were detected by western blot. The endogenous Uev1 was monitored by LN2B antibody. d Growth curve of non-specific target (shCK) and shUEV1 MDA-MB-231 cell lines (shUEV1-1 and shUEV1-2) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1 b, c. e Growth curve of non-specific target (shCK) and shUEV1 MCF7 cell line (shUEV1-8) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1 b, c. f Two shUEV1 MDA-MB-231 cell lines were transfected with the pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-1 + UEV1A and shUEV1-2 + UEV1A). Non-specific shRNA targeted MDA-MB-231 cells were also transfected with the pcDNA4.0/TO/HA(+) vector to serve as a control. Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1 b, c. g The MCF7 shUEV1-8 cell line was transfected with pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-8 + UEV1A). The non-specific shRNA targeted MCF7 cells were transfected with the vector alone (shCK + CK). Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1 b, c. Each sample was measured in triplicate and repeated at least 2 times. * P
Figure Legend Snippet: Uev1 depletion inactivates AKT pathway signaling and reduces cell survival under serum starvation conditions in breast cancer cells. MDA-MB-231 or MCF7 cells were transfected with shRNA lentiviral particles against UEV1 (sh UEV1 ) or non-specific target (shCK). shUEV1-1 and shUEV1-2 represent two independent stable shUEV1 MDA-MB-231 cell lines. shUEV1-8 represents a stable shUEV1 MCF7 cell line. a UEV1A transcript levels in shCK and shUEV1 lines were determined by qRT-PCR in MDA-MB-231 cells. b UEV1A transcript levels in shCK and shUEV1 line were determined by qRT-PCR in MCF7 cells. c AKT pathway proteins in whole-cell extracts (WCE) or nuclear fractions (N) were detected by western blot. The endogenous Uev1 was monitored by LN2B antibody. d Growth curve of non-specific target (shCK) and shUEV1 MDA-MB-231 cell lines (shUEV1-1 and shUEV1-2) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1 b, c. e Growth curve of non-specific target (shCK) and shUEV1 MCF7 cell line (shUEV1-8) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1 b, c. f Two shUEV1 MDA-MB-231 cell lines were transfected with the pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-1 + UEV1A and shUEV1-2 + UEV1A). Non-specific shRNA targeted MDA-MB-231 cells were also transfected with the pcDNA4.0/TO/HA(+) vector to serve as a control. Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1 b, c. g The MCF7 shUEV1-8 cell line was transfected with pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-8 + UEV1A). The non-specific shRNA targeted MCF7 cells were transfected with the vector alone (shCK + CK). Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1 b, c. Each sample was measured in triplicate and repeated at least 2 times. * P

Techniques Used: Multiple Displacement Amplification, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Expressing, Cell Culture

Uev1A promotes cell survival under serum starvation conditions through the AKT but not NF-κB pathway in breast cancer cells. a UEV1A overexpressed MDA-MB-231-TR cells (left panel) and MCF7 cells (right panel) were treated with LY294002. After 24 h, the AKT pathway proteins were examined by western blot in the whole-cell extract (WCE) or nuclear fraction (N) in UEV1A overexpressed cells alone (UEV1A) or treated with 10 μM LY294002 (1A + LY), or vector only (CK). The expression levels of ectopic Uev1A were monitored by an anti-HA antibody. b , c Growth curve of control (CK) and UEV1A -overexpressed MDA-MB-231-TR ( b ) and MCF7 ( c ) cells under serum-deprived conditions. Experimental conditions were as described in Fig. 2 c except that some cells were treated with 10 μM LY294002 (LY+). d , e Effects of UEV1A overexpression on the expression of selected FOXO1 target genes. Transcript levels of putative FOXO1 target genes in MDA-MB-231-TR ( d ) or MCF7 ( e ) cells overexpressing UEV1A as determined by qRT-PCR. f , g Growth curve of control (CK) and UEV1A -overexpressed MDA-MB-231-TR ( f ) and MCF7 ( g ) cells under serum-deprived conditions. Experimental conditions were as described in Fig. 2 c except that some cells were treated with 40 μM Bay11-7082 (Bay+). Each sample was measured in triplicate and repeated at least 2 times. * P
Figure Legend Snippet: Uev1A promotes cell survival under serum starvation conditions through the AKT but not NF-κB pathway in breast cancer cells. a UEV1A overexpressed MDA-MB-231-TR cells (left panel) and MCF7 cells (right panel) were treated with LY294002. After 24 h, the AKT pathway proteins were examined by western blot in the whole-cell extract (WCE) or nuclear fraction (N) in UEV1A overexpressed cells alone (UEV1A) or treated with 10 μM LY294002 (1A + LY), or vector only (CK). The expression levels of ectopic Uev1A were monitored by an anti-HA antibody. b , c Growth curve of control (CK) and UEV1A -overexpressed MDA-MB-231-TR ( b ) and MCF7 ( c ) cells under serum-deprived conditions. Experimental conditions were as described in Fig. 2 c except that some cells were treated with 10 μM LY294002 (LY+). d , e Effects of UEV1A overexpression on the expression of selected FOXO1 target genes. Transcript levels of putative FOXO1 target genes in MDA-MB-231-TR ( d ) or MCF7 ( e ) cells overexpressing UEV1A as determined by qRT-PCR. f , g Growth curve of control (CK) and UEV1A -overexpressed MDA-MB-231-TR ( f ) and MCF7 ( g ) cells under serum-deprived conditions. Experimental conditions were as described in Fig. 2 c except that some cells were treated with 40 μM Bay11-7082 (Bay+). Each sample was measured in triplicate and repeated at least 2 times. * P

Techniques Used: Multiple Displacement Amplification, Western Blot, Plasmid Preparation, Expressing, Over Expression, Quantitative RT-PCR

49) Product Images from "Identification and Comparative Analysis of Cadmium Tolerance-Associated miRNAs and Their Targets in Two Soybean Genotypes"

Article Title: Identification and Comparative Analysis of Cadmium Tolerance-Associated miRNAs and Their Targets in Two Soybean Genotypes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081471

Expression patterns of 16 Cd-responsive miRNAs confirmed by qRT-PCR. The y -axis represents the expression levels of Cd/CK. The dark grey shading bar represents the relative expression level of miRNAs in HX3 measured by microarray in responsive to Cd stress. The dark grey diagonal stripe shading bar represents the relative expression level of miRNAs in ZH24 measured by microarray in responsive to Cd stress. The french grey shading bar represents the relative expression level of miRNAs in HX3 measured by qRT-PCR in responsive to Cd stress. The french grey diagonal stripe shading bar represents the relative expression level of miRNAs in ZH24 measured by qRT-PCR in responsive to Cd stress. The results of qRT-PCR are mean ± SD of duplicates of three biological replicates. Significance of the changes between HX3 and ZH24 at the levels of Cd/CK was checked with Student's t-test at the level of P≤0.01 (shown as “**”).
Figure Legend Snippet: Expression patterns of 16 Cd-responsive miRNAs confirmed by qRT-PCR. The y -axis represents the expression levels of Cd/CK. The dark grey shading bar represents the relative expression level of miRNAs in HX3 measured by microarray in responsive to Cd stress. The dark grey diagonal stripe shading bar represents the relative expression level of miRNAs in ZH24 measured by microarray in responsive to Cd stress. The french grey shading bar represents the relative expression level of miRNAs in HX3 measured by qRT-PCR in responsive to Cd stress. The french grey diagonal stripe shading bar represents the relative expression level of miRNAs in ZH24 measured by qRT-PCR in responsive to Cd stress. The results of qRT-PCR are mean ± SD of duplicates of three biological replicates. Significance of the changes between HX3 and ZH24 at the levels of Cd/CK was checked with Student's t-test at the level of P≤0.01 (shown as “**”).

Techniques Used: Expressing, Quantitative RT-PCR, Microarray

50) Product Images from "Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease"

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.024745

Elevated IL10 expression and STAT3 activation in ADPKD kidney macrophages. (A) Elevated IL10 expression in human ADPKD kidneys. RNA was isolated from NHKs and non-cystic regions of ADPKD kidneys (three of each). IL10 expression was analyzed by performing qRT-PCR. The line indicates the mean relative value. (B) IL-10 is present in ADPKD cyst fluid. IL-10 in pooled cyst fluid collected from ADPKD kidneys from 12 different individuals was measured by using ELISA. Data are mean±s.e.m. (C-G) Phospho-STAT3 was present in macrophages of cystic ADPKD kidneys. Formalin-fixed, paraffin-embedded tissues from ADPKD kidneys were serially sectioned (C-E), and consecutive sections were stained by using immunohistochemistry with antibodies against the M2 macrophage marker CD163 (C), phospho-STAT3 (D) or secondary antibody only (E, No Ab) as a control. Arrows indicate phospho-STAT3-positive cells in macrophage-rich regions of interstitium. (F,G) Sections of ADPKD kidneys that had been fixed and prepared in a similar manner were co-stained with anti-CD163 (green) and anti-phospho-STAT3 (red) antibodies, followed by incubation with two distinct fluorescence-labeled secondary antibodies, each with the appropriate antibody specificity (F), and with DAPI (G). Arrowheads indicate co-staining cells. n =3 biological replicates. hIL-10, human IL-10.
Figure Legend Snippet: Elevated IL10 expression and STAT3 activation in ADPKD kidney macrophages. (A) Elevated IL10 expression in human ADPKD kidneys. RNA was isolated from NHKs and non-cystic regions of ADPKD kidneys (three of each). IL10 expression was analyzed by performing qRT-PCR. The line indicates the mean relative value. (B) IL-10 is present in ADPKD cyst fluid. IL-10 in pooled cyst fluid collected from ADPKD kidneys from 12 different individuals was measured by using ELISA. Data are mean±s.e.m. (C-G) Phospho-STAT3 was present in macrophages of cystic ADPKD kidneys. Formalin-fixed, paraffin-embedded tissues from ADPKD kidneys were serially sectioned (C-E), and consecutive sections were stained by using immunohistochemistry with antibodies against the M2 macrophage marker CD163 (C), phospho-STAT3 (D) or secondary antibody only (E, No Ab) as a control. Arrows indicate phospho-STAT3-positive cells in macrophage-rich regions of interstitium. (F,G) Sections of ADPKD kidneys that had been fixed and prepared in a similar manner were co-stained with anti-CD163 (green) and anti-phospho-STAT3 (red) antibodies, followed by incubation with two distinct fluorescence-labeled secondary antibodies, each with the appropriate antibody specificity (F), and with DAPI (G). Arrowheads indicate co-staining cells. n =3 biological replicates. hIL-10, human IL-10.

Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Marker, Incubation, Fluorescence, Labeling

51) Product Images from "Structural, Evolutionary, and Functional Analysis of the Class III Peroxidase Gene Family in Chinese Pear (Pyrus bretschneideri)"

Article Title: Structural, Evolutionary, and Functional Analysis of the Class III Peroxidase Gene Family in Chinese Pear (Pyrus bretschneideri)

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2016.01874

Heat map representation of PbPRX genes the eight stages of pear fruit development, 15 days after flowering (DAF), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF, and 145 DAF . These expression profile data were obtained using qRT-PCR, and relative expression was log2 transformed, thereby values −2, −1, 0, 1, and 2 represent low, intermediate and high expression, respectively.
Figure Legend Snippet: Heat map representation of PbPRX genes the eight stages of pear fruit development, 15 days after flowering (DAF), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF, and 145 DAF . These expression profile data were obtained using qRT-PCR, and relative expression was log2 transformed, thereby values −2, −1, 0, 1, and 2 represent low, intermediate and high expression, respectively.

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

52) Product Images from "Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza"

Article Title: Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

Journal: PeerJ

doi: 10.7717/peerj.4461

Transcript abundance of SmMET1 (A), SmCMT1 (B), SmCMT2a (C), SmCMT2b (D), SmCMT3 (E), SmDRM1 (F), SmDRM2 (G), and SmDNMT2 (H) in roots (Rt), Stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza . Transcript level was analyzed using quantitative real time RT-PCR. SmUBQ10 was used as a reference. qRT-PCR was performed in triplicates for each independent biological replicate. Transcript levels in roots were arbitrarily set to 1 and the levels in other tissues were given relative to this. One-way ANOVA was performed using IBM SPSS 20 software. P
Figure Legend Snippet: Transcript abundance of SmMET1 (A), SmCMT1 (B), SmCMT2a (C), SmCMT2b (D), SmCMT3 (E), SmDRM1 (F), SmDRM2 (G), and SmDNMT2 (H) in roots (Rt), Stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza . Transcript level was analyzed using quantitative real time RT-PCR. SmUBQ10 was used as a reference. qRT-PCR was performed in triplicates for each independent biological replicate. Transcript levels in roots were arbitrarily set to 1 and the levels in other tissues were given relative to this. One-way ANOVA was performed using IBM SPSS 20 software. P

Techniques Used: Quantitative RT-PCR, Software

53) Product Images from "The small molecule NSM00191 specifically represses the TNF-α/NF-кB axis in foot and ankle rheumatoid arthritis"

Article Title: The small molecule NSM00191 specifically represses the TNF-α/NF-кB axis in foot and ankle rheumatoid arthritis

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.24232

miR-7-5p specifically targeted p65. (A) The 3′-UTR of p65 contains a putative miR-7-5p binding site. The seed binding sequence of miR-7-5p in the WT 3′-UTR of p65 is indicated in red, while it is indicated in blue for mutant 3′-UTR. (B) The mutant of p65 3′-UTR failed to bind miR-7-5p. The miR-7-5p-mimic or its negative control (miR-NC) was co-transfected with pMIR-p65-3′-UTR-WT or pMIR-p65-3′-UTR-mutant into HFLS-RA cells for 24 h. The luciferase activity was quantified using the dual-luciferase assay reporter system by normalizing the signal to the Renilla activity. (C-F) The expression of miR-7-5p was negatively correlated with the p65 level. The miR-7-5p-mimic, anti-miR-5p or miR-NC was transfected into SW982 or HFLS-RA (HFLS) cells. The expression of miR-7-5p and p65 was determined in these cells. (G) The overexpression or downregulation of miR-7-5p affected the expression of NF-κB downstream targets. The cells used in C-F were subjected to qRT-PCR to examine the expression of NF-κB downstream targets, including TNF-α, IL-6, MCP-1 and CCL5. ** P
Figure Legend Snippet: miR-7-5p specifically targeted p65. (A) The 3′-UTR of p65 contains a putative miR-7-5p binding site. The seed binding sequence of miR-7-5p in the WT 3′-UTR of p65 is indicated in red, while it is indicated in blue for mutant 3′-UTR. (B) The mutant of p65 3′-UTR failed to bind miR-7-5p. The miR-7-5p-mimic or its negative control (miR-NC) was co-transfected with pMIR-p65-3′-UTR-WT or pMIR-p65-3′-UTR-mutant into HFLS-RA cells for 24 h. The luciferase activity was quantified using the dual-luciferase assay reporter system by normalizing the signal to the Renilla activity. (C-F) The expression of miR-7-5p was negatively correlated with the p65 level. The miR-7-5p-mimic, anti-miR-5p or miR-NC was transfected into SW982 or HFLS-RA (HFLS) cells. The expression of miR-7-5p and p65 was determined in these cells. (G) The overexpression or downregulation of miR-7-5p affected the expression of NF-κB downstream targets. The cells used in C-F were subjected to qRT-PCR to examine the expression of NF-κB downstream targets, including TNF-α, IL-6, MCP-1 and CCL5. ** P

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Negative Control, Transfection, Luciferase, Activity Assay, Expressing, Over Expression, Quantitative RT-PCR

NSM00191 significantly improved the inflammatory response in vivo . (A-B) NSM00191 significantly inhibited the expression of NF-κB downstream targets. The DBA/1 mice ( A , n=12) and TNF-α-overexpressing mice ( B , n=12) were divided into two groups. One group of mice was treated with DMSO; the other group mice were treated with 20 μM NSM00191 for 7 days. Then, the joint tissues were collected, and their RNAs were isolated. The RNAs from each treatment mice were equally combined and subjected into qRT-PCR analyses to examine the expression of TNF-α, IL-6, MCP-1, CCL5, COX-2, VCAM-1, CD40 and MHC-1. (C) NSM00191 inhibited the interaction between TRADD and TRAF2 in vivo . The joint samples used in A and B were subjected to Western blot analysis to determine the protein levels of TNFR1, TRADD, TRAF2, IKK2 and p65. GAPDH was used as a loading control. (D) NSM00191 decreased the p65 levels in vivo. One pair of joint samples used in A and B were subjected to IHC staining to determine the protein levels of TNFR1, TRADD, TRAF2, IKK and p65.
Figure Legend Snippet: NSM00191 significantly improved the inflammatory response in vivo . (A-B) NSM00191 significantly inhibited the expression of NF-κB downstream targets. The DBA/1 mice ( A , n=12) and TNF-α-overexpressing mice ( B , n=12) were divided into two groups. One group of mice was treated with DMSO; the other group mice were treated with 20 μM NSM00191 for 7 days. Then, the joint tissues were collected, and their RNAs were isolated. The RNAs from each treatment mice were equally combined and subjected into qRT-PCR analyses to examine the expression of TNF-α, IL-6, MCP-1, CCL5, COX-2, VCAM-1, CD40 and MHC-1. (C) NSM00191 inhibited the interaction between TRADD and TRAF2 in vivo . The joint samples used in A and B were subjected to Western blot analysis to determine the protein levels of TNFR1, TRADD, TRAF2, IKK2 and p65. GAPDH was used as a loading control. (D) NSM00191 decreased the p65 levels in vivo. One pair of joint samples used in A and B were subjected to IHC staining to determine the protein levels of TNFR1, TRADD, TRAF2, IKK and p65.

Techniques Used: In Vivo, Expressing, Mouse Assay, Isolation, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining

The inflammatory targets of NF-κB were activated in p65-overexpressing cells. The heat maps of the consistently altered genes in p65-overexpressing and knockdown cells are shown. The RNA from SW982 cells, SW982 cells overexpressing p65 (S-OE), SW982 cells with knockdown of p65 (S-KD), HFLS-RA cells (HFLS), HFLS-RA cells overexpressing p65 (H-OE), and HFLS-RA cells with knockdown of p65 (H-KD) were subjected to microarray analysis. The elevated genes are indicated in red, and the downregulated genes are shown in green. qRT-PCR was performed to verify the expression of TNF-α (B) , IL-6 (C) , COX-2 (D) , IRF-1 (E) , CCL5 (F) , and CCL20 (G) in SW982, S-OE, S-KD, HFLS, H-OE and H-KD cells. * P
Figure Legend Snippet: The inflammatory targets of NF-κB were activated in p65-overexpressing cells. The heat maps of the consistently altered genes in p65-overexpressing and knockdown cells are shown. The RNA from SW982 cells, SW982 cells overexpressing p65 (S-OE), SW982 cells with knockdown of p65 (S-KD), HFLS-RA cells (HFLS), HFLS-RA cells overexpressing p65 (H-OE), and HFLS-RA cells with knockdown of p65 (H-KD) were subjected to microarray analysis. The elevated genes are indicated in red, and the downregulated genes are shown in green. qRT-PCR was performed to verify the expression of TNF-α (B) , IL-6 (C) , COX-2 (D) , IRF-1 (E) , CCL5 (F) , and CCL20 (G) in SW982, S-OE, S-KD, HFLS, H-OE and H-KD cells. * P

Techniques Used: Microarray, Quantitative RT-PCR, Expressing

miR-7-5p was significantly downregulated in RA patients. (A) The heat maps of the consistently altered miRNAs in RA patients are shown. RNA samples from three paired joint tissue specimens (HC-4, -5, -6 and RA-4, 5, -6) were subjected to miRNA microarray analysis. The elevated miRNAs are indicated in red, and the downregulated miRNAs are shown in green. qRT-PCR was performed to verify the expression of miR-7-5p (B) , miR-124a (C) , miR-338 (D) , miR-568 (E) , miR-575 (F) , miR-148b (G) , miR-177 (H) , miR-198 (I) and miR-600 (J) in the RNA samples used in A. * P
Figure Legend Snippet: miR-7-5p was significantly downregulated in RA patients. (A) The heat maps of the consistently altered miRNAs in RA patients are shown. RNA samples from three paired joint tissue specimens (HC-4, -5, -6 and RA-4, 5, -6) were subjected to miRNA microarray analysis. The elevated miRNAs are indicated in red, and the downregulated miRNAs are shown in green. qRT-PCR was performed to verify the expression of miR-7-5p (B) , miR-124a (C) , miR-338 (D) , miR-568 (E) , miR-575 (F) , miR-148b (G) , miR-177 (H) , miR-198 (I) and miR-600 (J) in the RNA samples used in A. * P

Techniques Used: Microarray, Quantitative RT-PCR, Expressing

54) Product Images from "Heterochromatin Formation Promotes Longevity and Represses Ribosomal RNA Synthesis"

Article Title: Heterochromatin Formation Promotes Longevity and Represses Ribosomal RNA Synthesis

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002473

Heterochromatin controls rRNA transcription. (A) Schematic representation of a pre-rRNA transcript, with an R2 element inserted into the 28S gene. Arrows above the 5′ETS region represent PCR primers used for qPCR analysis. (B) RNA was isolated from 3 rd instar larvae of indicated genotypes and was subjected to Northern blotting with an R2 5′ antisense probe. Transcripts from the Adh gene were used as a loading control. Levels of the transcripts were quantified with a phospho-imager. The full-length (FL) R2 transcript is 3.6 kb. The lower bands are all degradation products, which appear soon after transcription [42] . A density plot is shown to the right with arbitrary units (a.u.). (C) The levels of pre-rRNA in 2-day-old male flies of indicated genotypes were measured by qRT-PCR. Relative pre-rRNA levels are shown with standard deviations. (D) Representative larvae of indicated genotypes. Su(var)205 −/− were transheterozygous for Su(var)205 2 and Su(var)205 5 . (E) Flies of indicated genotypes were outcrossed to minimize genetic background effects and were raised in parallel at 25°C with similar larval density. Top: Representative male flies of indicated genotypes. Bottom: The fly body weight was measured as the average of 10 2-day old male flies. Su(var)205 2/+ and Su(var)205 5/+ male flies had similar body weights, the results were combined and shown as Su(var)205 +/− . Standard deviations and p values (compared with wild-type control; Student's t -Test) are shown.
Figure Legend Snippet: Heterochromatin controls rRNA transcription. (A) Schematic representation of a pre-rRNA transcript, with an R2 element inserted into the 28S gene. Arrows above the 5′ETS region represent PCR primers used for qPCR analysis. (B) RNA was isolated from 3 rd instar larvae of indicated genotypes and was subjected to Northern blotting with an R2 5′ antisense probe. Transcripts from the Adh gene were used as a loading control. Levels of the transcripts were quantified with a phospho-imager. The full-length (FL) R2 transcript is 3.6 kb. The lower bands are all degradation products, which appear soon after transcription [42] . A density plot is shown to the right with arbitrary units (a.u.). (C) The levels of pre-rRNA in 2-day-old male flies of indicated genotypes were measured by qRT-PCR. Relative pre-rRNA levels are shown with standard deviations. (D) Representative larvae of indicated genotypes. Su(var)205 −/− were transheterozygous for Su(var)205 2 and Su(var)205 5 . (E) Flies of indicated genotypes were outcrossed to minimize genetic background effects and were raised in parallel at 25°C with similar larval density. Top: Representative male flies of indicated genotypes. Bottom: The fly body weight was measured as the average of 10 2-day old male flies. Su(var)205 2/+ and Su(var)205 5/+ male flies had similar body weights, the results were combined and shown as Su(var)205 +/− . Standard deviations and p values (compared with wild-type control; Student's t -Test) are shown.

Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Isolation, Northern Blot, Quantitative RT-PCR

55) Product Images from "Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate"

Article Title: Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Journal: Oncotarget

doi:

Effects of UA and a series of related triterpenes found in P. frutescens on TPA-induced inflammatory gene expression Female ICR mice (7–9 weeks of age; n = 4/group) were shaved on the dorsal skin and the two days later pretreated with acetone vehicle (0.2 ml), UA (2 μmol), OA (2 μmol), AA (2 umol), CA (2 μmol), 3-epiCA (2 μmol), MA (2 μmol) or 3-epiMA (2 μmol) before TPA treatment. All treatments were given twice-weekly for two weeks. Mice were sacrificed 6 hrs after the last TPA treatment, and epidermal RNA was isolated to be subjected to qRT-PCR analysis. mRNA levels of Cox-2, Il17a, Il22, Cxcl1, Cxcl2, and Vegfa were normalized to Gapdh. The graphs represent mean ± SEM. * p ≤ 0.05 when compared to acetone-treated group; ** p ≤ 0.05 when compared to TPA-treated group; and # p ≤ 0.05 when compared to UA + TPA-treated group. Mann-Whitney U test was used for statistical analysis.
Figure Legend Snippet: Effects of UA and a series of related triterpenes found in P. frutescens on TPA-induced inflammatory gene expression Female ICR mice (7–9 weeks of age; n = 4/group) were shaved on the dorsal skin and the two days later pretreated with acetone vehicle (0.2 ml), UA (2 μmol), OA (2 μmol), AA (2 umol), CA (2 μmol), 3-epiCA (2 μmol), MA (2 μmol) or 3-epiMA (2 μmol) before TPA treatment. All treatments were given twice-weekly for two weeks. Mice were sacrificed 6 hrs after the last TPA treatment, and epidermal RNA was isolated to be subjected to qRT-PCR analysis. mRNA levels of Cox-2, Il17a, Il22, Cxcl1, Cxcl2, and Vegfa were normalized to Gapdh. The graphs represent mean ± SEM. * p ≤ 0.05 when compared to acetone-treated group; ** p ≤ 0.05 when compared to TPA-treated group; and # p ≤ 0.05 when compared to UA + TPA-treated group. Mann-Whitney U test was used for statistical analysis.

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

56) Product Images from "Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells"

Article Title: Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells

Journal: American Journal of Cancer Research

doi:

FOXO3a binds to the promoter of PUMA and positively regulates its expression. (A) FOXO3a is not able to directly bind to the promoters of the genes involved in autophagic signaling. MG63-R12 and MG63-R12+FOXO3a cells were subjected to a ChIP assay using the FOXO3a antibody, and the binding of autophagic signaling genes, including LC3, SIRT1, Beclin-1, ATG5 and ATG7 , was assessed by qRT-PCR. The expression levels of each gene were normalized against the control vector. (B) FOXO3a specifically binds to the promoter of PUMA . The same DNA in (A) was used to examine the binding of apoptotic signaling genes, including p53, PUMA, Bax, Caspase-3 and Apaf-1 . (C) A schematic diagram of the PUMA promoter. The promoter region (-1 - -1500) of PUMA is indicated, and the consensus sequence of the FOXO3a binding site is indicated in red. The mutant FOXO3a binding site is indicated in blue. (D) Luciferase assay. MG63-R12, MG63-R12+FOXO3a, U2OS-R5, and U2OS-R5+FOXO3a cells were co-transfected the firefly luciferase reporter vector pGL4.26-P PUMA-WT -Luc or pGL4.26-P PUMA-Mutant -Luc with the Renilla luciferase vector pRL-TK, respectively. After incubation at 37°C for 24 hrs, the cells were subjected to a luciferase assay using the Dual Luciferase Reporter Assay System. The luciferase activities in MG63-R12+FOXO3a and U2OS-R5+FOXO3a cells were normalized against MG63-R12 and U2OS-R5 cells, respectively (** P
Figure Legend Snippet: FOXO3a binds to the promoter of PUMA and positively regulates its expression. (A) FOXO3a is not able to directly bind to the promoters of the genes involved in autophagic signaling. MG63-R12 and MG63-R12+FOXO3a cells were subjected to a ChIP assay using the FOXO3a antibody, and the binding of autophagic signaling genes, including LC3, SIRT1, Beclin-1, ATG5 and ATG7 , was assessed by qRT-PCR. The expression levels of each gene were normalized against the control vector. (B) FOXO3a specifically binds to the promoter of PUMA . The same DNA in (A) was used to examine the binding of apoptotic signaling genes, including p53, PUMA, Bax, Caspase-3 and Apaf-1 . (C) A schematic diagram of the PUMA promoter. The promoter region (-1 - -1500) of PUMA is indicated, and the consensus sequence of the FOXO3a binding site is indicated in red. The mutant FOXO3a binding site is indicated in blue. (D) Luciferase assay. MG63-R12, MG63-R12+FOXO3a, U2OS-R5, and U2OS-R5+FOXO3a cells were co-transfected the firefly luciferase reporter vector pGL4.26-P PUMA-WT -Luc or pGL4.26-P PUMA-Mutant -Luc with the Renilla luciferase vector pRL-TK, respectively. After incubation at 37°C for 24 hrs, the cells were subjected to a luciferase assay using the Dual Luciferase Reporter Assay System. The luciferase activities in MG63-R12+FOXO3a and U2OS-R5+FOXO3a cells were normalized against MG63-R12 and U2OS-R5 cells, respectively (** P

Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Plasmid Preparation, Sequencing, Mutagenesis, Luciferase, Transfection, Incubation, Reporter Assay

57) Product Images from "GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞"

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M707117200

FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.
Figure Legend Snippet: FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.

Techniques Used: Expressing, Quantitative RT-PCR, Activity Assay, Transduction, Infection, Western Blot, Transfection, Plasmid Preparation

58) Product Images from "Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants 1. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants 1 [OA]"

Article Title: Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants 1. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants 1 [OA]

Journal: Plant Physiology

doi: 10.1104/pp.106.090894

RNA Isolation and qRT-PCR Analysis
Figure Legend Snippet: RNA Isolation and qRT-PCR Analysis

Techniques Used: Isolation, Quantitative RT-PCR

59) Product Images from "Placenta Growth Factor (PlGF), a Novel Inducer of Plasminogen Activator Inhibitor-1 (PAI-1) in Sickle Cell Disease (SCD) *"

Article Title: Placenta Growth Factor (PlGF), a Novel Inducer of Plasminogen Activator Inhibitor-1 (PAI-1) in Sickle Cell Disease (SCD) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.101691

PAI-1 expression in mice and human subjects. A and B , plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi , hemizygous Berkeley mice; Berk-SS , Berkeley SS mice. C , plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D , immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown ; nuclei are stained red ). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E , the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F , qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects ( n = 9) and normal controls ( n = 9). RQ , relative quantification. G and H , HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G , total RNA was subjected to RPA for the expression of indicated genes. H , the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p
Figure Legend Snippet: PAI-1 expression in mice and human subjects. A and B , plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi , hemizygous Berkeley mice; Berk-SS , Berkeley SS mice. C , plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D , immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown ; nuclei are stained red ). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E , the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F , qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects ( n = 9) and normal controls ( n = 9). RQ , relative quantification. G and H , HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G , total RNA was subjected to RPA for the expression of indicated genes. H , the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p

Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Recombinant, Recombinase Polymerase Amplification

60) Product Images from "TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis"

Article Title: TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis

Journal: BMC Plant Biology

doi: 10.1186/s12870-015-0644-9

Expression pattern of relevant genes ( a RD29b , b SAG13 , c SAG113 , d AIB1 , e ERD11 , and f ABI5 ) in WT and TaNAC29 -overexpression (OE) plants. Seedlings of WT and OE were treated with 250 mM salt stress for 10 d and drought stress for 17 d, respectively. Total RNAs were extracted from leaves, and qRT-PCR analysis was performed. The 2 −ΔΔCT method was used in qRT-PCR analysis. Values are means ± SE of three replicates. Asterisks indicate statistically significant differences from WT (* P
Figure Legend Snippet: Expression pattern of relevant genes ( a RD29b , b SAG13 , c SAG113 , d AIB1 , e ERD11 , and f ABI5 ) in WT and TaNAC29 -overexpression (OE) plants. Seedlings of WT and OE were treated with 250 mM salt stress for 10 d and drought stress for 17 d, respectively. Total RNAs were extracted from leaves, and qRT-PCR analysis was performed. The 2 −ΔΔCT method was used in qRT-PCR analysis. Values are means ± SE of three replicates. Asterisks indicate statistically significant differences from WT (* P

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

Expression patterns of TaNAC29 in wheat after stress treatments. Expression patterns of TaNAC29 in wheat leaves and roots after NaCl, PEG6000, ABA and H 2 O 2 treatments by qRT-PCR analysis. Leaf and root were collected after different stress treatment. The 2 −ΔΔCT method was used in qRT-PCR analysis. Transcript levels were normalized to TaActin . Values are means ± SE of three replicates. Asterisks indicate statistically significant differences from mock (* P
Figure Legend Snippet: Expression patterns of TaNAC29 in wheat after stress treatments. Expression patterns of TaNAC29 in wheat leaves and roots after NaCl, PEG6000, ABA and H 2 O 2 treatments by qRT-PCR analysis. Leaf and root were collected after different stress treatment. The 2 −ΔΔCT method was used in qRT-PCR analysis. Transcript levels were normalized to TaActin . Values are means ± SE of three replicates. Asterisks indicate statistically significant differences from mock (* P

Techniques Used: Expressing, Quantitative RT-PCR

61) Product Images from "Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1"

Article Title: Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

Journal: Nature Communications

doi: 10.1038/ncomms11150

Differential miR21 expression in ovarian tumour tissue and cancer cell lines. ( a ) qRT–PCR analysis of miR21 expression in cultured normal ovarian fibroblasts, CAFs, normal adipocytes, CAAs and ovarian cancer cell lines. ( b ) qRT–PCR analysis of miR21 expression in microdissected normal adipocytes ( n =5), CAAs ( n =9), normal fibroblasts ( n =5), CAFs ( n =5) and ovarian cancer cells from omental ( n =10) and primary ovarian sites ( n =10). *** P
Figure Legend Snippet: Differential miR21 expression in ovarian tumour tissue and cancer cell lines. ( a ) qRT–PCR analysis of miR21 expression in cultured normal ovarian fibroblasts, CAFs, normal adipocytes, CAAs and ovarian cancer cell lines. ( b ) qRT–PCR analysis of miR21 expression in microdissected normal adipocytes ( n =5), CAAs ( n =9), normal fibroblasts ( n =5), CAFs ( n =5) and ovarian cancer cells from omental ( n =10) and primary ovarian sites ( n =10). *** P

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

Increased miR21 expression in exosomes isolated from CAAs. ( a ) Electron micrograph showing whole-mount exosomes isolated from CAA-conditioned medium. Scale bar, 100 nm. ( b ) Histogram showing the particle diameter (nm) of the population of small vesicles collected from CAA-conditioned medium using the standard exosome isolation method of ultracentrifugation and quantified using qNano. The amount of exosomes secreted into the conditioned media was normalized with the number of cells in the culture for conditioned media collection. The amount of miR-21 in the exosomes was then normalized with the input amount of RNA for reverse transcription and the subsequent qRT–PCR analysis, to determine the relative miR-21 expression level in exosomes. ( c ) Western blot analysis showing exosome marker CD63 in the exosome-enriched conditioned medium but not in the cell lysate. In contrast, cis -Golgi matrix protein (GM130) was only observed in the cell lysate and not in the exosome fraction. Another exosome marker, 70-kDa heat shock protein (HSP70), was also observed in the exosomes. ( d ) The heat map showing the relative expression of small RNAs in exosomes isolated from ovarian cancer cell lines ( n =4), normal ovarian fibroblasts ( n =2), CAFs ( n =3), normal adipocytes ( n =2) and CAAs ( n =2). ( e , f ) Relative normalized expression of mature 5′, mature 5′ super variant and precursor variant of miR21 in exosomes isolated from ovarian cancer cell lines ( n =4), normal ovarian fibroblasts ( n =2), CAFs ( n =3), normal adipocytes ( n =2) and CAAs ( n =2) examined using miRNA-sequencing ( e ) and qRT–PCR ( f ) analyses.
Figure Legend Snippet: Increased miR21 expression in exosomes isolated from CAAs. ( a ) Electron micrograph showing whole-mount exosomes isolated from CAA-conditioned medium. Scale bar, 100 nm. ( b ) Histogram showing the particle diameter (nm) of the population of small vesicles collected from CAA-conditioned medium using the standard exosome isolation method of ultracentrifugation and quantified using qNano. The amount of exosomes secreted into the conditioned media was normalized with the number of cells in the culture for conditioned media collection. The amount of miR-21 in the exosomes was then normalized with the input amount of RNA for reverse transcription and the subsequent qRT–PCR analysis, to determine the relative miR-21 expression level in exosomes. ( c ) Western blot analysis showing exosome marker CD63 in the exosome-enriched conditioned medium but not in the cell lysate. In contrast, cis -Golgi matrix protein (GM130) was only observed in the cell lysate and not in the exosome fraction. Another exosome marker, 70-kDa heat shock protein (HSP70), was also observed in the exosomes. ( d ) The heat map showing the relative expression of small RNAs in exosomes isolated from ovarian cancer cell lines ( n =4), normal ovarian fibroblasts ( n =2), CAFs ( n =3), normal adipocytes ( n =2) and CAAs ( n =2). ( e , f ) Relative normalized expression of mature 5′, mature 5′ super variant and precursor variant of miR21 in exosomes isolated from ovarian cancer cell lines ( n =4), normal ovarian fibroblasts ( n =2), CAFs ( n =3), normal adipocytes ( n =2) and CAAs ( n =2) examined using miRNA-sequencing ( e ) and qRT–PCR ( f ) analyses.

Techniques Used: Expressing, Isolation, Cellular Antioxidant Activity Assay, Quantitative RT-PCR, Western Blot, Marker, Variant Assay, Sequencing

62) Product Images from "Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver"

Article Title: Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver

Journal: Xenotransplantation

doi: 10.1111/xen.12512

Albumin production and gene expression. A, Expression of albumin gene was examined for each condition by qRT-PCR after24, 48, and 72 h. Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. B, The concentration of porcine albumin was determined by enzyme-linked immunosorbent assay (ELISA) for each condition after 24, 48, and 72 h. # P
Figure Legend Snippet: Albumin production and gene expression. A, Expression of albumin gene was examined for each condition by qRT-PCR after24, 48, and 72 h. Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. B, The concentration of porcine albumin was determined by enzyme-linked immunosorbent assay (ELISA) for each condition after 24, 48, and 72 h. # P

Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Ammonia clearance and expression of liver-specific genes. A, Detoxification of ammonia for each cold storage conditionafter 24, 48, and 72 h. B, Expressions of the six genes of the urea cycle including arginase 1 (Arg1), argininosuccinate synthase 1 (Ass1), argininosuccinate lyase (Asl), carbamoyl phosphate synthase 1 (Cps1), N -acetylglutamate synthase (Nags), and ornithine transcarbamylase (Otc) were examined by qRT-PCR for each group after 24, 48, and 72 h. Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. C, Expressions of liver-related genes including Cyp1a2, Cyp2e1, and hepatocyte nuclear factor 4 (Hnf4) were examined by qRT-PCR for each group after 24, 48, and 72 h Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. D, The slope of ammonia clearance in hepatocyte spheroids was calculated. Clearance slope = C Final − C lnitial t # P
Figure Legend Snippet: Ammonia clearance and expression of liver-specific genes. A, Detoxification of ammonia for each cold storage conditionafter 24, 48, and 72 h. B, Expressions of the six genes of the urea cycle including arginase 1 (Arg1), argininosuccinate synthase 1 (Ass1), argininosuccinate lyase (Asl), carbamoyl phosphate synthase 1 (Cps1), N -acetylglutamate synthase (Nags), and ornithine transcarbamylase (Otc) were examined by qRT-PCR for each group after 24, 48, and 72 h. Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. C, Expressions of liver-related genes including Cyp1a2, Cyp2e1, and hepatocyte nuclear factor 4 (Hnf4) were examined by qRT-PCR for each group after 24, 48, and 72 h Control group 0-h values served as calibrators to determine the relative expression of gene at each time point and group. D, The slope of ammonia clearance in hepatocyte spheroids was calculated. Clearance slope = C Final − C lnitial t # P

Techniques Used: Expressing, Quantitative RT-PCR

63) Product Images from "In Planta Preliminary Screening of ER Glycoprotein Folding Quality Control (ERQC) Modulators"

Article Title: In Planta Preliminary Screening of ER Glycoprotein Folding Quality Control (ERQC) Modulators

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19072135

Transcription of defense genes. Arabidopsis thaliana WT (wild-type, empty bars), WT grown in the presence of N B-DNJ (70 µM, light grey bars) and psl5-1 (dark grey bars) 10-day-old seedlings (20 seedlings in each experiment) were treated with water or elf18 and flg22 elicitors. Transcription of PHI1 and RET-OX genes were determined by qRT-PCR 30 min after treatment. Transcription levels are shown as the mean of at least three independent experiments (± s.e.; n = 3) normalized to UBQ5 (ubiquitin 5) used as a reference. In both ( a ) and ( b ), asterisks indicate statistically significant differences between elf18-treated seedlings (WT + N B-DNJ and psl5-1 ) and corresponding treatment of the wild type according to Student’s t -test (3 asterisks = p
Figure Legend Snippet: Transcription of defense genes. Arabidopsis thaliana WT (wild-type, empty bars), WT grown in the presence of N B-DNJ (70 µM, light grey bars) and psl5-1 (dark grey bars) 10-day-old seedlings (20 seedlings in each experiment) were treated with water or elf18 and flg22 elicitors. Transcription of PHI1 and RET-OX genes were determined by qRT-PCR 30 min after treatment. Transcription levels are shown as the mean of at least three independent experiments (± s.e.; n = 3) normalized to UBQ5 (ubiquitin 5) used as a reference. In both ( a ) and ( b ), asterisks indicate statistically significant differences between elf18-treated seedlings (WT + N B-DNJ and psl5-1 ) and corresponding treatment of the wild type according to Student’s t -test (3 asterisks = p

Techniques Used: Quantitative RT-PCR

64) Product Images from "Uev1A-Ubc13 promotes colorectal cancer metastasis through regulating CXCL1 expression via NF-кB activation"

Article Title: Uev1A-Ubc13 promotes colorectal cancer metastasis through regulating CXCL1 expression via NF-кB activation

Journal: Oncotarget

doi: 10.18632/oncotarget.24640

Uev1A positively regulates CXCL1 and MMP9 expression (A) Transcript levels of selected putative NF-κB target genes in HCT116-TR cells expressing different UEV s as determined by qRT-PCR. (B) Elevated CXCL1 and MMP9 protein levels in UEV1A -overexpressed HCT116 cells as determined by western blot. Actin serves as a loading control and HA-tag shows ectopic UEV1A overexpression. (C) Relative transcript levels of CXCL1 and MMP9 in two independent shUEV1-transfected HCT116 cell lines as determined by qRT-PCR. (D) Relative protein levels of CXCL1 and MMP9 in two independent shUEV1-transfected HCT116 cell lines as determined by western blot. ** P
Figure Legend Snippet: Uev1A positively regulates CXCL1 and MMP9 expression (A) Transcript levels of selected putative NF-κB target genes in HCT116-TR cells expressing different UEV s as determined by qRT-PCR. (B) Elevated CXCL1 and MMP9 protein levels in UEV1A -overexpressed HCT116 cells as determined by western blot. Actin serves as a loading control and HA-tag shows ectopic UEV1A overexpression. (C) Relative transcript levels of CXCL1 and MMP9 in two independent shUEV1-transfected HCT116 cell lines as determined by qRT-PCR. (D) Relative protein levels of CXCL1 and MMP9 in two independent shUEV1-transfected HCT116 cell lines as determined by western blot. ** P

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

Manipulation of UEV expression levels in HCT116 cells cDNA4.0/TO/HA(+) vector expressing UEV1A , UEV1C , MMS2, mutated UEV1A ( 1A-F38E ) or vector only (CK) was stably transfected into HCT116-TR cells, with or without Dox treatment. The level of ectopic gene expression was monitored (A) by qRT-PCR and (B) by western blot against an anti-HA antibody. (C) UEV1A and UEV1C transcript levels in shCK and shUEV1 lines were determined by qRT-PCR. HCT116 cells were transfected with shRNA lentiviral particles against UEV1 (sh UEV1 ) or non-specific target (shCK). 20 single colonies were picked and subcultured. shUEV1-1, shUEV1-2 and shUEV1-5 represent three independent stable shUEV1 lines. (D) Uev1C and Mms2 levels in shCK and shUEV1 lines were determined by western blot against anti-Uev1 (LN2B) and MMS2+Uev1 (LN3) antibodies. * P
Figure Legend Snippet: Manipulation of UEV expression levels in HCT116 cells cDNA4.0/TO/HA(+) vector expressing UEV1A , UEV1C , MMS2, mutated UEV1A ( 1A-F38E ) or vector only (CK) was stably transfected into HCT116-TR cells, with or without Dox treatment. The level of ectopic gene expression was monitored (A) by qRT-PCR and (B) by western blot against an anti-HA antibody. (C) UEV1A and UEV1C transcript levels in shCK and shUEV1 lines were determined by qRT-PCR. HCT116 cells were transfected with shRNA lentiviral particles against UEV1 (sh UEV1 ) or non-specific target (shCK). 20 single colonies were picked and subcultured. shUEV1-1, shUEV1-2 and shUEV1-5 represent three independent stable shUEV1 lines. (D) Uev1C and Mms2 levels in shCK and shUEV1 lines were determined by western blot against anti-Uev1 (LN2B) and MMS2+Uev1 (LN3) antibodies. * P

Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Quantitative RT-PCR, Western Blot, shRNA

UEV1A is overexpressed in human colon cancer cell lines and tumor samples (A) Relative transcript levels of UEV1A and UEV1C variants in human colon cancer cell lines as determined by qRT-PCR.( * P
Figure Legend Snippet: UEV1A is overexpressed in human colon cancer cell lines and tumor samples (A) Relative transcript levels of UEV1A and UEV1C variants in human colon cancer cell lines as determined by qRT-PCR.( * P

Techniques Used: Quantitative RT-PCR

65) Product Images from "MicroRNA-300 Regulates the Ubiquitination of PTEN through the CRL4BDCAF13 E3 Ligase in Osteosarcoma Cells"

Article Title: MicroRNA-300 Regulates the Ubiquitination of PTEN through the CRL4BDCAF13 E3 Ligase in Osteosarcoma Cells

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.12.010

Expression of miR-300 Was Downregulated in Osteosarcoma Cell Lines and in the Majority of Cancerous Tissues from Patients with Osteosarcoma (A and B) Both the CUL4B protein (A) and mRNA (B) levels were upregulated in osteosarcoma cell lines. The hFOB1.19, U2OS, Saos-2, MG63, and HOS cell lines were subjected to immunoblotting and qRT-PCR analyses to determine CUL4B protein and mRNA levels. (C and D) Expression of 20 miRNAs that were predicted to target the 3′ UTR of CUL4B in hFOB1.19, U2OS and MG63 cells. (C) miR-4251, miR-4531, miR-3977, miR-6830, miR-4659a, miR-545, miR-153, miR-4659b, miR-561 and miR-381. (D) miR-300, miR-4776, miR-6885, miR-7159, miR-8060, miR-3691, miR-3664, miR-4451, miR-3671 and miR-4482. (E and F) Expression of miR-300 in osteosarcoma cancerous tissues is shown. Relative expression of miR-300 in osteosarcoma tumors (n = 48) was normalized to corresponding adjacent normal tissues (n = 48). (E) Log2 fold change; (F) absolute fold change. p
Figure Legend Snippet: Expression of miR-300 Was Downregulated in Osteosarcoma Cell Lines and in the Majority of Cancerous Tissues from Patients with Osteosarcoma (A and B) Both the CUL4B protein (A) and mRNA (B) levels were upregulated in osteosarcoma cell lines. The hFOB1.19, U2OS, Saos-2, MG63, and HOS cell lines were subjected to immunoblotting and qRT-PCR analyses to determine CUL4B protein and mRNA levels. (C and D) Expression of 20 miRNAs that were predicted to target the 3′ UTR of CUL4B in hFOB1.19, U2OS and MG63 cells. (C) miR-4251, miR-4531, miR-3977, miR-6830, miR-4659a, miR-545, miR-153, miR-4659b, miR-561 and miR-381. (D) miR-300, miR-4776, miR-6885, miR-7159, miR-8060, miR-3691, miR-3664, miR-4451, miR-3671 and miR-4482. (E and F) Expression of miR-300 in osteosarcoma cancerous tissues is shown. Relative expression of miR-300 in osteosarcoma tumors (n = 48) was normalized to corresponding adjacent normal tissues (n = 48). (E) Log2 fold change; (F) absolute fold change. p

Techniques Used: Expressing, Quantitative RT-PCR

miR-300 Directly Targeted CUL4B (A) Schematic representation of the 3′ UTR of CUL4B shows a putative miR-300 binding site. The seed location for WT 3′ UTR of CUL4B is indicated in red, whereas the mutant 3′ UTR is indicated in blue. (B) The expression of miR-300 was decreased in osteosarcoma cell lines. The hFOB1.19, U2OS, Saos-2, MG63, and HOS cells were subjected to qRT-PCR to determine the miR-300 level. RNU6B was selected as an internal control for normalization, and the resulting ratio in hFOB1.19 cells was arbitrarily defined as 1-fold. (C) Detection of the overexpression of miR-300 in hFOB1.19, U2OS, and MG63 cells. The miR-300-mimic or its negative control miR-NC was transfected into hFOB1.19, U2OS, and MG63 cells for 24 hr. Expression of miR-300 was determined by qRT-PCR. (D and E) Overexpression of miR-300 caused the downregulation of CUL4B at both the mRNA and protein levels. Cells used in (C) were used to determine the expression of CUL4B by qRT-PCR (D) and western blot (E). (F) The CUL4B 3′ UTR mutant failed to bind miR-300. The miR-300-mimic or miR-NC was co-transfected with pMIR-CUL4B-3 ′ -UTR-WT or pMIR-CUL4B-3 ′ -UTR mutant into U2OS cells for 24 hr. Luciferase activity was quantified using the dual-luciferase assay reporter system by normalizing to Renilla activity. (G) Overexpression of miR-300 inhibited osteosarcoma cell proliferation. Cells used in (C) were subject to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to evaluate the degree of cell proliferation, and cell viability was determined at 490 nm. (H) Overexpression of miR-300 inhibited osteosarcoma cell colony formation ability. Cells used in (C) were seeded onto six-well plates with a density of 1 × 10 3 cells/well and cultured with 0.1 mL of DMEM medium for 2 weeks. Then, the cells were stained with 0.5% crystal violet, and the number of colonies was counted. (I) Overexpression of miR-300 inhibited osteosarcoma cell invasion. Cells used in (C) were used to determine invasion ability with Boyden chamber assays. *p
Figure Legend Snippet: miR-300 Directly Targeted CUL4B (A) Schematic representation of the 3′ UTR of CUL4B shows a putative miR-300 binding site. The seed location for WT 3′ UTR of CUL4B is indicated in red, whereas the mutant 3′ UTR is indicated in blue. (B) The expression of miR-300 was decreased in osteosarcoma cell lines. The hFOB1.19, U2OS, Saos-2, MG63, and HOS cells were subjected to qRT-PCR to determine the miR-300 level. RNU6B was selected as an internal control for normalization, and the resulting ratio in hFOB1.19 cells was arbitrarily defined as 1-fold. (C) Detection of the overexpression of miR-300 in hFOB1.19, U2OS, and MG63 cells. The miR-300-mimic or its negative control miR-NC was transfected into hFOB1.19, U2OS, and MG63 cells for 24 hr. Expression of miR-300 was determined by qRT-PCR. (D and E) Overexpression of miR-300 caused the downregulation of CUL4B at both the mRNA and protein levels. Cells used in (C) were used to determine the expression of CUL4B by qRT-PCR (D) and western blot (E). (F) The CUL4B 3′ UTR mutant failed to bind miR-300. The miR-300-mimic or miR-NC was co-transfected with pMIR-CUL4B-3 ′ -UTR-WT or pMIR-CUL4B-3 ′ -UTR mutant into U2OS cells for 24 hr. Luciferase activity was quantified using the dual-luciferase assay reporter system by normalizing to Renilla activity. (G) Overexpression of miR-300 inhibited osteosarcoma cell proliferation. Cells used in (C) were subject to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to evaluate the degree of cell proliferation, and cell viability was determined at 490 nm. (H) Overexpression of miR-300 inhibited osteosarcoma cell colony formation ability. Cells used in (C) were seeded onto six-well plates with a density of 1 × 10 3 cells/well and cultured with 0.1 mL of DMEM medium for 2 weeks. Then, the cells were stained with 0.5% crystal violet, and the number of colonies was counted. (I) Overexpression of miR-300 inhibited osteosarcoma cell invasion. Cells used in (C) were used to determine invasion ability with Boyden chamber assays. *p

Techniques Used: Binding Assay, Mutagenesis, Expressing, Quantitative RT-PCR, Over Expression, Negative Control, Transfection, Western Blot, Luciferase, Activity Assay, MTT Assay, Cell Culture, Staining

DNA Hypermethylation Caused the Downregulation of miR-300 in Osteosarcoma Cells (A) The promoter region of miR-300 contained one CpG island. CpG island prediction was performed using a database ( http://www.urogene.org ). The predicted CpG island and the miR-300 locus are indicated. (B and C) Effects of AZA and TSA on miR-300 expression in the U2OS and MG63 cell lines. U2OS (B) and MG63 (C) cells were primarily treated with DMSO, AZA (1 μM), or TSA (300 nM), followed by determination of miR-300 expression via qRT-PCR. (D and E) Effects of AZA and TSA on CUL4B expression in (D) U2OS and (E) MG63 cells. The cells used in (A) and (B) were used to determine the expression of CUL4B by qRT-PCR. (F and G) AZA treatment caused the inhibition of osteosarcoma cell proliferation. U2OS (F) and MG63 (G) cells were treated with DMSO or AZA (1 μM), followed by the determination of cell viability at 490 nm. (H) Expression levels of the methylated CpG island with or without AZA treatment. The hFOB1.19, U2OS, and MG63 cell lines were treated with or without 1 μM AZA; then the expression levels of the methylated CpG island were determined by qMSP analysis. *p
Figure Legend Snippet: DNA Hypermethylation Caused the Downregulation of miR-300 in Osteosarcoma Cells (A) The promoter region of miR-300 contained one CpG island. CpG island prediction was performed using a database ( http://www.urogene.org ). The predicted CpG island and the miR-300 locus are indicated. (B and C) Effects of AZA and TSA on miR-300 expression in the U2OS and MG63 cell lines. U2OS (B) and MG63 (C) cells were primarily treated with DMSO, AZA (1 μM), or TSA (300 nM), followed by determination of miR-300 expression via qRT-PCR. (D and E) Effects of AZA and TSA on CUL4B expression in (D) U2OS and (E) MG63 cells. The cells used in (A) and (B) were used to determine the expression of CUL4B by qRT-PCR. (F and G) AZA treatment caused the inhibition of osteosarcoma cell proliferation. U2OS (F) and MG63 (G) cells were treated with DMSO or AZA (1 μM), followed by the determination of cell viability at 490 nm. (H) Expression levels of the methylated CpG island with or without AZA treatment. The hFOB1.19, U2OS, and MG63 cell lines were treated with or without 1 μM AZA; then the expression levels of the methylated CpG island were determined by qMSP analysis. *p

Techniques Used: Expressing, Quantitative RT-PCR, Inhibition, Methylation

66) Product Images from "High-glucose-induced miR-214-3p inhibits BMSCs osteogenic differentiation in type 1 diabetes mellitus"

Article Title: High-glucose-induced miR-214-3p inhibits BMSCs osteogenic differentiation in type 1 diabetes mellitus

Journal: Cell Death Discovery

doi: 10.1038/s41420-019-0223-1

High-glucose-induced miR-214-3p functionally targets β-catenin. a Schematic diagram illustrating the design of luciferase reporters with the β-catenin 3′-UTR. The sequences of miR-214-3p and miR-214-3p-Mut are also shown. b The effect of AgomiR-214-3p, AgomiR-N.C and AgomiR-214-3p-Mut on luciferase activity in BMSCs after transfection with the β-catenin 3′-UTR reporter ( n = 5). c The effect of AntagomiR-214-3p, AntagomiR-N.C and AntagomiR-214-3p-Mut on luciferase activity in BMSCs after transfection with the β-catenin 3′-UTR reporter ( n = 5). d Western blot analysis of β-catenin protein levels in BMSCs after treatment with AgomiR-214-3p, AntagomiR-214-3p and their corresponding negative controls. e The effect of AntagomiR-214-3p and AntagomiR-N.C on luciferase activity in BMSCs supplemented with high glucose ( n = 5). f Western blot analysis of β-catenin protein levels in BMSCs supplemented with high glucose after treatment with AntagomiR-214-3p or AntagomiR-N.C. g qRT-PCR analysis of Bglap and Alp in BMSCs after treatment with β-catenin siRNA and AntagomiR-214-3p in osteogenic medium supplemented with high glucose ( n = 3). h Alizarin red staining of calcium deposition in BMSCs after treatment with β-catenin siRNA and AntagomiR-214-3p in osteogenic medium supplemented with high glucose. All data are expressed as mean ± SEM, ** p
Figure Legend Snippet: High-glucose-induced miR-214-3p functionally targets β-catenin. a Schematic diagram illustrating the design of luciferase reporters with the β-catenin 3′-UTR. The sequences of miR-214-3p and miR-214-3p-Mut are also shown. b The effect of AgomiR-214-3p, AgomiR-N.C and AgomiR-214-3p-Mut on luciferase activity in BMSCs after transfection with the β-catenin 3′-UTR reporter ( n = 5). c The effect of AntagomiR-214-3p, AntagomiR-N.C and AntagomiR-214-3p-Mut on luciferase activity in BMSCs after transfection with the β-catenin 3′-UTR reporter ( n = 5). d Western blot analysis of β-catenin protein levels in BMSCs after treatment with AgomiR-214-3p, AntagomiR-214-3p and their corresponding negative controls. e The effect of AntagomiR-214-3p and AntagomiR-N.C on luciferase activity in BMSCs supplemented with high glucose ( n = 5). f Western blot analysis of β-catenin protein levels in BMSCs supplemented with high glucose after treatment with AntagomiR-214-3p or AntagomiR-N.C. g qRT-PCR analysis of Bglap and Alp in BMSCs after treatment with β-catenin siRNA and AntagomiR-214-3p in osteogenic medium supplemented with high glucose ( n = 3). h Alizarin red staining of calcium deposition in BMSCs after treatment with β-catenin siRNA and AntagomiR-214-3p in osteogenic medium supplemented with high glucose. All data are expressed as mean ± SEM, ** p

Techniques Used: Luciferase, Activity Assay, Transfection, Western Blot, Quantitative RT-PCR, ALP Assay, Staining

miR-214-3p and β-catenin expression in diabetic osteoporosis patients. a qRT-PCR analysis of miR-214-3p mRNA levels in human bone specimens from healthy people or patients with T1DM ( n = 3). b qRT-PCR analysis of miR-214-3p mRNA levels in BMSCs from healthy people or patients with T1DM ( n = 3). c Western blot analysis of β-catenin in the bone specimens isolated from patients with T1DM or healthy people. d Western blot analysis of β-catenin in the BMSCs isolated from patients with T1DM or healthy people. All data are expressed as mean ± SEM, * p
Figure Legend Snippet: miR-214-3p and β-catenin expression in diabetic osteoporosis patients. a qRT-PCR analysis of miR-214-3p mRNA levels in human bone specimens from healthy people or patients with T1DM ( n = 3). b qRT-PCR analysis of miR-214-3p mRNA levels in BMSCs from healthy people or patients with T1DM ( n = 3). c Western blot analysis of β-catenin in the bone specimens isolated from patients with T1DM or healthy people. d Western blot analysis of β-catenin in the BMSCs isolated from patients with T1DM or healthy people. All data are expressed as mean ± SEM, * p

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

Identification of differentially expressed miRNAs in bone tissues from WT and diabetic mice. a The measurement of blood glucose concentrations in STZ-induced diabetic mice and control WT mice ( n = 5). b Representative dual-energy X-ray images of mouse femur from STZ-induced diabetic mice or control mice. c Bone mineral density (BMD) of STZ-induced diabetic mice or control mice ( n = 5). d Heatmap of differentially expressed miRNAs of bone specimens from STZ-induced diabetic mice or normal mice. e , f qRT-PCR analysis of miRNA expression in bone specimens from STZ-induced diabetic mice or control mice ( n = 3). All data are expressed as mean ± SEM, * p
Figure Legend Snippet: Identification of differentially expressed miRNAs in bone tissues from WT and diabetic mice. a The measurement of blood glucose concentrations in STZ-induced diabetic mice and control WT mice ( n = 5). b Representative dual-energy X-ray images of mouse femur from STZ-induced diabetic mice or control mice. c Bone mineral density (BMD) of STZ-induced diabetic mice or control mice ( n = 5). d Heatmap of differentially expressed miRNAs of bone specimens from STZ-induced diabetic mice or normal mice. e , f qRT-PCR analysis of miRNA expression in bone specimens from STZ-induced diabetic mice or control mice ( n = 3). All data are expressed as mean ± SEM, * p

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

High-glucose-induced miR-214-3p inhibits BMSCs osteogenic differentiation. a qRT-PCR analysis of Bglap , Alp , Opn in BMSCs after treatment with high glucose for 3 weeks ( n = 3). b Alizarin red staining of calcium deposition in BMSCs after treatment with high glucose for 3 weeks. c , d qRT-PCR analysis of miR-214-3p, Bglap and Alp mRNA levels in BMSCs after treatment with 200 μM AgomiR-214-3p or AgomiR-N.C in osteogenic medium for 3 weeks ( n = 3). e Alizarin red staining of calcium deposition in BMSCs after treatment with 200 μM AgomiR-214-3p or AgomiR-N.C in osteogenic medium for 3 weeks. f , g qRT-PCR analysis of miR-214-3p, Bglap and Alp mRNA levels in BMSCs after treatment with high glucose together with or without 200 μM AntagomiR-214-3p and AntagomiR-N.C for 3 weeks ( n = 3). h Alizarin red staining of BMSCs after treatment with high glucose together with 200 μM AntagomiR-214-3p or AntagomiR-N.C for 3 weeks. All data are expressed as mean ± SEM, * p
Figure Legend Snippet: High-glucose-induced miR-214-3p inhibits BMSCs osteogenic differentiation. a qRT-PCR analysis of Bglap , Alp , Opn in BMSCs after treatment with high glucose for 3 weeks ( n = 3). b Alizarin red staining of calcium deposition in BMSCs after treatment with high glucose for 3 weeks. c , d qRT-PCR analysis of miR-214-3p, Bglap and Alp mRNA levels in BMSCs after treatment with 200 μM AgomiR-214-3p or AgomiR-N.C in osteogenic medium for 3 weeks ( n = 3). e Alizarin red staining of calcium deposition in BMSCs after treatment with 200 μM AgomiR-214-3p or AgomiR-N.C in osteogenic medium for 3 weeks. f , g qRT-PCR analysis of miR-214-3p, Bglap and Alp mRNA levels in BMSCs after treatment with high glucose together with or without 200 μM AntagomiR-214-3p and AntagomiR-N.C for 3 weeks ( n = 3). h Alizarin red staining of BMSCs after treatment with high glucose together with 200 μM AntagomiR-214-3p or AntagomiR-N.C for 3 weeks. All data are expressed as mean ± SEM, * p

Techniques Used: Quantitative RT-PCR, ALP Assay, Staining

High glucose induces miR-214-3p expression in BMSCs. a qRT-PCR analysis of miR-214-3p levels in bone specimens from mice treated with or without STZ ( n = 3). b qRT-PCR analysis of miR-214-3p levels in BMSCs extracted from the mice treated with or without STZ ( n = 3). c qRT-PCR analysis of miR-214-3p levels in BMSCs treated with glucose at different concentrations for 24 h ( n = 3). d qRT-PCR analysis of miR-214-3p expression in BMSCs at the indicated time points after treatment with glucose at 16.5 mM ( n = 3). All data are expressed as mean ± SEM, * p
Figure Legend Snippet: High glucose induces miR-214-3p expression in BMSCs. a qRT-PCR analysis of miR-214-3p levels in bone specimens from mice treated with or without STZ ( n = 3). b qRT-PCR analysis of miR-214-3p levels in BMSCs extracted from the mice treated with or without STZ ( n = 3). c qRT-PCR analysis of miR-214-3p levels in BMSCs treated with glucose at different concentrations for 24 h ( n = 3). d qRT-PCR analysis of miR-214-3p expression in BMSCs at the indicated time points after treatment with glucose at 16.5 mM ( n = 3). All data are expressed as mean ± SEM, * p

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

67) Product Images from "Initial Spore Density Has an Influence on Ochratoxin A Content in Aspergillus ochraceus CGMCC 3.4412 in PDB and Its Interaction with Seeds"

Article Title: Initial Spore Density Has an Influence on Ochratoxin A Content in Aspergillus ochraceus CGMCC 3.4412 in PDB and Its Interaction with Seeds

Journal: Toxins

doi: 10.3390/toxins9040146

High ISD repressed the expressions of OTA biosynthesis genes in A. ochraceus . qRT-PCR (quantitative reverse transcription PCR) was used to analyze expressions of OTA biosynthesis genes ( pks , p450-b03 , p450-h11 ) by A. ochraceus CGMCC 3.4412 cultured in PDB media for six days with ISD of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 spores/mL. The relative expressions were quantified by the expression level of the Ao18s gene. Data are the means of three separate experiments.
Figure Legend Snippet: High ISD repressed the expressions of OTA biosynthesis genes in A. ochraceus . qRT-PCR (quantitative reverse transcription PCR) was used to analyze expressions of OTA biosynthesis genes ( pks , p450-b03 , p450-h11 ) by A. ochraceus CGMCC 3.4412 cultured in PDB media for six days with ISD of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 spores/mL. The relative expressions were quantified by the expression level of the Ao18s gene. Data are the means of three separate experiments.

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, Cell Culture, Expressing

68) Product Images from "Epigenetic silencing of SALL2 confers tamoxifen resistance in breast cancer"

Article Title: Epigenetic silencing of SALL2 confers tamoxifen resistance in breast cancer

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201910638

SALL 2 transcriptionally upregulates ESR 1 in breast cancer RNA‐seq analysis of ESR1 mRNA levels in 9 paired pre‐tamoxifen‐treated primary breast cancer tissues and relapsed tamoxifen‐resistant breast cancer tissues. qRT–PCR analysis of ESR1 expression in 9 paired pre‐tamoxifen‐treated primary breast cancer tissues and relapsed tamoxifen‐resistant breast cancer tissues. GAPDH was used as an internal control. WB analysis of SALL2 (A) and NKX3‐1 (B) expression in the indicated cells transfected with Ri‐Vector (V‐Ri) or shRNAs (Ri#1/2) against SALL2 or NKX3‐1. α‐Tubulin was used as the loading control. qRT–PCR analysis of ESR1 expression in the indicated cells transfected with Ri‐Vector or shRNAs (Ri#1/2) against SALL2 or NKX3‐1. Data information: In (A), P ‐values were determined by two‐tailed paired Student's t ‐test. In (B), data are presented as mean ± SD, and P ‐values were determined by two‐tailed unpaired Student's t ‐test, n = 3. In (E and F), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. * P
Figure Legend Snippet: SALL 2 transcriptionally upregulates ESR 1 in breast cancer RNA‐seq analysis of ESR1 mRNA levels in 9 paired pre‐tamoxifen‐treated primary breast cancer tissues and relapsed tamoxifen‐resistant breast cancer tissues. qRT–PCR analysis of ESR1 expression in 9 paired pre‐tamoxifen‐treated primary breast cancer tissues and relapsed tamoxifen‐resistant breast cancer tissues. GAPDH was used as an internal control. WB analysis of SALL2 (A) and NKX3‐1 (B) expression in the indicated cells transfected with Ri‐Vector (V‐Ri) or shRNAs (Ri#1/2) against SALL2 or NKX3‐1. α‐Tubulin was used as the loading control. qRT–PCR analysis of ESR1 expression in the indicated cells transfected with Ri‐Vector or shRNAs (Ri#1/2) against SALL2 or NKX3‐1. Data information: In (A), P ‐values were determined by two‐tailed paired Student's t ‐test. In (B), data are presented as mean ± SD, and P ‐values were determined by two‐tailed unpaired Student's t ‐test, n = 3. In (E and F), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. * P

Techniques Used: Electron Paramagnetic Resonance, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test

SALL 2 transcriptionally activates ESR 1 qRT–PCR analysis of ESR1 expression in SALL2‐silenced, SALL2‐overexpressing, and control cells. GAPDH was used as an internal control. WB analysis of expression of SALL2 and ERα in the indicated cells. α‐Tubulin was used as a loading control. Upper panel: schematic illustration of the predicted binding site for SALL2 in the indicated ESR1 promoter regions (upper panel). Lower left panel: schematic illustration of the wild‐type or mutant ESR1 promoter regions cloned into the pGL3 luciferase reporter plasmid; lower right panel: quantification of luciferase activity of the ESR1 promoter reporter was examined in the indicated cells (lower panel). Putative SALL2‐binding sites are shown as red filled circles, and the blue filled box shows the mutated site. Red letters in each binding region indicate the putative or mutated SALL2‐binding sequences. Vct, empty vector; Wt, wild‐type; Mut, mutant. Schematic illustration of the human ESR1 gene promoter (upper panel) and ChIP analysis of enrichment of SALL2 on the ESR1 promoter (lower panel). IgG was used as a negative control. The red squares represent the qRT–PCR region. ChIP assays were performed in the indicated cells using anti‐p300 acetyltransferase, anti‐RNA POL II (RNAP II), and anti‐H3K4me3 antibodies. ERE luciferase assays were performed to assess ERα activation in the indicated cells transfected with ERE‐luc (3×ERE) plasmid and then treated with E2 (10 nM) or TAM (1 μM) for 24 h. qRT–PCR analysis of TFF1 and PGR expression in the indicated cell lines treated with or without E2 (10 nM) or TAM (1 μM) for 24 h. GAPDH was used as an internal control. Data information: In (A, left; C–E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (A, middle and right), P ‐values were determined by two‐tailed Student's t ‐test, n = 3. In (F and G), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test, n = 3. * P
Figure Legend Snippet: SALL 2 transcriptionally activates ESR 1 qRT–PCR analysis of ESR1 expression in SALL2‐silenced, SALL2‐overexpressing, and control cells. GAPDH was used as an internal control. WB analysis of expression of SALL2 and ERα in the indicated cells. α‐Tubulin was used as a loading control. Upper panel: schematic illustration of the predicted binding site for SALL2 in the indicated ESR1 promoter regions (upper panel). Lower left panel: schematic illustration of the wild‐type or mutant ESR1 promoter regions cloned into the pGL3 luciferase reporter plasmid; lower right panel: quantification of luciferase activity of the ESR1 promoter reporter was examined in the indicated cells (lower panel). Putative SALL2‐binding sites are shown as red filled circles, and the blue filled box shows the mutated site. Red letters in each binding region indicate the putative or mutated SALL2‐binding sequences. Vct, empty vector; Wt, wild‐type; Mut, mutant. Schematic illustration of the human ESR1 gene promoter (upper panel) and ChIP analysis of enrichment of SALL2 on the ESR1 promoter (lower panel). IgG was used as a negative control. The red squares represent the qRT–PCR region. ChIP assays were performed in the indicated cells using anti‐p300 acetyltransferase, anti‐RNA POL II (RNAP II), and anti‐H3K4me3 antibodies. ERE luciferase assays were performed to assess ERα activation in the indicated cells transfected with ERE‐luc (3×ERE) plasmid and then treated with E2 (10 nM) or TAM (1 μM) for 24 h. qRT–PCR analysis of TFF1 and PGR expression in the indicated cell lines treated with or without E2 (10 nM) or TAM (1 μM) for 24 h. GAPDH was used as an internal control. Data information: In (A, left; C–E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (A, middle and right), P ‐values were determined by two‐tailed Student's t ‐test, n = 3. In (F and G), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test, n = 3. * P

Techniques Used: Electron Paramagnetic Resonance, Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Mutagenesis, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Activation Assay, Transfection, Two Tailed Test

Establishing MCF 7/tamoxifen‐resistant ( MCF 7‐ TMR ) cell line Representative images (left panel) and quantification (right panel) of colony formation by MCF7 and MCF7‐TMR cell lines with increasing doses of tamoxifen (TAM) treatment. qRT–PCR analysis of SALL2 and ESR1 expression in MCF7 and MCF7‐TMR cell lines. GAPDH was used as an internal control. WB analysis of SALL2 and ERα expression in MCF7 and MCF7‐TMR cell lines. α‐Tubulin was used as a loading control. Data information: In (A), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (B), data are presented as mean ± SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 3. *** P
Figure Legend Snippet: Establishing MCF 7/tamoxifen‐resistant ( MCF 7‐ TMR ) cell line Representative images (left panel) and quantification (right panel) of colony formation by MCF7 and MCF7‐TMR cell lines with increasing doses of tamoxifen (TAM) treatment. qRT–PCR analysis of SALL2 and ESR1 expression in MCF7 and MCF7‐TMR cell lines. GAPDH was used as an internal control. WB analysis of SALL2 and ERα expression in MCF7 and MCF7‐TMR cell lines. α‐Tubulin was used as a loading control. Data information: In (A), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (B), data are presented as mean ± SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 3. *** P

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Silencing SALL 2 promotes tamoxifen resistance via downregulation of ESR 1 Schematic illustration of in vivo models of tamoxifen therapy in formed by MCF7 cells (upper panel). Tumor growth curves of the indicated xenograft tumors ( n = 8/group) (lower panel). Quantification of xenograft tumor weight at the end of the experiment shown in (A) ( n = 8/group). Representative IHC images of SALL2 and ERα in the E2‐ and TAM‐treated xenografts at the end of the experiment shown in (A). Scale bars: 50 μm. Tumor growth curves of the indicated xenograft tumors ( n = 8/group) (left panel) and representative tumor images (right panel). Representative IHC images of Ki67 and ERα in the xenografts. Scale bars: 50 μm (left and right panels) and 500 μm (middle panel). WB analysis of the indicated protein expression in the MCF7‐TMR/control and MCF7‐TMR/SALL2‐Dox cells treated with or without doxycycline (Dox); α‐tubulin was used as a loading control. qRT–PCR analysis of expression of multiple ERα downstream target genes in the MCF7‐TMR/control and MCF7‐TMR/SALL2‐Dox cells treated with vehicle (Veh) or doxycycline (DOX). GAPDH was used as an internal control. Quantification of crystal violet‐stained colony formed by the indicated cells treated with or without Dox. Tumor growth curves of the indicated xenograft tumors ( n = 8/group) upon doxycycline and 5‐Aza‐dC treatment. 5‐Aza‐dC treatment was started when the tumors reached approximately 200 mm 3 . Data information: In (A, D, H, and I), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test, n = 3 in (H). In (B and G), data are presented as mean ± SD, and P ‐values were determined by 1‐way ANOVA, n = 3 in (G). *** P
Figure Legend Snippet: Silencing SALL 2 promotes tamoxifen resistance via downregulation of ESR 1 Schematic illustration of in vivo models of tamoxifen therapy in formed by MCF7 cells (upper panel). Tumor growth curves of the indicated xenograft tumors ( n = 8/group) (lower panel). Quantification of xenograft tumor weight at the end of the experiment shown in (A) ( n = 8/group). Representative IHC images of SALL2 and ERα in the E2‐ and TAM‐treated xenografts at the end of the experiment shown in (A). Scale bars: 50 μm. Tumor growth curves of the indicated xenograft tumors ( n = 8/group) (left panel) and representative tumor images (right panel). Representative IHC images of Ki67 and ERα in the xenografts. Scale bars: 50 μm (left and right panels) and 500 μm (middle panel). WB analysis of the indicated protein expression in the MCF7‐TMR/control and MCF7‐TMR/SALL2‐Dox cells treated with or without doxycycline (Dox); α‐tubulin was used as a loading control. qRT–PCR analysis of expression of multiple ERα downstream target genes in the MCF7‐TMR/control and MCF7‐TMR/SALL2‐Dox cells treated with vehicle (Veh) or doxycycline (DOX). GAPDH was used as an internal control. Quantification of crystal violet‐stained colony formed by the indicated cells treated with or without Dox. Tumor growth curves of the indicated xenograft tumors ( n = 8/group) upon doxycycline and 5‐Aza‐dC treatment. 5‐Aza‐dC treatment was started when the tumors reached approximately 200 mm 3 . Data information: In (A, D, H, and I), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test, n = 3 in (H). In (B and G), data are presented as mean ± SD, and P ‐values were determined by 1‐way ANOVA, n = 3 in (G). *** P

Techniques Used: Electron Paramagnetic Resonance, In Vivo, Immunohistochemistry, Western Blot, Expressing, Quantitative RT-PCR, Staining

Overexpression of SALL 2 restores sensitivity of resistant breast cancer cells to tamoxifen qRT–PCR analysis of ESR1 expression in the MCF7‐TMR/SALL2‐Ri#1 cells (A) and MCF7/SALL2‐Ri#1 cells (B) with the indicated treatments. GAPDH was used as an internal control. Tumor growth curves of the indicated MCF7/SALL2‐Ri#1/xenograft tumors ( n = 8/group) upon 5‐Aza‐dC and TAM treatment. 5‐Aza‐dC treatment was started when the tumors reached approximately 200 mm 3 . Quantification of xenograft tumor weight at the end of the experiment shown in (C) ( n = 8/group). The proliferation index was determined using the percentage of Ki67‐positive cells, and the apoptosis index was determined using the percentage of TUNEL‐positive cells, in the MCF7/SALL2‐Ri xenograft tumors upon 5‐Aza‐dC and TAM treatment ( n = 8/group). Data information: In (A, B, D, and E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3 in (A and B). In (C), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test. n.s., no significance. Exact P ‐values are specified in Appendix Table S10 .
Figure Legend Snippet: Overexpression of SALL 2 restores sensitivity of resistant breast cancer cells to tamoxifen qRT–PCR analysis of ESR1 expression in the MCF7‐TMR/SALL2‐Ri#1 cells (A) and MCF7/SALL2‐Ri#1 cells (B) with the indicated treatments. GAPDH was used as an internal control. Tumor growth curves of the indicated MCF7/SALL2‐Ri#1/xenograft tumors ( n = 8/group) upon 5‐Aza‐dC and TAM treatment. 5‐Aza‐dC treatment was started when the tumors reached approximately 200 mm 3 . Quantification of xenograft tumor weight at the end of the experiment shown in (C) ( n = 8/group). The proliferation index was determined using the percentage of Ki67‐positive cells, and the apoptosis index was determined using the percentage of TUNEL‐positive cells, in the MCF7/SALL2‐Ri xenograft tumors upon 5‐Aza‐dC and TAM treatment ( n = 8/group). Data information: In (A, B, D, and E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3 in (A and B). In (C), data are presented as mean ± SD, and P ‐values were determined by two‐way ANOVA test. n.s., no significance. Exact P ‐values are specified in Appendix Table S10 .

Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, TUNEL Assay

SALL 2 promoter is methylated in tamoxifen‐resistant breast cancer Color coding of the ChromHMM regions in MCF7 cells obtained from GEO (GSE69118): yellow, promoter; green, enhancer; light blue, transcribed; and blue, un‐transcribed. BSP primers were designed to amplify the sequence in the predicted CpG island region. qRT–PCR analysis of SALL2 expression in the indicated cells treated with vehicle or 5‐Aza‐dC. GAPDH was used as an internal control. BSP analysis (left panel) and quantification (right panel) of the methylation status of SALL2 gene in the indicated cells. ChIP analyses of enrichment of 5mc (D) and DNMTs and histone modifications (E) on the SALL2 promoter. qRT–PCR analysis of SALL2 expression (upper panel) and BSP analysis of SALL2 methylation status (lower panel) in 6 ER + breast cancer cell lines. GAPDH was used as an internal control. Bar graph showing negative correlation of SALL2 DNA methylation level with SALL2 expression analyzed by IHC staining. Kaplan–Meier analysis of DFS (left panel) or OS (right panel) curves in tamoxifen‐treated patients with SALL2‐hypomethylated and SALL2‐hypermethylated ER + breast cancer. Data information: In (B–E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (F), data are presented as mean ± SD, n = 3. In (G), n = 90, P ‐values were determined by χ 2 test. In (H), n = 90, log‐rank test. * P
Figure Legend Snippet: SALL 2 promoter is methylated in tamoxifen‐resistant breast cancer Color coding of the ChromHMM regions in MCF7 cells obtained from GEO (GSE69118): yellow, promoter; green, enhancer; light blue, transcribed; and blue, un‐transcribed. BSP primers were designed to amplify the sequence in the predicted CpG island region. qRT–PCR analysis of SALL2 expression in the indicated cells treated with vehicle or 5‐Aza‐dC. GAPDH was used as an internal control. BSP analysis (left panel) and quantification (right panel) of the methylation status of SALL2 gene in the indicated cells. ChIP analyses of enrichment of 5mc (D) and DNMTs and histone modifications (E) on the SALL2 promoter. qRT–PCR analysis of SALL2 expression (upper panel) and BSP analysis of SALL2 methylation status (lower panel) in 6 ER + breast cancer cell lines. GAPDH was used as an internal control. Bar graph showing negative correlation of SALL2 DNA methylation level with SALL2 expression analyzed by IHC staining. Kaplan–Meier analysis of DFS (left panel) or OS (right panel) curves in tamoxifen‐treated patients with SALL2‐hypomethylated and SALL2‐hypermethylated ER + breast cancer. Data information: In (B–E), data are presented as mean ± SD, and P ‐values were determined by one‐way ANOVA test, n = 3. In (F), data are presented as mean ± SD, n = 3. In (G), n = 90, P ‐values were determined by χ 2 test. In (H), n = 90, log‐rank test. * P

Techniques Used: Methylation, Sequencing, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, DNA Methylation Assay, Immunohistochemistry, Staining

69) Product Images from "Global Transcriptional Response of Nitrosomonas europaea to Chloroform and Chloromethane ▿ to Chloroform and Chloromethane ▿ †"

Article Title: Global Transcriptional Response of Nitrosomonas europaea to Chloroform and Chloromethane ▿ to Chloroform and Chloromethane ▿ †

Journal:

doi: 10.1128/AEM.02831-06

Comparison of the expression of selected genes by qRT-PCR (black bars) and microarray (white bars) methods. (Top) CM treatment; (bottom) CF treatment. Error bars represent standard errors of the means.
Figure Legend Snippet: Comparison of the expression of selected genes by qRT-PCR (black bars) and microarray (white bars) methods. (Top) CM treatment; (bottom) CF treatment. Error bars represent standard errors of the means.

Techniques Used: Expressing, Quantitative RT-PCR, Microarray

70) Product Images from "Analysis of changes to lncRNAs and their target mRNAs in murine jejunum after radiation treatment, et al. Analysis of changes to lncRNAs and their target mRNAs in murine jejunum after radiation treatment"

Article Title: Analysis of changes to lncRNAs and their target mRNAs in murine jejunum after radiation treatment, et al. Analysis of changes to lncRNAs and their target mRNAs in murine jejunum after radiation treatment

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13940

qRT ‐ PCR validations of two up‐regulated and two down‐regulated lnc RNA s and mRNA s. ENSMUST 00000173070 and AK 157361 are two up‐regulated lnc RNA s, AK 083183 and AK 038898 are two down‐regulated lnc RNA s, Mboat1 and Nek10 are two up‐regulated mRNA s, Ccl24 and Cyp2c55 are two down‐regulated mRNA s. * P
Figure Legend Snippet: qRT ‐ PCR validations of two up‐regulated and two down‐regulated lnc RNA s and mRNA s. ENSMUST 00000173070 and AK 157361 are two up‐regulated lnc RNA s, AK 083183 and AK 038898 are two down‐regulated lnc RNA s, Mboat1 and Nek10 are two up‐regulated mRNA s, Ccl24 and Cyp2c55 are two down‐regulated mRNA s. * P

Techniques Used: Quantitative RT-PCR

71) Product Images from "Receptor-Like Kinase LYK9 in Pisum sativum L. Is the CERK1-Like Receptor that Controls Both Plant Immunity and AM Symbiosis Development"

Article Title: Receptor-Like Kinase LYK9 in Pisum sativum L. Is the CERK1-Like Receptor that Controls Both Plant Immunity and AM Symbiosis Development

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19010008

Relative expression of type-A cytokinin response regulator genes PsRR4 , PsRR6 , PsRR8 , PsRR9 was determined using qRT-PCR. ( A ) Analysis of pea roots on 14 and 28 days after inoculation with Rhizophagus irregularis (dai). NI, not inoculated; ( B ) Effect was estimated in pea roots treated with 10 −5 M CO5 for 24 h. As a control mock-treated roots were used. The relative expression was normalized against the constitutively expressed ubiquitin and actin genes. Asterisks indicate statistically significant differences compared with control (not inoculated or not treated plants): *** p
Figure Legend Snippet: Relative expression of type-A cytokinin response regulator genes PsRR4 , PsRR6 , PsRR8 , PsRR9 was determined using qRT-PCR. ( A ) Analysis of pea roots on 14 and 28 days after inoculation with Rhizophagus irregularis (dai). NI, not inoculated; ( B ) Effect was estimated in pea roots treated with 10 −5 M CO5 for 24 h. As a control mock-treated roots were used. The relative expression was normalized against the constitutively expressed ubiquitin and actin genes. Asterisks indicate statistically significant differences compared with control (not inoculated or not treated plants): *** p

Techniques Used: Expressing, Quantitative RT-PCR

72) Product Images from "Involvement of COX2-Thromboxane Pathway in TCDD-Induced Precardiac Edema in Developing Zebrafish"

Article Title: Involvement of COX2-Thromboxane Pathway in TCDD-Induced Precardiac Edema in Developing Zebrafish

Journal: Aquatic toxicology (Amsterdam, Netherlands)

doi: 10.1016/j.aquatox.2014.04.025

Induction of COX2b but not COX2a by TCDD in whole zebrafish eleutheroembryos. Expression levels of COX2a (A) and COX2b (B) transcripts in whole zebrafish eleutheroembryos at 55 hpf and 72 hpf were determined by qRT-PCR. Embryos were exposed to 1 ppb TCDD
Figure Legend Snippet: Induction of COX2b but not COX2a by TCDD in whole zebrafish eleutheroembryos. Expression levels of COX2a (A) and COX2b (B) transcripts in whole zebrafish eleutheroembryos at 55 hpf and 72 hpf were determined by qRT-PCR. Embryos were exposed to 1 ppb TCDD

Techniques Used: Expressing, Quantitative RT-PCR

COX2b expression in early life stage of zebrafish. Expression levels of COX2b transcripts in various developmental stages of whole zebrafish eleutheroembryos (from 0 to 96 hpf) were determined by qRT-PCR. N =4 or 5 per group. Thirty to fifty whole embryos
Figure Legend Snippet: COX2b expression in early life stage of zebrafish. Expression levels of COX2b transcripts in various developmental stages of whole zebrafish eleutheroembryos (from 0 to 96 hpf) were determined by qRT-PCR. N =4 or 5 per group. Thirty to fifty whole embryos

Techniques Used: Expressing, Quantitative RT-PCR

73) Product Images from "Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1"

Article Title: Facilitation of Rice Stripe Virus Accumulation in the Insect Vector by Himetobi P Virus VP1

Journal: Viruses

doi: 10.3390/v7031492

Detection of RSV accumulation levels in SBPHs after feeding-based RNA interference (RNAi). ( A ) The levels of HiPV VP1 and RSV RNP transcripts in insects after feeding them artificial diets without dsRNA (CK), dsGFP and dsVP1 (with 150 ng/μl dsRNA concentration for 6 days). After qRT-PCR, the levels of VP1 and RNP transcripts were normalized relative to the β -actin transcript according to the ΔC T algorithm, and the resulting 2 −ΔCt values were used to plot with different feeding treatments as the abscissa. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP . Each histogram bar represents the mean (±SE) from four repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p
Figure Legend Snippet: Detection of RSV accumulation levels in SBPHs after feeding-based RNA interference (RNAi). ( A ) The levels of HiPV VP1 and RSV RNP transcripts in insects after feeding them artificial diets without dsRNA (CK), dsGFP and dsVP1 (with 150 ng/μl dsRNA concentration for 6 days). After qRT-PCR, the levels of VP1 and RNP transcripts were normalized relative to the β -actin transcript according to the ΔC T algorithm, and the resulting 2 −ΔCt values were used to plot with different feeding treatments as the abscissa. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP . Each histogram bar represents the mean (±SE) from four repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p

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

RSV-acquisition ability of SBPH after feeding-based RNAi. After qRT-PCR, the levels of VP1 transcripts in SBPH via different feeding treatments (CK, dsGFP and dsVP1; 150 ng/μL dsRNA concentration for 6 days) were normalized relative to the β -actin transcript according to the ΔC T algorithm. The virus-acquisition rate of SBPH was detected using the DIBA method after a 10-day latent period. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates RSV-acquisition rate of SBPH. Each histogram bar represents the mean (±SE) from three (CK and dsGFP) or five (dsVP1) repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p
Figure Legend Snippet: RSV-acquisition ability of SBPH after feeding-based RNAi. After qRT-PCR, the levels of VP1 transcripts in SBPH via different feeding treatments (CK, dsGFP and dsVP1; 150 ng/μL dsRNA concentration for 6 days) were normalized relative to the β -actin transcript according to the ΔC T algorithm. The virus-acquisition rate of SBPH was detected using the DIBA method after a 10-day latent period. The left ordinate indicates the expression levels of VP1 , and the right ordinate indicates RSV-acquisition rate of SBPH. Each histogram bar represents the mean (±SE) from three (CK and dsGFP) or five (dsVP1) repeats, and the different letters above the error bars indicate significant difference as per Tukey’s honest significant difference (HSD) test ( p

Techniques Used: Quantitative RT-PCR, Concentration Assay, Dot Immunobinding, Expressing

Titers of HiPV and RSV in single SBPH. After qRT-PCR, the levels of HiPV VP1 and RSV RNP transcripts in single SBPH were normalized relative to the β -actin transcript according to the ΔC T algorithm. The 118 individual SBPHs are arranged according to descending VP1 expression quantity as the abscissa; the left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP .
Figure Legend Snippet: Titers of HiPV and RSV in single SBPH. After qRT-PCR, the levels of HiPV VP1 and RSV RNP transcripts in single SBPH were normalized relative to the β -actin transcript according to the ΔC T algorithm. The 118 individual SBPHs are arranged according to descending VP1 expression quantity as the abscissa; the left ordinate indicates the expression levels of VP1 , and the right ordinate indicates the expression levels of RNP .

Techniques Used: Quantitative RT-PCR, Expressing

74) Product Images from "Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets"

Article Title: Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets

Journal: PeerJ

doi: 10.7717/peerj.4535

Pairwise variation ( V n / V n +1 ) values analysis in all the seven experimental subsets calculated using geNorm. The cut-off value to determine the optimal number of RGs for qRT-PCR normalization is 0.15.
Figure Legend Snippet: Pairwise variation ( V n / V n +1 ) values analysis in all the seven experimental subsets calculated using geNorm. The cut-off value to determine the optimal number of RGs for qRT-PCR normalization is 0.15.

Techniques Used: Quantitative RT-PCR

75) Product Images from "Sialoglycoprotein from Gadous morhua eggs improve high bone turnover activity via down-regulating BMP-2/Smads and Wnt/β-catenin signal pathways"

Article Title: Sialoglycoprotein from Gadous morhua eggs improve high bone turnover activity via down-regulating BMP-2/Smads and Wnt/β-catenin signal pathways

Journal: Food Science and Biotechnology

doi: 10.1007/s10068-018-0379-0

Effects of Gm -SGP on the transcription of Runx2 and Osx. RNA and protein were extracted from distal femurs and the mRNA expression of Runx2 and Osx were measured by qRT-PCR ( A ); protein expression of Runx2 was measured by western blot ( B ). Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P
Figure Legend Snippet: Effects of Gm -SGP on the transcription of Runx2 and Osx. RNA and protein were extracted from distal femurs and the mRNA expression of Runx2 and Osx were measured by qRT-PCR ( A ); protein expression of Runx2 was measured by western blot ( B ). Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P

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

Effects of Gm -SGP on gene expression of osteogenesis markers ALP, CoL1, and OCN. RNA was extracted from distal femurs and the mRNA expression of ALP, CoL1, and OCN were measured by qRT-PCR. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P
Figure Legend Snippet: Effects of Gm -SGP on gene expression of osteogenesis markers ALP, CoL1, and OCN. RNA was extracted from distal femurs and the mRNA expression of ALP, CoL1, and OCN were measured by qRT-PCR. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P

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

Effects of Gm -SGP on mRNA and protein expressions of key genes in the Wnt/β-catenin signaling pathway. RNA and protein were extracted from distal femurs and the mRNA expression of Lrp-5 and β-catenin were measured by qRT-PCR ( A ); protein expression of total β-catenin ( B ) and intranuclear β-catenin ( C ) were measured by western blot. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P
Figure Legend Snippet: Effects of Gm -SGP on mRNA and protein expressions of key genes in the Wnt/β-catenin signaling pathway. RNA and protein were extracted from distal femurs and the mRNA expression of Lrp-5 and β-catenin were measured by qRT-PCR ( A ); protein expression of total β-catenin ( B ) and intranuclear β-catenin ( C ) were measured by western blot. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P

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

Effects of Gm -SGP on mRNA and protein expressions of key genes in the BMP-2/Smads signaling pathway. RNA and protein were extracted from distal femurs and the mRNA expression including BMP-2, Smad-1 and Smad-4 were measured by qRT-PCR ( A ); protein expression of BMP-2 ( B ), Smad1, p-Smad1 ( C ) and Smad4 ( D ) were measured by western blot. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P
Figure Legend Snippet: Effects of Gm -SGP on mRNA and protein expressions of key genes in the BMP-2/Smads signaling pathway. RNA and protein were extracted from distal femurs and the mRNA expression including BMP-2, Smad-1 and Smad-4 were measured by qRT-PCR ( A ); protein expression of BMP-2 ( B ), Smad1, p-Smad1 ( C ) and Smad4 ( D ) were measured by western blot. Data are presented as mean ± SD (n = 8). Multiple comparisons were done using one-way ANOVA analyses. a P

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

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Article Snippet: .. The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method. ..

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo
Article Snippet: .. Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from liver tissue using methods previously described [ ] using TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized using M-MLV reverse transcriptase (Promega Co., Madison, WI) and qRT-PCR analysis was performed using 2× iQ™ SYBR® Green supermix PCR Master Mix (BioRad, Hercules, CA). ..

Article Title: p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans
Article Snippet: .. This cDNA was then subjected to qRT-PCR analysis using SYBR green detection on an iCycler machine (Bio-Rad, http://www.bio-rad.com ). .. Primers for qRT-PCR were designed using Primer3 (Massachusetts Institute of Technology), checked for specificity against the C. elegans genome and tested for efficiency with a dilution series of template.

Article Title: Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress
Article Snippet: .. The qRT-PCR analysis was performed with a CFX96 Real Time PCR Detection System (Bio-Rad). .. For rice, reactions (10 μL) included SsoFast EvaGreen Supermix Master Mix (Bio-Rad) and 0.6 μM gene-specific primers ( ).

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: .. For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System. .. Relative mRNA levels were calculated through comparison with GAPDH amplification values.

Article Title: RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A
Article Snippet: .. qRT-PCR analysis of ATP synthase genes Relative quantification of five genes in the three cotton lines were conducted by real-time qRT-PCR analysis with a C1000 TouchTM Thermal Cycler (Bio-Rad, USA) with TransStartR Tip Green qPCR SuperMix (Trans, China). ..

Article Title: Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation
Article Snippet: .. Then, a total of 500 ng RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR analysis was performed in triplicates with the SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). .. The expression level of SETDB1 mRNA was calculated using 2−ΔΔCt algorithm and normalized to that of GAPDH.

Article Title: EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways
Article Snippet: .. Total RNA was extracted using TRI Reagent (MRC), and reverse transcribed using the Superscript III kit (Invitrogen). cDNA was subjected to qRT-PCR analysis using SYBR green detection (Bio-Rad) on an iCycler machine (Eppendorf). .. Primers for qRT-PCR were usually designed to span an intron, using the Primer-BLAST tool of the National Center for Biotechnology Information of the National Institutes of Health ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and checked for specificity against the C. elegans genome.

Article Title: RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)
Article Snippet: .. The qRT-PCR analysis was performed with SsoFast EvaGreen supermix (Bio-Rad, ON, Canada) according to the manufacturer’s instructions using a Bio-Rad CFX96TM system. ..

SYBR Green Assay:

Article Title: In Vivo Determination of Direct Targets of the Nonsense-Mediated Decay Pathway in Drosophila
Article Snippet: .. All qRT-PCR analysis was conducted using iQ SYBR Green master mix and a MyiQ thermal cycler running iQ5 v2.0 software (Bio-Rad, Hercules, CA). ..

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: .. RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad. .. Expression of human genes was normalized to that of human OAZ1.

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression
Article Snippet: .. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) Total RNA isolated from PCa cell lines with Aurum total RNA mini kit (Bio-Rad, Hercules, CA) was subjected to qRT-PCR analysis using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad), according to the manufacturer’s instructions. ..

Article Title: The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis
Article Snippet: .. For mRNA quantification, cDNA was synthesized using iScript RT Supermix (Bio-Rad), following the manufacturer's protocol. qRT-PCR analysis was performed in triplicate using iQ SYBR green Supermix (BioRad) on an iCycler Real-Time Detection System (Eppendorf). ..

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: .. Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Reactions were carried out using a iCycler iQ system (BioRAD) for 30 cycles (95°C for 15 s, 72°C for 30 s).

Article Title: CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression
Article Snippet: .. 1 μg of total RNA was used to obtain the cDNA with the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR analysis was carried out in 96-well plates with the CFX 96 thermocycler (Bio-Rad, Hercules, CA) by using 20 ng of each cDNA and Sybr Green (Applied Biosystems, Foster City, CA) or Real Master Mix (5Prime Inc., Gaithersburg, MD). .. For human SPP1, CBX7 and GAPDH amplification we used TaqMan gene expression assays (Applied Biosystems, CBX7: Hs00545603_m1; SPP1: Hs00959010_m1; GAPDH: Hs02758991_g1).

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma
Article Snippet: .. The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method. ..

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo
Article Snippet: .. Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from liver tissue using methods previously described [ ] using TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized using M-MLV reverse transcriptase (Promega Co., Madison, WI) and qRT-PCR analysis was performed using 2× iQ™ SYBR® Green supermix PCR Master Mix (BioRad, Hercules, CA). ..

Article Title: p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans
Article Snippet: .. This cDNA was then subjected to qRT-PCR analysis using SYBR green detection on an iCycler machine (Bio-Rad, http://www.bio-rad.com ). .. Primers for qRT-PCR were designed using Primer3 (Massachusetts Institute of Technology), checked for specificity against the C. elegans genome and tested for efficiency with a dilution series of template.

Article Title: EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways
Article Snippet: .. Total RNA was extracted using TRI Reagent (MRC), and reverse transcribed using the Superscript III kit (Invitrogen). cDNA was subjected to qRT-PCR analysis using SYBR green detection (Bio-Rad) on an iCycler machine (Eppendorf). .. Primers for qRT-PCR were usually designed to span an intron, using the Primer-BLAST tool of the National Center for Biotechnology Information of the National Institutes of Health ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and checked for specificity against the C. elegans genome.

Microarray:

Article Title: p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans
Article Snippet: Animals were treated as described for samples used in microarray analysis. .. This cDNA was then subjected to qRT-PCR analysis using SYBR green detection on an iCycler machine (Bio-Rad, http://www.bio-rad.com ).

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: The microarray data for this study are available with accession number GSE45583 through NCBI’s Gene Expression Omnibus (GEO). .. For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System.

Incubation:

Article Title: EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways
Article Snippet: For S. aureus infection assays, infected samples were compared with parallel samples feeding on E. coli OP50, heat-killed by 30 min incubation at 95°C, plated on the same TSA medium. .. Total RNA was extracted using TRI Reagent (MRC), and reverse transcribed using the Superscript III kit (Invitrogen). cDNA was subjected to qRT-PCR analysis using SYBR green detection (Bio-Rad) on an iCycler machine (Eppendorf).

Significance Assay:

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: Corrected p-values of 0.05 were used as the cutoff for significance, corresponding to a significance threshold of |fold change| > 1.4, adjusted p-value < 0.05, using a moderated t-statistic (LIMMA). .. For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System.

Expressing:

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad. .. Expression of human genes was normalized to that of human OAZ1.

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression
Article Snippet: Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) Total RNA isolated from PCa cell lines with Aurum total RNA mini kit (Bio-Rad, Hercules, CA) was subjected to qRT-PCR analysis using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad), according to the manufacturer’s instructions. .. The primers used for the estimation of GPR158, AR, PSA and NSE expression are shown in .

Article Title: CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression
Article Snippet: 1 μg of total RNA was used to obtain the cDNA with the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR analysis was carried out in 96-well plates with the CFX 96 thermocycler (Bio-Rad, Hercules, CA) by using 20 ng of each cDNA and Sybr Green (Applied Biosystems, Foster City, CA) or Real Master Mix (5Prime Inc., Gaithersburg, MD). .. For human SPP1, CBX7 and GAPDH amplification we used TaqMan gene expression assays (Applied Biosystems, CBX7: Hs00545603_m1; SPP1: Hs00959010_m1; GAPDH: Hs02758991_g1).

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma
Article Snippet: .. The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method. ..

Article Title: p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans
Article Snippet: We did not find a substantial difference in gene expression between the daf-2;daf-16 preps of slightly different ages for the genes we analyzed). .. This cDNA was then subjected to qRT-PCR analysis using SYBR green detection on an iCycler machine (Bio-Rad, http://www.bio-rad.com ).

Article Title: Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress
Article Snippet: Paragraph title: 4.7. Gene Expression Analysis Using qRT-PCR ... The qRT-PCR analysis was performed with a CFX96 Real Time PCR Detection System (Bio-Rad).

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: .. For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System. .. Relative mRNA levels were calculated through comparison with GAPDH amplification values.

Article Title: RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A
Article Snippet: qRT-PCR analysis of ATP synthase genes Relative quantification of five genes in the three cotton lines were conducted by real-time qRT-PCR analysis with a C1000 TouchTM Thermal Cycler (Bio-Rad, USA) with TransStartR Tip Green qPCR SuperMix (Trans, China). .. The relative expression level was calculated by the 2−∆∆ Ct method with three replicates [ ].

Article Title: Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation
Article Snippet: Then, a total of 500 ng RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR analysis was performed in triplicates with the SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). .. The expression level of SETDB1 mRNA was calculated using 2−ΔΔCt algorithm and normalized to that of GAPDH.

Article Title: RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)
Article Snippet: The gene expression levels of nine flowering related DEGs and three randomly selected DEGs at the VS stage between the early and late maturing lines were chosen for validation using qRT-PCR. .. The qRT-PCR analysis was performed with SsoFast EvaGreen supermix (Bio-Rad, ON, Canada) according to the manufacturer’s instructions using a Bio-Rad CFX96TM system.

Transfection:

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: .. Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Reactions were carried out using a iCycler iQ system (BioRAD) for 30 cycles (95°C for 15 s, 72°C for 30 s).

Sequencing:

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: Total RNA from these lysates, as well as from cell samples, was purified according to the RNeasy Miniprep kit directions and then analyzed by the KUMC Genome Sequencing Facility for quality, determined with an Agilent 2100 Bioanalyzer. .. RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad.

Article Title: RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)
Article Snippet: Four plants, used for Sanger sequencing were employed for qRT-PCR validation. .. The qRT-PCR analysis was performed with SsoFast EvaGreen supermix (Bio-Rad, ON, Canada) according to the manufacturer’s instructions using a Bio-Rad CFX96TM system.

Infection:

Article Title: EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways
Article Snippet: For S. aureus infection assays, infected samples were compared with parallel samples feeding on E. coli OP50, heat-killed by 30 min incubation at 95°C, plated on the same TSA medium. .. Total RNA was extracted using TRI Reagent (MRC), and reverse transcribed using the Superscript III kit (Invitrogen). cDNA was subjected to qRT-PCR analysis using SYBR green detection (Bio-Rad) on an iCycler machine (Eppendorf).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression
Article Snippet: .. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) Total RNA isolated from PCa cell lines with Aurum total RNA mini kit (Bio-Rad, Hercules, CA) was subjected to qRT-PCR analysis using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad), according to the manufacturer’s instructions. ..

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma
Article Snippet: RNA was reversely transcribed into cDNA using prime SCRIPT™ RT-PCR kit (TaKaRa, Dalian, China). .. The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method.

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo
Article Snippet: Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from liver tissue using methods previously described [ ] using TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized using M-MLV reverse transcriptase (Promega Co., Madison, WI) and qRT-PCR analysis was performed using 2× iQ™ SYBR® Green supermix PCR Master Mix (BioRad, Hercules, CA). .. Primers were tested by agarose gel electrophoresis following RT-PCR reaction to assure the expected transcript sizes.

Isolation:

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression
Article Snippet: .. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) Total RNA isolated from PCa cell lines with Aurum total RNA mini kit (Bio-Rad, Hercules, CA) was subjected to qRT-PCR analysis using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad), according to the manufacturer’s instructions. ..

Article Title: The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis
Article Snippet: Paragraph title: RNA isolation and qRT-PCR ... For mRNA quantification, cDNA was synthesized using iScript RT Supermix (Bio-Rad), following the manufacturer's protocol. qRT-PCR analysis was performed in triplicate using iQ SYBR green Supermix (BioRad) on an iCycler Real-Time Detection System (Eppendorf).

Purification:

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: Total RNA from these lysates, as well as from cell samples, was purified according to the RNeasy Miniprep kit directions and then analyzed by the KUMC Genome Sequencing Facility for quality, determined with an Agilent 2100 Bioanalyzer. .. RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad.

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: .. Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Reactions were carried out using a iCycler iQ system (BioRAD) for 30 cycles (95°C for 15 s, 72°C for 30 s).

Article Title: Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation
Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from tissues and cells using TRIzol™ plus RNA Purification Kit (Invitrogen) according to the manual of application. .. Then, a total of 500 ng RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR analysis was performed in triplicates with the SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems).

Polymerase Chain Reaction:

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. The purity of the PCR products was determined by melt curve analysis.

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo
Article Snippet: .. Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from liver tissue using methods previously described [ ] using TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized using M-MLV reverse transcriptase (Promega Co., Madison, WI) and qRT-PCR analysis was performed using 2× iQ™ SYBR® Green supermix PCR Master Mix (BioRad, Hercules, CA). ..

Plasmid Preparation:

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: .. Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Reactions were carried out using a iCycler iQ system (BioRAD) for 30 cycles (95°C for 15 s, 72°C for 30 s).

Software:

Article Title: In Vivo Determination of Direct Targets of the Nonsense-Mediated Decay Pathway in Drosophila
Article Snippet: .. All qRT-PCR analysis was conducted using iQ SYBR Green master mix and a MyiQ thermal cycler running iQ5 v2.0 software (Bio-Rad, Hercules, CA). ..

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Data analysis was completed using the iCycler IQ system software v.3.1.7050 (BioRAD).

Real-time Polymerase Chain Reaction:

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: .. RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad. .. Expression of human genes was normalized to that of human OAZ1.

Article Title: Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression
Article Snippet: .. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) Total RNA isolated from PCa cell lines with Aurum total RNA mini kit (Bio-Rad, Hercules, CA) was subjected to qRT-PCR analysis using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad), according to the manufacturer’s instructions. ..

Article Title: Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors
Article Snippet: .. Quantitative reverse transcriptase real-time PCR Total cellular RNA was purified from stably transfected HEK-293 Flp-In cells using GenElute mammalian total RNA purification kit (Sigma) according to the manufacturer’s instructions, followed by DNase treatment (Invitrogen). cDNA was synthesized using a gene-specific primer for the pcDNA5/FRT vector (SMN1cDNA) and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using isoform-specific primers ( Supplementary Tables S3 and S4 ) where each reaction contained 1 µl template cDNA, 10 pmol of each primer and 1X iQ SYBR green supermix (BioRAD) to a final volume of 25 µl. .. Reactions were carried out using a iCycler iQ system (BioRAD) for 30 cycles (95°C for 15 s, 72°C for 30 s).

Article Title: Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress
Article Snippet: .. The qRT-PCR analysis was performed with a CFX96 Real Time PCR Detection System (Bio-Rad). .. For rice, reactions (10 μL) included SsoFast EvaGreen Supermix Master Mix (Bio-Rad) and 0.6 μM gene-specific primers ( ).

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: .. For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System. .. Relative mRNA levels were calculated through comparison with GAPDH amplification values.

Article Title: RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A
Article Snippet: .. qRT-PCR analysis of ATP synthase genes Relative quantification of five genes in the three cotton lines were conducted by real-time qRT-PCR analysis with a C1000 TouchTM Thermal Cycler (Bio-Rad, USA) with TransStartR Tip Green qPCR SuperMix (Trans, China). ..

Article Title: Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation
Article Snippet: .. Then, a total of 500 ng RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR analysis was performed in triplicates with the SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). .. The expression level of SETDB1 mRNA was calculated using 2−ΔΔCt algorithm and normalized to that of GAPDH.

RNA Extraction:

Article Title: CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression
Article Snippet: Paragraph title: RNA extraction and quantitative (q)RT-PCR ... 1 μg of total RNA was used to obtain the cDNA with the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR analysis was carried out in 96-well plates with the CFX 96 thermocycler (Bio-Rad, Hercules, CA) by using 20 ng of each cDNA and Sybr Green (Applied Biosystems, Foster City, CA) or Real Master Mix (5Prime Inc., Gaithersburg, MD).

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma
Article Snippet: Paragraph title: RNA Extraction and qRT-PCR ... The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method.

RNA Expression:

Article Title: A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice
Article Snippet: Paragraph title: RNA Expression Analysis ... For qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using iScript (BioRad) according to the manufacturer’s instructions. qPCR reactions were then performed using TaqMan Gene Expression Assays (Applied Biosystems; for primers used see ) and an ABI7500 Fast Real-Time PCR System.

Agarose Gel Electrophoresis:

Article Title: Inflammation rapidly modulates the expression of ALDH1A1 (RALDH1) and vimentin in the liver and hepatic macrophages of rats in vivo
Article Snippet: Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from liver tissue using methods previously described [ ] using TRIzol reagent (Life Technologies, Carlsbad, CA). cDNA was synthesized using M-MLV reverse transcriptase (Promega Co., Madison, WI) and qRT-PCR analysis was performed using 2× iQ™ SYBR® Green supermix PCR Master Mix (BioRad, Hercules, CA). .. Primers were tested by agarose gel electrophoresis following RT-PCR reaction to assure the expected transcript sizes.

Concentration Assay:

Article Title: High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma
Article Snippet: Purity and concentration of RNA were detected by NanoDrop ND 1000. .. The qRT-PCR analysis was performed on an IQ5 system (Bio-Rad, USA) with SYBR Green reagents (Boster, Wuhan, China) according to the manufacturer’s instructions. β-actin was used as an internal reference for normalization and the relative expression of MLL2 mRNA was evaluated by the 2− ΔΔCT method.

Lysis:

Article Title: Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease
Article Snippet: qRT-PCR Using a mortar and pestle, renal tissues, stored at −80°C, were ground to a fine powder under liquid N2 and lysed immediately by the addition of RLT lysis buffer from the RNeasy Miniprep kit (Qiagen, Valencia, CA). .. RNAs with an RNA integrity number (RIN) of at least 8 were used for qRT-PCR analysis. cDNA was synthesized using a kit (#1725037) from Bio-Rad (Hercules, CA), and qRT-PCR analysis was performed on a Bio-Rad CFX96 real-time PCR machine using Sybr Green mix (#1725271) from Bio-Rad.

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  • 99
    Bio-Rad qrt pcr analysis
    Validation of RNA-seq results by <t>qRT-PCR.</t> Left and right y-axes indicate FPKM values from RNA-seq (blue bar) and relative transcript abundance from qRT-PCR (orange bar). D and S on the x-axis represent Daewonkong and SS0903-2B-21-1-2, respectively. Bars indicate means and standard deviation of three biological replicates. Asterisk above each bar indicates statistical difference between genotypes, as determined by Student’s t-test ( p
    Qrt Pcr Analysis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr analysis/product/Bio-Rad
    Average 99 stars, based on 368 article reviews
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    qrt pcr analysis - by Bioz Stars, 2020-02
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    79
    Bio-Rad real time quantitative reverse transcription pcr qrt pcr analysis qrt pcr
    Real-time <t>qRT-PCR</t> analysis of DEGs encoding respiration and detoxification-related enzymes in S . zeamais after oil-fumigation. The gene expression (mean ± SD) quantified as a relative fold change was carried out using the 2 −ΔΔCT method. The asterisks indicate significant differences in the expression level of DEGs between oil and no-oil treated samples (* p value
    Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analysis Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative reverse transcription pcr qrt pcr analysis qrt pcr/product/Bio-Rad
    Average 79 stars, based on 1 article reviews
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    78
    Bio-Rad quatitative real time polymerase chain reaction qrt pcr analysis
    Type I interferon (IFN) did not contribute to poly(I:C)-induced apoptosis of gastric adenocarcinoma cells. (A) BGC-823 cells were transfected with 1 μg/mL poly(I:C) for 4 h. <t>qRT-PCR</t> was performed to analyze type I IFN pathway-associated
    Quatitative Real Time Polymerase Chain Reaction Qrt Pcr Analysis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quatitative real time polymerase chain reaction qrt pcr analysis/product/Bio-Rad
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quatitative real time polymerase chain reaction qrt pcr analysis - by Bioz Stars, 2020-02
    78/100 stars
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    Validation of RNA-seq results by qRT-PCR. Left and right y-axes indicate FPKM values from RNA-seq (blue bar) and relative transcript abundance from qRT-PCR (orange bar). D and S on the x-axis represent Daewonkong and SS0903-2B-21-1-2, respectively. Bars indicate means and standard deviation of three biological replicates. Asterisk above each bar indicates statistical difference between genotypes, as determined by Student’s t-test ( p

    Journal: Scientific Reports

    Article Title: Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen

    doi: 10.1038/s41598-018-38219-6

    Figure Lengend Snippet: Validation of RNA-seq results by qRT-PCR. Left and right y-axes indicate FPKM values from RNA-seq (blue bar) and relative transcript abundance from qRT-PCR (orange bar). D and S on the x-axis represent Daewonkong and SS0903-2B-21-1-2, respectively. Bars indicate means and standard deviation of three biological replicates. Asterisk above each bar indicates statistical difference between genotypes, as determined by Student’s t-test ( p

    Article Snippet: qRT-PCR validation of DEGs Gene-specific primers for qRT-PCR analysis were designed based on the nucleotide sequences of selected DEGs using Primer3 ( http://primer3plus.com/ ) (Supplementary Table ). cDNA was synthesized using an iScript™ cDNA Synthesis Kit (Cat. 170-8891; Bio-Rad, Hercules, CA, USA). qRT-PCR was conducted using an iQ™ SYBR Green Supermix kit (Cat. 170-8882; Bio-Rad) on a LightCycler® 480 (Roche Diagnostics, Laval, QC, Canada).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation

    Genetic organization of flhBAFG and fliA genes, and flhF gene expression at 28°C. A . Genetic organization and transcriptional units of flhBAFG and fliA , with arrows showing the direction of transcription. Recognition sites for Eco RI and Stu I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn 3 - gusA insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. cDNA synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. B . Photographs of swim assay plates showing the swimming motility of the BGR1 mutant fliA ::Tn 3 - gusA45 and complementation with pBGFA carrying the fliA gene at 28°C and 37°C. C . Expression levels of the flhF gene in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9, and the flhC mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.

    Journal: PLoS ONE

    Article Title: Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

    doi: 10.1371/journal.pone.0084831

    Figure Lengend Snippet: Genetic organization of flhBAFG and fliA genes, and flhF gene expression at 28°C. A . Genetic organization and transcriptional units of flhBAFG and fliA , with arrows showing the direction of transcription. Recognition sites for Eco RI and Stu I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn 3 - gusA insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. cDNA synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. B . Photographs of swim assay plates showing the swimming motility of the BGR1 mutant fliA ::Tn 3 - gusA45 and complementation with pBGFA carrying the fliA gene at 28°C and 37°C. C . Expression levels of the flhF gene in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9, and the flhC mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.

    Article Snippet: For qRT-PCR analysis, the primers of flhC-cDNA, flhF-cDNA, flhC-qRTF, flhC-qRTR, flhF-qRTF, and flhF-qRTR were used ( ). qRT-PCR analysis was performed according to the manufacturer's instructions except using SsoFast™ EvaGreen Supermix (Bio-Rad) with 25 ng of cDNA as template. qRT-PCR was performed using a thermal cycler (Model C1000™; Bio-Rad) under the following conditions: 98°C for 2 min, followed by 35 cycles at 98°C for 20 s, 65°C for 30 s, and 72°C for 30 s. The 16S rRNA gene was used for data normalization.

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Mutagenesis, Quantitative RT-PCR

    Expression of flhC and other flagellar genes in the QS mutants at 28°C and 37°C. A . Expression levels of flhC in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments. B . Expression levels of fliC and flgK genes were assessed in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9 at 28°C and 37°C. BGF49, BGR1 fliC ::Tn 3 - gusA49 ; S2F49, BGS2 fliC ::Tn 3 - gusA49 ; S9F49, BGS9 fliC ::Tn 3 - gusA49 ; BGF28, BGR1 flgK ::Tn 3 - gusA28 ; S2F28, BGS2 flgK ::Tn 3 - gusA28 ; S9F28, BGS9 flgK ::Tn 3 - gusA28 .

    Journal: PLoS ONE

    Article Title: Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

    doi: 10.1371/journal.pone.0084831

    Figure Lengend Snippet: Expression of flhC and other flagellar genes in the QS mutants at 28°C and 37°C. A . Expression levels of flhC in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments. B . Expression levels of fliC and flgK genes were assessed in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9 at 28°C and 37°C. BGF49, BGR1 fliC ::Tn 3 - gusA49 ; S2F49, BGS2 fliC ::Tn 3 - gusA49 ; S9F49, BGS9 fliC ::Tn 3 - gusA49 ; BGF28, BGR1 flgK ::Tn 3 - gusA28 ; S2F28, BGS2 flgK ::Tn 3 - gusA28 ; S9F28, BGS9 flgK ::Tn 3 - gusA28 .

    Article Snippet: For qRT-PCR analysis, the primers of flhC-cDNA, flhF-cDNA, flhC-qRTF, flhC-qRTR, flhF-qRTF, and flhF-qRTR were used ( ). qRT-PCR analysis was performed according to the manufacturer's instructions except using SsoFast™ EvaGreen Supermix (Bio-Rad) with 25 ng of cDNA as template. qRT-PCR was performed using a thermal cycler (Model C1000™; Bio-Rad) under the following conditions: 98°C for 2 min, followed by 35 cycles at 98°C for 20 s, 65°C for 30 s, and 72°C for 30 s. The 16S rRNA gene was used for data normalization.

    Techniques: Expressing, Mutagenesis, Quantitative RT-PCR

    Real-time qRT-PCR analysis of DEGs encoding respiration and detoxification-related enzymes in S . zeamais after oil-fumigation. The gene expression (mean ± SD) quantified as a relative fold change was carried out using the 2 −ΔΔCT method. The asterisks indicate significant differences in the expression level of DEGs between oil and no-oil treated samples (* p value

    Journal: PLoS ONE

    Article Title: Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation

    doi: 10.1371/journal.pone.0167748

    Figure Lengend Snippet: Real-time qRT-PCR analysis of DEGs encoding respiration and detoxification-related enzymes in S . zeamais after oil-fumigation. The gene expression (mean ± SD) quantified as a relative fold change was carried out using the 2 −ΔΔCT method. The asterisks indicate significant differences in the expression level of DEGs between oil and no-oil treated samples (* p value

    Article Snippet: Real time quantitative reverse transcription PCR (qRT-PCR) analysis qRT-PCR was performed on a Bio-Rad iCycler iQ Real-time Detection System (Bio-Rad, Hercules, CA, USA).

    Techniques: Quantitative RT-PCR, Expressing

    Type I interferon (IFN) did not contribute to poly(I:C)-induced apoptosis of gastric adenocarcinoma cells. (A) BGC-823 cells were transfected with 1 μg/mL poly(I:C) for 4 h. qRT-PCR was performed to analyze type I IFN pathway-associated

    Journal: Journal of Interferon & Cytokine Research

    Article Title: Intracellular Poly(I:C) Initiated Gastric Adenocarcinoma Cell Apoptosis and Subsequently Ameliorated NK Cell Functions

    doi: 10.1089/jir.2012.0118

    Figure Lengend Snippet: Type I interferon (IFN) did not contribute to poly(I:C)-induced apoptosis of gastric adenocarcinoma cells. (A) BGC-823 cells were transfected with 1 μg/mL poly(I:C) for 4 h. qRT-PCR was performed to analyze type I IFN pathway-associated

    Article Snippet: Quatitative real-time polymerase chain reaction (qRT-PCR) analysis was detected with iCycleriQ real-time PCR system (Bio-Rad).

    Techniques: Transfection, Quantitative RT-PCR