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ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by <t>qRT-PCR,</t> of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.
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1) Product Images from "Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean"

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084416

ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.
Figure Legend Snippet: ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

Techniques Used: Expressing, Fluorescence, Quantitative RT-PCR

Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.
Figure Legend Snippet: Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

Techniques Used: Expressing, Northern Blot, Hybridization, Quantitative RT-PCR

Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.
Figure Legend Snippet: Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.

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

Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.
Figure Legend Snippet: Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.

Techniques Used: Over Expression, Transgenic Assay, Transformation Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR

Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.
Figure Legend Snippet: Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

Techniques Used: Expressing, Fluorescence, Quantitative RT-PCR

Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.
Figure Legend Snippet: Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.

Techniques Used: Expressing, Infection, Quantitative RT-PCR

2) Product Images from "Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy"

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy

Journal: Nature microbiology

doi: 10.1038/s41564-017-0016-3

Different lineages of ZIKV infection of whole blood elicit differential immunomodulatory responses Mock- or ZIKV-infected PBMCs and plasma were isolated from whole blood of equal number of male and female donors ( n = 8) at 24 hpi. a , Representative heat map of 44 immune mediator profiles in PBMCs. b , Plasma IFN-β level was determined by ELISA. Gene expression profiles of c , type I IFN signaling genes ( STAT1/2 , OAS1/3 and viperin ), d , M1-skewed pro-inflammatory genes ( CXCL10 , IL-23A , CD64 , CD80 , IL-18 , IDO , SOCS1 and CCR7 ), and e , M2-skewed immunosuppression genes ( IL-10 , Arg-1 , CD200R , CD163 , CD23 , CCL22 and VEGF ) were normalized to GAPDH and expressed as fold changes relative to mock controls. f , CXCL10 and IL-10 protein expressions within isolated plasma specimens ( n = 8) were determined using ELISA. g , CXCL10 and IL-10 mRNA expressions within sorted immune subsets from isolated PBMCs ( n = 4), using qRT-PCR. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P
Figure Legend Snippet: Different lineages of ZIKV infection of whole blood elicit differential immunomodulatory responses Mock- or ZIKV-infected PBMCs and plasma were isolated from whole blood of equal number of male and female donors ( n = 8) at 24 hpi. a , Representative heat map of 44 immune mediator profiles in PBMCs. b , Plasma IFN-β level was determined by ELISA. Gene expression profiles of c , type I IFN signaling genes ( STAT1/2 , OAS1/3 and viperin ), d , M1-skewed pro-inflammatory genes ( CXCL10 , IL-23A , CD64 , CD80 , IL-18 , IDO , SOCS1 and CCR7 ), and e , M2-skewed immunosuppression genes ( IL-10 , Arg-1 , CD200R , CD163 , CD23 , CCL22 and VEGF ) were normalized to GAPDH and expressed as fold changes relative to mock controls. f , CXCL10 and IL-10 protein expressions within isolated plasma specimens ( n = 8) were determined using ELISA. g , CXCL10 and IL-10 mRNA expressions within sorted immune subsets from isolated PBMCs ( n = 4), using qRT-PCR. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P

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

Asian ZIKV preferentially targets non-classical monocytes, driving specific IL-10 expression PBMCs were isolated from whole blood derived from healthy donors ( n = 6 – 11), followed by African ZIKV (MR766) or Asian ZIKV (H/PF/2013) infections at MOI 1 for 2, 5 or 8 dpi. a , Schematic representation of experimental set-up. b , Viral burden of total monocytes ( n = 6) during longitudinal infections detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. c , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets of mock- and ZIKV-infected monocytes were expressed as percentage change relative to mock controls, or presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells). d , At 2 dpi, total monocytes ( n = 11) were harvested to determine infectivity of total monocytes or e , gated monocyte subsets using intracellular ZIKV Env antigen staining by FACS. f , Viral burden were determined within sorted monocyte subsets using viral load qRT-PCR. g , CXCL10 and h , IL-10 mRNA expressions within sorted monocyte subsets were determined using qRT-PCR. i , Cell viability of total monocytes were determined using live/dead staining by FACS analysis. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P
Figure Legend Snippet: Asian ZIKV preferentially targets non-classical monocytes, driving specific IL-10 expression PBMCs were isolated from whole blood derived from healthy donors ( n = 6 – 11), followed by African ZIKV (MR766) or Asian ZIKV (H/PF/2013) infections at MOI 1 for 2, 5 or 8 dpi. a , Schematic representation of experimental set-up. b , Viral burden of total monocytes ( n = 6) during longitudinal infections detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. c , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets of mock- and ZIKV-infected monocytes were expressed as percentage change relative to mock controls, or presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells). d , At 2 dpi, total monocytes ( n = 11) were harvested to determine infectivity of total monocytes or e , gated monocyte subsets using intracellular ZIKV Env antigen staining by FACS. f , Viral burden were determined within sorted monocyte subsets using viral load qRT-PCR. g , CXCL10 and h , IL-10 mRNA expressions within sorted monocyte subsets were determined using qRT-PCR. i , Cell viability of total monocytes were determined using live/dead staining by FACS analysis. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P

Techniques Used: Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Infection, Staining, FACS

Pregnancy is associated with enhanced ZIKV infection and profound monocyte subset shift Whole blood was obtained from healthy non-pregnant ( n = 10) and pregnant women ( n = 5 per trimester) who were in the first (1 st ), second (2 nd ) or third (3 rd ) trimester of pregnancy. ZIKV infections were performed at MOI 1 and PBMCs were isolated at 40 hpi. Viral burdens within total PBMCs of a , non-pregnant and pregnant subjects or b , non-pregnant and each trimester were detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. c , Intracellular ZIKV E antigen in various blood subsets (CD45 + CD14 + monocytes, CD45 + CD56 + NK cells, CD45 + CD3 + T cells and CD45 + CD19 + B cells) were determined using FACS. d , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets present within mock- and ZIKV-infected PBMCs were presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells), or e , expressed as percentage change relative to mock controls. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P
Figure Legend Snippet: Pregnancy is associated with enhanced ZIKV infection and profound monocyte subset shift Whole blood was obtained from healthy non-pregnant ( n = 10) and pregnant women ( n = 5 per trimester) who were in the first (1 st ), second (2 nd ) or third (3 rd ) trimester of pregnancy. ZIKV infections were performed at MOI 1 and PBMCs were isolated at 40 hpi. Viral burdens within total PBMCs of a , non-pregnant and pregnant subjects or b , non-pregnant and each trimester were detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. c , Intracellular ZIKV E antigen in various blood subsets (CD45 + CD14 + monocytes, CD45 + CD56 + NK cells, CD45 + CD3 + T cells and CD45 + CD19 + B cells) were determined using FACS. d , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets present within mock- and ZIKV-infected PBMCs were presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells), or e , expressed as percentage change relative to mock controls. Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P

Techniques Used: Infection, Isolation, Quantitative RT-PCR, FACS, Flow Cytometry, Cytometry

ZIKV infects CD14 + monocytes and drives monocyte subset shift to CD16 + non-classical subset during whole blood infection Whole blood derived from healthy donors ( n = 8) were infected with African ZIKV (MR766) or Asian ZIKV (H/PF/2013) at MOI 1 for 24 h. PBMCs were isolated at 24 hpi for fluorescence-activated cell sorting (FACS) or RNA extraction. a , Schematic representation of experimental set-up. b , Viral burden of total PBMCs or c , sorted immune subsets (CD45 + CD14 + monocytes, CD45 + CD56 + NK cells, CD45 + CD3 + T cells and CD45 + CD19 + B cells) were detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. d , Intracellular ZIKV Env antigen in various blood subsets were determined using FACS. e , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets of mock- and ZIKV-infected PBMCs were expressed as percentage change relative to mock controls, or presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells). Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P
Figure Legend Snippet: ZIKV infects CD14 + monocytes and drives monocyte subset shift to CD16 + non-classical subset during whole blood infection Whole blood derived from healthy donors ( n = 8) were infected with African ZIKV (MR766) or Asian ZIKV (H/PF/2013) at MOI 1 for 24 h. PBMCs were isolated at 24 hpi for fluorescence-activated cell sorting (FACS) or RNA extraction. a , Schematic representation of experimental set-up. b , Viral burden of total PBMCs or c , sorted immune subsets (CD45 + CD14 + monocytes, CD45 + CD56 + NK cells, CD45 + CD3 + T cells and CD45 + CD19 + B cells) were detected using viral load qRT-PCR with the specific probes and primers against the ZIKV NS1 RNA. d , Intracellular ZIKV Env antigen in various blood subsets were determined using FACS. e , Flow cytometry profiling of classical (CD14 + CD16 − ), intermediate (CD14 hi CD16 + ) and non-classical (CD14 lo CD16 + ) monocyte subsets of mock- and ZIKV-infected PBMCs were expressed as percentage change relative to mock controls, or presented as no. of cells per subset within a gated CD45 hi SSC hi myeloid population (10 5 cells). Data (mean ± SEM) were presented in box plot showing upper (75%) and lower (25%) quartiles, with horizontal line as median and whiskers as maximum and minimum values observed. * P

Techniques Used: Infection, Derivative Assay, Isolation, Fluorescence, FACS, RNA Extraction, Quantitative RT-PCR, Flow Cytometry, Cytometry

3) Product Images from "Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish"

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish

Journal: Genetics

doi: 10.1534/genetics.116.199133

Expression characterization of zebrafish foxl2a-1 , foxl2a-2 , and foxl2b during ovary development and maintenance. (A) Sexually dimorphic expression between mature ovary and testis. (B) Sequential and divergent expression during ovary development and maintenance from 15 to 270 dpf. The qRT-PCR quantification of gene expression was normalized to actb1 . The relative expression values were first calculated against actb1 and then against that of foxl2a-1 at 25 dpf. (C and D) Ovary section in situ localization at (C) 35 dpf and (D) 105 dpf. Bars are shown at bottom-right of the images.
Figure Legend Snippet: Expression characterization of zebrafish foxl2a-1 , foxl2a-2 , and foxl2b during ovary development and maintenance. (A) Sexually dimorphic expression between mature ovary and testis. (B) Sequential and divergent expression during ovary development and maintenance from 15 to 270 dpf. The qRT-PCR quantification of gene expression was normalized to actb1 . The relative expression values were first calculated against actb1 and then against that of foxl2a-1 at 25 dpf. (C and D) Ovary section in situ localization at (C) 35 dpf and (D) 105 dpf. Bars are shown at bottom-right of the images.

Techniques Used: Expressing, Quantitative RT-PCR, In Situ

Foxl2a and foxl2b cooperatively regulate ovary differentiation and maintenance in zebrafish. (A–V) Relative expression qRT-PCR detection of the indicated genes in ovary (O), ovotestis (O/T), and testis (T) of homozygous double mutants ( foxl2a −/− /foxl2b −/− ) at 60 dpf ( n = 3). Gene names are indicated at the top. (W–Y) Relative expression qRT-PCR detection of (W) foxl2a-1 , (X) foxl2a-2 , and (Y) foxl2b in the ovaries of WT and foxl2b mutants ( foxl2b −/− ) or foxl2a mutants ( foxl2a −/− ) from 35 to 270 dpf, respectively ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).
Figure Legend Snippet: Foxl2a and foxl2b cooperatively regulate ovary differentiation and maintenance in zebrafish. (A–V) Relative expression qRT-PCR detection of the indicated genes in ovary (O), ovotestis (O/T), and testis (T) of homozygous double mutants ( foxl2a −/− /foxl2b −/− ) at 60 dpf ( n = 3). Gene names are indicated at the top. (W–Y) Relative expression qRT-PCR detection of (W) foxl2a-1 , (X) foxl2a-2 , and (Y) foxl2b in the ovaries of WT and foxl2b mutants ( foxl2b −/− ) or foxl2a mutants ( foxl2a −/− ) from 35 to 270 dpf, respectively ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).

Techniques Used: Expressing, Quantitative RT-PCR

Transcriptome profiling changes of cyp genes in the ovaries of foxl2a −/− and foxl2b −/− mutants. (A) Relatively transcribed changes of nine cyp genes between foxl2a −/− and WT ovaries or foxl2b −/− and WT ovaries at 150 dpf. (B–J) Relative expression qRT-PCR detection of nine cyp genes in WT, foxl2a −/− , and foxl2b −/− ovaries from 105 to 270 dpf ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).
Figure Legend Snippet: Transcriptome profiling changes of cyp genes in the ovaries of foxl2a −/− and foxl2b −/− mutants. (A) Relatively transcribed changes of nine cyp genes between foxl2a −/− and WT ovaries or foxl2b −/− and WT ovaries at 150 dpf. (B–J) Relative expression qRT-PCR detection of nine cyp genes in WT, foxl2a −/− , and foxl2b −/− ovaries from 105 to 270 dpf ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).

Techniques Used: Expressing, Quantitative RT-PCR

Transcriptome profiling changes of gonadal development-related genes in ovaries of foxl2a −/− and foxl2b −/− mutants. (A) Relatively transcribed changes of several kinds of gonadal development-related genes between foxl2a −/− and WT ovaries or foxl2b −/− and WT ovaries at 150 dpf. (B–N) Relative expression qRT-PCR detection of the indicated genes in WT, foxl2a −/− , and foxl2b −/− ovaries from 105 to 270 dpf ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).
Figure Legend Snippet: Transcriptome profiling changes of gonadal development-related genes in ovaries of foxl2a −/− and foxl2b −/− mutants. (A) Relatively transcribed changes of several kinds of gonadal development-related genes between foxl2a −/− and WT ovaries or foxl2b −/− and WT ovaries at 150 dpf. (B–N) Relative expression qRT-PCR detection of the indicated genes in WT, foxl2a −/− , and foxl2b −/− ovaries from 105 to 270 dpf ( n = 3). The qRT-PCR quantification of each gene expression is normalized to actb1 , and the data are presented as mean ± SEM * P ≤ 0.05 (Duncan’s multiple range test).

Techniques Used: Expressing, Quantitative RT-PCR

4) Product Images from "Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs"

Article Title: Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2013.00211

Age-dependent changes in DNA damage and repair. (A) mRNA fold expression changes normalized to a set of housekeeping genes and the expression in young animals as determined by qRT-PCR. The bars represent means ± 95% confidence intervals. (B) Protein expression levels for selected genes measured by Western blot. The bars represent means normalized to the Actin expression levels and the expression in young animals ± standard deviation. (C) Western blot images. (D) Olive comet tail lengths determined by the comet assay. The data points represent averages of 300–400 comets per age, the error bars show standard deviation. (E) Percentage of cells positive for γH2AX. The bars represent averages of 300–400 cells per age and organ ± standard deviations. * Indicates significance based on one-way ANOVA ( p
Figure Legend Snippet: Age-dependent changes in DNA damage and repair. (A) mRNA fold expression changes normalized to a set of housekeeping genes and the expression in young animals as determined by qRT-PCR. The bars represent means ± 95% confidence intervals. (B) Protein expression levels for selected genes measured by Western blot. The bars represent means normalized to the Actin expression levels and the expression in young animals ± standard deviation. (C) Western blot images. (D) Olive comet tail lengths determined by the comet assay. The data points represent averages of 300–400 comets per age, the error bars show standard deviation. (E) Percentage of cells positive for γH2AX. The bars represent averages of 300–400 cells per age and organ ± standard deviations. * Indicates significance based on one-way ANOVA ( p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Single Cell Gel Electrophoresis

Age-dependent changes in the activity of the apoptotic pathway. (A) mRNA expression levels of genes involved in the apoptotic pathway by qRT-PCR. The data points represent means normalized to the expression in young animals ± 95% confidence intervals. (B) Western blot images and quantification of cleaved CASP3 band intensity normalized to Actin levels and protein levels detected in tissues from young animals. Each data point represents mean ± standard deviation. (C) Plots of cell populations were measured by flow cytometry, the PerCP-Cy5 channel detected PI-positive cells, and the FITC channel detected Annexin V-FITC positive cells. (D) Quantification of flow cytometer analysis of Annexin V/PI staining. The data points represent averages of 6–8 animals with 10,000 events detected per animal. The asterisks on bars indicate significant differences between young animals, and the asterisks above lines indicate significant differences between the two samples. * Indicates significance based on one-way ANOVA ( p
Figure Legend Snippet: Age-dependent changes in the activity of the apoptotic pathway. (A) mRNA expression levels of genes involved in the apoptotic pathway by qRT-PCR. The data points represent means normalized to the expression in young animals ± 95% confidence intervals. (B) Western blot images and quantification of cleaved CASP3 band intensity normalized to Actin levels and protein levels detected in tissues from young animals. Each data point represents mean ± standard deviation. (C) Plots of cell populations were measured by flow cytometry, the PerCP-Cy5 channel detected PI-positive cells, and the FITC channel detected Annexin V-FITC positive cells. (D) Quantification of flow cytometer analysis of Annexin V/PI staining. The data points represent averages of 6–8 animals with 10,000 events detected per animal. The asterisks on bars indicate significant differences between young animals, and the asterisks above lines indicate significant differences between the two samples. * Indicates significance based on one-way ANOVA ( p

Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Flow Cytometry, Cytometry, Staining

The effects of age on cell cycle and DNA replication in the spleen and thymus of rats . (A) Fold changes of mRNA expression as measured by qRT-PCR standardized to gene expression in young animals. The bars represent mean ± 95% confidence interval. (B) Western blots showing protein levels of CDK2, p16, and PCNA. (C) Protein expression levels of selected genes standardized to the expression in young animals and normalized to the expression of Actin. The error bars represent standard deviations. * Indicates significance based on Student's t-test ( p
Figure Legend Snippet: The effects of age on cell cycle and DNA replication in the spleen and thymus of rats . (A) Fold changes of mRNA expression as measured by qRT-PCR standardized to gene expression in young animals. The bars represent mean ± 95% confidence interval. (B) Western blots showing protein levels of CDK2, p16, and PCNA. (C) Protein expression levels of selected genes standardized to the expression in young animals and normalized to the expression of Actin. The error bars represent standard deviations. * Indicates significance based on Student's t-test ( p

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

Changes in epigenetic regulation in the thymus and spleen of aging rats . (A) Protein levels of DNMT1 and DNMT3a. The bars represent means normalized to Actin expression and expression in young animals ± standard deviation. (B) Global DNA methylation levels as measured by the Cytosine Extension assay. The bars represent mean ± standard deviations. (C) RNA and protein expression levels of histone modifying enzymes as measured by qRT-PCR or Western blot. For qRT-PCR graphs, the bars represent mean standardized to expression levels in young animals. The error bars represent 95% confidence intervals. For Western blots, the bars represent means normalized to Actin bands and expression in young animals ± standard deviation. (D) Quantification of histones and histone modifications by Western blot. The bars represent means normalized to Actin bands or Coomassie loading controls and expression levels in young animals ± standard deviations. (E) Western blot images. * Indicates significance based on Student's t-test ( p
Figure Legend Snippet: Changes in epigenetic regulation in the thymus and spleen of aging rats . (A) Protein levels of DNMT1 and DNMT3a. The bars represent means normalized to Actin expression and expression in young animals ± standard deviation. (B) Global DNA methylation levels as measured by the Cytosine Extension assay. The bars represent mean ± standard deviations. (C) RNA and protein expression levels of histone modifying enzymes as measured by qRT-PCR or Western blot. For qRT-PCR graphs, the bars represent mean standardized to expression levels in young animals. The error bars represent 95% confidence intervals. For Western blots, the bars represent means normalized to Actin bands and expression in young animals ± standard deviation. (D) Quantification of histones and histone modifications by Western blot. The bars represent means normalized to Actin bands or Coomassie loading controls and expression levels in young animals ± standard deviations. (E) Western blot images. * Indicates significance based on Student's t-test ( p

Techniques Used: Expressing, Standard Deviation, DNA Methylation Assay, Quantitative RT-PCR, Western Blot

5) Product Images from "RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family"

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family

Journal: Nature Communications

doi: 10.1038/s41467-018-08035-7

HSPA1A inhibits chondrocyte apoptosis. a qRT-PCR analysis ( n = 7) of matrix-degrading enzymes in chondrocytes treated with IL-1β and infected with 400 MOI of control virus or the indicated MOIs of Ad-shZFP36L1. b , c Chondrocytes were infected with Ad-C or Ad-HSPA1A ( b ) or transfected with empty vector (EV, 1 μg) or vectors encoding WT-HSPA1A or K71E-HSPA1A ( c ) and left untreated or exposed to the NO donor, SNP, for 6 h. Apoptotic chondrocytes were identified and quantified by TUNEL staining ( n = 5). d – f Detection and quantitation of apoptotic chondrocytes in calcified cartilage (CC) and articular cartilage (AC) of sham- or DMM-operated mice subjected to IA injection with Ad-C or Ad-HSPA1A ( n = 5 mice per group) ( d ), sham- or DMM-operated Zfp36l1 +/− and WT mice ( e ; n = 6 mice per group), or sham- or DMM-operated mice subjected to IA injection with Ad-shC or Ad-shZFP36L1 ( f ; n = 5 mice per group). Means ± s.e.m. with one-way ANOVA ( a – c ) and two-tailed t -test ( d – f ). * P
Figure Legend Snippet: HSPA1A inhibits chondrocyte apoptosis. a qRT-PCR analysis ( n = 7) of matrix-degrading enzymes in chondrocytes treated with IL-1β and infected with 400 MOI of control virus or the indicated MOIs of Ad-shZFP36L1. b , c Chondrocytes were infected with Ad-C or Ad-HSPA1A ( b ) or transfected with empty vector (EV, 1 μg) or vectors encoding WT-HSPA1A or K71E-HSPA1A ( c ) and left untreated or exposed to the NO donor, SNP, for 6 h. Apoptotic chondrocytes were identified and quantified by TUNEL staining ( n = 5). d – f Detection and quantitation of apoptotic chondrocytes in calcified cartilage (CC) and articular cartilage (AC) of sham- or DMM-operated mice subjected to IA injection with Ad-C or Ad-HSPA1A ( n = 5 mice per group) ( d ), sham- or DMM-operated Zfp36l1 +/− and WT mice ( e ; n = 6 mice per group), or sham- or DMM-operated mice subjected to IA injection with Ad-shC or Ad-shZFP36L1 ( f ; n = 5 mice per group). Means ± s.e.m. with one-way ANOVA ( a – c ) and two-tailed t -test ( d – f ). * P

Techniques Used: Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Quantitation Assay, Mouse Assay, IA, Injection, Two Tailed Test

Upregulation of ZFP36L1 in chondrocytes stimulated with OA-associated catabolic regulators. a Microarray analysis of AU-rich element (ARE)-binding proteins in chondrocytes treated with IL-1β or infected with Ad-HIF-2α or Ad-ZIP8. b , c qRT-PCR analyses of ZFP36 family members in chondrocytes treated with IL-1β or infected with 800 MOI of control virus (Ad-C) or the indicated MOIs of Ad-HIF-2α or Ad-ZIP8 ( n = 3 or 5). d Representative images of Alcian blue staining and ZFP36L1 immunostaining in undamaged and damaged regions of OA cartilage from the same patient ( n = 7 patients). e Representative images of Safranin-O staining and ZFP36L1 immunostaining in mouse OA cartilage caused by DMM surgery ( n = 5 mice per group) or by IA injection of Ad-HIF-2α or Ad-ZIP8 ( n = 5 mice per group). Means ± s.e.m. with one-way ANOVA (* P
Figure Legend Snippet: Upregulation of ZFP36L1 in chondrocytes stimulated with OA-associated catabolic regulators. a Microarray analysis of AU-rich element (ARE)-binding proteins in chondrocytes treated with IL-1β or infected with Ad-HIF-2α or Ad-ZIP8. b , c qRT-PCR analyses of ZFP36 family members in chondrocytes treated with IL-1β or infected with 800 MOI of control virus (Ad-C) or the indicated MOIs of Ad-HIF-2α or Ad-ZIP8 ( n = 3 or 5). d Representative images of Alcian blue staining and ZFP36L1 immunostaining in undamaged and damaged regions of OA cartilage from the same patient ( n = 7 patients). e Representative images of Safranin-O staining and ZFP36L1 immunostaining in mouse OA cartilage caused by DMM surgery ( n = 5 mice per group) or by IA injection of Ad-HIF-2α or Ad-ZIP8 ( n = 5 mice per group). Means ± s.e.m. with one-way ANOVA (* P

Techniques Used: Microarray, Binding Assay, Infection, Quantitative RT-PCR, Staining, Immunostaining, Mouse Assay, IA, Injection

ZFP36L1 targets HSP70 family members in chondrocytes. a Microarray analysis of chondrocytes infected with 400 MOI of Ad-shZFP36L1 to knock down ZFP36L1 (3 replicates). b , c qRT-PCR ( b ) and RT-PCR and western blot analyses ( c ) of ZFP36L1, HSPA1A, HSPA1B, and HSP70 in chondrocytes infected with Ad-C (800 MOI) or the indicated MOIs of Ad-shZFP36L1 or Ad-ZFP36L1 for 36 h ( n = 4). Representative images are presented ( c ). d RNA binding of ZFP36L1 in chondrocytes infected with Ad-C or Ad-ZFP36L1. The binding of ZFP36L1 to the 3′-UTR of HSPA1A was determined by RT-PCR of immunoprecipitates obtained using anti-ZFP36L1 or IgG. Representative images are presented from five biologically independent samples. e mRNA decay assay. Chondrocytes were infected with Ad-HSPA1A with or without Ad- ZFP36L1 for 12 h, and then exposed to Actinomycin D (1 μg ml − 1 ) for the indicated time periods. The mRNA levels of HSPA1A were quantified by qRT-PCR analysis ( n = 9). Representative western blot images of HSP70 and ZFP36L1 proteins. Means ± s.e.m. with one-way ANOVA (* P
Figure Legend Snippet: ZFP36L1 targets HSP70 family members in chondrocytes. a Microarray analysis of chondrocytes infected with 400 MOI of Ad-shZFP36L1 to knock down ZFP36L1 (3 replicates). b , c qRT-PCR ( b ) and RT-PCR and western blot analyses ( c ) of ZFP36L1, HSPA1A, HSPA1B, and HSP70 in chondrocytes infected with Ad-C (800 MOI) or the indicated MOIs of Ad-shZFP36L1 or Ad-ZFP36L1 for 36 h ( n = 4). Representative images are presented ( c ). d RNA binding of ZFP36L1 in chondrocytes infected with Ad-C or Ad-ZFP36L1. The binding of ZFP36L1 to the 3′-UTR of HSPA1A was determined by RT-PCR of immunoprecipitates obtained using anti-ZFP36L1 or IgG. Representative images are presented from five biologically independent samples. e mRNA decay assay. Chondrocytes were infected with Ad-HSPA1A with or without Ad- ZFP36L1 for 12 h, and then exposed to Actinomycin D (1 μg ml − 1 ) for the indicated time periods. The mRNA levels of HSPA1A were quantified by qRT-PCR analysis ( n = 9). Representative western blot images of HSP70 and ZFP36L1 proteins. Means ± s.e.m. with one-way ANOVA (* P

Techniques Used: Microarray, Infection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, RNA Binding Assay, Binding Assay, Mrna Decay Assay

6) Product Images from "Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology"

Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology

Journal: Life Science Alliance

doi: 10.26508/lsa.201800268

Knockdown efficiency of different shRNAs. (A–C) qRT-PCR showing the mRNA expression of CANP1 (A), CAPN7 (B), and CTSL (C) in HEK293 cells after transfection of shRNA targeting CAPN1 , CAPN7 , and CTSL , respectively. Two shRNAs were used for each gene, which all resulted in significant knockdown of the gene expression. Data were presented as mean ± SEM, n = 3. ** P
Figure Legend Snippet: Knockdown efficiency of different shRNAs. (A–C) qRT-PCR showing the mRNA expression of CANP1 (A), CAPN7 (B), and CTSL (C) in HEK293 cells after transfection of shRNA targeting CAPN1 , CAPN7 , and CTSL , respectively. Two shRNAs were used for each gene, which all resulted in significant knockdown of the gene expression. Data were presented as mean ± SEM, n = 3. ** P

Techniques Used: Quantitative RT-PCR, Expressing, Transfection, shRNA

Effects of Z-FA-FMK on SMN protein expression in type II SMA patient fibroblast cells (GM22592). (A, B) In SMA type II fibroblast cells, vehicle (DMSO) or Z-FA-FMK were applied for 2 d; Western blot analysis revealed a dose-dependent increase in the expression of SMN proteins after treatment with Z-FA-FMK. n = 4. (C) qRT-PCR showing the mRNA expression in fibroblast cells after treatment with different concentrations of Z-FA-FMK. Data were presented as mean ± SEM, n = 3. * P
Figure Legend Snippet: Effects of Z-FA-FMK on SMN protein expression in type II SMA patient fibroblast cells (GM22592). (A, B) In SMA type II fibroblast cells, vehicle (DMSO) or Z-FA-FMK were applied for 2 d; Western blot analysis revealed a dose-dependent increase in the expression of SMN proteins after treatment with Z-FA-FMK. n = 4. (C) qRT-PCR showing the mRNA expression in fibroblast cells after treatment with different concentrations of Z-FA-FMK. Data were presented as mean ± SEM, n = 3. * P

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

Identification of Z-FA-FMK as a potent compound that increases SMN protein expression in HEK293 and patient fibroblast cells. (A) Fluorescent images showed the increased GFP fluorescence at 2 d after treatment with different concentrations of Z-FA-FMK. Scale bar, 50 μm. (B, C) Western blot analysis of these samples revealed a dose-dependent increase in the expression of SMNΔ7-GFP proteins after the treatment of Z-FA-FMK. (D) qRT-PCR showing the mRNA expression in SMN2-GFP cells after treatment with different concentrations of Z-FA-FMK. n = 3. (E–G) In SMA type I fibroblast cells, vehicle (DMSO) or Z-FA-FMK were applied for 2 d, followed by analysis of the protein (E, F) and mRNA (G) expression in these cells. n = 3. Data were presented as mean ± SEM, * P
Figure Legend Snippet: Identification of Z-FA-FMK as a potent compound that increases SMN protein expression in HEK293 and patient fibroblast cells. (A) Fluorescent images showed the increased GFP fluorescence at 2 d after treatment with different concentrations of Z-FA-FMK. Scale bar, 50 μm. (B, C) Western blot analysis of these samples revealed a dose-dependent increase in the expression of SMNΔ7-GFP proteins after the treatment of Z-FA-FMK. (D) qRT-PCR showing the mRNA expression in SMN2-GFP cells after treatment with different concentrations of Z-FA-FMK. n = 3. (E–G) In SMA type I fibroblast cells, vehicle (DMSO) or Z-FA-FMK were applied for 2 d, followed by analysis of the protein (E, F) and mRNA (G) expression in these cells. n = 3. Data were presented as mean ± SEM, * P

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

Examination of gene copy numbers in fibroblast cells of WT (normal), SMA patients (GM03813 and GM22592), and patient father (GM03815). Notes: (1) The SMN gene copy numbers were measured using real-time TaqMan PCR method as described before ( Gomez-Curet et al, 2007 ). Detailed information about the sequences of primers, probers, and blockers can be found in the article ( Gomez-Curet et al, 2007 ). (2) For SMN1 assay, the PCR reactions (total 15 μl) contained 25 ng of genomic DNA, TaqMan Gene Expression Master Mix (Applied Biosystems), 250 nM of SMN probe, and 300 nM of SMN1 primers. For SMN2 assay, the reactions consisted of the same DNA and master mix, the SMN probe (650 nM), SMN2 primer (450 nM), and SMN1 blocker (650 nM). CFTR (as Control) reactions contained 25 ng of genomic DNA, 1× TaqMan Gene Expression Master Mix, 450 nM CFTR primers, and 250 nM CFTR probe. (3) The qRT-PCR for SMN1 , SMN2 , and CFTR were performed using ABI QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). The qRT-PCR conditions were 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min. The value was calculated using the ΔΔCT method. Data were presented as mean ± SD, n = 3–6. The copy numbers for SMN1 and SMN2 are coinciding with what were reported on the Coriell Institute Biorepositories Web site.
Figure Legend Snippet: Examination of gene copy numbers in fibroblast cells of WT (normal), SMA patients (GM03813 and GM22592), and patient father (GM03815). Notes: (1) The SMN gene copy numbers were measured using real-time TaqMan PCR method as described before ( Gomez-Curet et al, 2007 ). Detailed information about the sequences of primers, probers, and blockers can be found in the article ( Gomez-Curet et al, 2007 ). (2) For SMN1 assay, the PCR reactions (total 15 μl) contained 25 ng of genomic DNA, TaqMan Gene Expression Master Mix (Applied Biosystems), 250 nM of SMN probe, and 300 nM of SMN1 primers. For SMN2 assay, the reactions consisted of the same DNA and master mix, the SMN probe (650 nM), SMN2 primer (450 nM), and SMN1 blocker (650 nM). CFTR (as Control) reactions contained 25 ng of genomic DNA, 1× TaqMan Gene Expression Master Mix, 450 nM CFTR primers, and 250 nM CFTR probe. (3) The qRT-PCR for SMN1 , SMN2 , and CFTR were performed using ABI QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). The qRT-PCR conditions were 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min. The value was calculated using the ΔΔCT method. Data were presented as mean ± SD, n = 3–6. The copy numbers for SMN1 and SMN2 are coinciding with what were reported on the Coriell Institute Biorepositories Web site.

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

7) Product Images from "Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development"

Article Title: Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.01604

TAG content in GmLEC2a over-expression Arabidopsis seeds. Ectopic expression of GmLEC2a in Arabidopsis alters TAG composition and enhance TAG content in mature seeds. (A) Expression of GmLEC2a in atlec2 mutant seeds by semi-qRT-PCR. (B) Composition of fatty acids in seeds of Arabidopsis atlec2 , wild-type WS-2 , and GmLEC2/atlect2 complementation plants. (C) Composition of fatty acids in seeds of Col-0 and GmLEC2aOE. (D) Over-expression of GmLEC2a in Col-0 ( GmLEC2aOE ) detected by using semi-qRT-PCR. (E ) TAG contents in seeds of Arabidopsis atlec2 , wild-type WS-2 , and GmLEC2/atlect2 complementation plants. (F) TAG content in seeds of wild-type Col-0 and GmLEC2a overexpression (GmLEC2aOE) plants. Transcript levels are expressed relatively to that of AtACTIN . All data are three biological replicates and are expressed as means ± SD. ∗∗ P
Figure Legend Snippet: TAG content in GmLEC2a over-expression Arabidopsis seeds. Ectopic expression of GmLEC2a in Arabidopsis alters TAG composition and enhance TAG content in mature seeds. (A) Expression of GmLEC2a in atlec2 mutant seeds by semi-qRT-PCR. (B) Composition of fatty acids in seeds of Arabidopsis atlec2 , wild-type WS-2 , and GmLEC2/atlect2 complementation plants. (C) Composition of fatty acids in seeds of Col-0 and GmLEC2aOE. (D) Over-expression of GmLEC2a in Col-0 ( GmLEC2aOE ) detected by using semi-qRT-PCR. (E ) TAG contents in seeds of Arabidopsis atlec2 , wild-type WS-2 , and GmLEC2/atlect2 complementation plants. (F) TAG content in seeds of wild-type Col-0 and GmLEC2a overexpression (GmLEC2aOE) plants. Transcript levels are expressed relatively to that of AtACTIN . All data are three biological replicates and are expressed as means ± SD. ∗∗ P

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

Validation of transcriptomic data with qRT-PCR. (A) Verification of transcription factors and few transporters transcriptomic data with qRT-PCR. (B) Verification of transcriptomic data of genes involved in TAG, fatty acid, sucrose biosynthesis and transporters of fatty acid, wax with qRT-PCR. Transcript levels are expressed relatively to that of GmACTIN . All data are three biological replicates and are expressed as means ± SD. ∗∗ P
Figure Legend Snippet: Validation of transcriptomic data with qRT-PCR. (A) Verification of transcription factors and few transporters transcriptomic data with qRT-PCR. (B) Verification of transcriptomic data of genes involved in TAG, fatty acid, sucrose biosynthesis and transporters of fatty acid, wax with qRT-PCR. Transcript levels are expressed relatively to that of GmACTIN . All data are three biological replicates and are expressed as means ± SD. ∗∗ P

Techniques Used: Quantitative RT-PCR

Ectopic expression of GmLEC2a in soybean hairy roots. (A,B) Hairy roots over-expressing GmLEC2a and GUS. The cotyledons of germinating soybean seeds were used for infection with Agrobacteria K599 harboring p B2GW7-GmLEC2a or GUS gene (control). The g enerated hairy roots over-expressing GUS (A) and GmLEC2a (B) were selected on MS medium containing ppt. (C,D) TLC analysis of neutral lipids extracted from hairy root over-expressing GmLEC2a (D) and GUS control (C) . (E) qRT-PCR conformation of GmLEC2a or GUS gene expression. Transcript levels are expressed relatively to that of GmACTIN. (F,G) Comparison of TAG composition (G) and content (F) in hairy roots over-expressing GmLEC2a and GUS control grown and extracted under the identical conditions. More than 5 independent transgenic hairy root lines were analyzed. Data are from three biological replicates. All data are three biological replicates and are expressed as means ± SD. ∗∗ P
Figure Legend Snippet: Ectopic expression of GmLEC2a in soybean hairy roots. (A,B) Hairy roots over-expressing GmLEC2a and GUS. The cotyledons of germinating soybean seeds were used for infection with Agrobacteria K599 harboring p B2GW7-GmLEC2a or GUS gene (control). The g enerated hairy roots over-expressing GUS (A) and GmLEC2a (B) were selected on MS medium containing ppt. (C,D) TLC analysis of neutral lipids extracted from hairy root over-expressing GmLEC2a (D) and GUS control (C) . (E) qRT-PCR conformation of GmLEC2a or GUS gene expression. Transcript levels are expressed relatively to that of GmACTIN. (F,G) Comparison of TAG composition (G) and content (F) in hairy roots over-expressing GmLEC2a and GUS control grown and extracted under the identical conditions. More than 5 independent transgenic hairy root lines were analyzed. Data are from three biological replicates. All data are three biological replicates and are expressed as means ± SD. ∗∗ P

Techniques Used: Expressing, Infection, Mass Spectrometry, Thin Layer Chromatography, Quantitative RT-PCR, Transgenic Assay

Identification of GmLEC2. (A) Phylogenetic analysis of GmLEC2s with other functionally characterized LEC2 proteins from Arabidopsis, castor bean, and maize. Rooted phylogenetic tree was constructed by using MEGA6 program through neighbor joining method. Bar shows 0.05 amino acid substitution. (B) Expression patterns of GmLEC2a in soybean tissues. Seeds, pods, nodules and flower from 8 to 11 weeks old plants, root, stem and leaf from 12 to 15 days old seedling were harvested for qRT-PCR. Transcript levels are expressed relative to that of GmACTIN . (C) Expression level of GmLEC2a in seed at six different developmental stages. Stages were classified on the basis of seed weight, as follows: stage 1 (S1). 40–70 mg; stage 2 (S2). 80–100 mg; stage 3 (S3).150–200 mg; stage 4 (Stage 4). 250–300 mg; stage 5 (S5). 350–430 mg; stage 6 (S6). 320–350 mg. Transcript levels are expressed relatively to that of GmACTIN .
Figure Legend Snippet: Identification of GmLEC2. (A) Phylogenetic analysis of GmLEC2s with other functionally characterized LEC2 proteins from Arabidopsis, castor bean, and maize. Rooted phylogenetic tree was constructed by using MEGA6 program through neighbor joining method. Bar shows 0.05 amino acid substitution. (B) Expression patterns of GmLEC2a in soybean tissues. Seeds, pods, nodules and flower from 8 to 11 weeks old plants, root, stem and leaf from 12 to 15 days old seedling were harvested for qRT-PCR. Transcript levels are expressed relative to that of GmACTIN . (C) Expression level of GmLEC2a in seed at six different developmental stages. Stages were classified on the basis of seed weight, as follows: stage 1 (S1). 40–70 mg; stage 2 (S2). 80–100 mg; stage 3 (S3).150–200 mg; stage 4 (Stage 4). 250–300 mg; stage 5 (S5). 350–430 mg; stage 6 (S6). 320–350 mg. Transcript levels are expressed relatively to that of GmACTIN .

Techniques Used: Construct, Expressing, Quantitative RT-PCR

8) Product Images from "RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family"

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family

Journal: Nature Communications

doi: 10.1038/s41467-018-08035-7

HSPA1A inhibits chondrocyte apoptosis. a qRT-PCR analysis ( n = 7) of matrix-degrading enzymes in chondrocytes treated with IL-1β and infected with 400 MOI of control virus or the indicated MOIs of Ad-shZFP36L1. b , c Chondrocytes were infected with Ad-C or Ad-HSPA1A ( b ) or transfected with empty vector (EV, 1 μg) or vectors encoding WT-HSPA1A or K71E-HSPA1A ( c ) and left untreated or exposed to the NO donor, SNP, for 6 h. Apoptotic chondrocytes were identified and quantified by TUNEL staining ( n = 5). d – f Detection and quantitation of apoptotic chondrocytes in calcified cartilage (CC) and articular cartilage (AC) of sham- or DMM-operated mice subjected to IA injection with Ad-C or Ad-HSPA1A ( n = 5 mice per group) ( d ), sham- or DMM-operated Zfp36l1 +/− and WT mice ( e ; n = 6 mice per group), or sham- or DMM-operated mice subjected to IA injection with Ad-shC or Ad-shZFP36L1 ( f ; n = 5 mice per group). Means ± s.e.m. with one-way ANOVA ( a – c ) and two-tailed t -test ( d – f ). * P
Figure Legend Snippet: HSPA1A inhibits chondrocyte apoptosis. a qRT-PCR analysis ( n = 7) of matrix-degrading enzymes in chondrocytes treated with IL-1β and infected with 400 MOI of control virus or the indicated MOIs of Ad-shZFP36L1. b , c Chondrocytes were infected with Ad-C or Ad-HSPA1A ( b ) or transfected with empty vector (EV, 1 μg) or vectors encoding WT-HSPA1A or K71E-HSPA1A ( c ) and left untreated or exposed to the NO donor, SNP, for 6 h. Apoptotic chondrocytes were identified and quantified by TUNEL staining ( n = 5). d – f Detection and quantitation of apoptotic chondrocytes in calcified cartilage (CC) and articular cartilage (AC) of sham- or DMM-operated mice subjected to IA injection with Ad-C or Ad-HSPA1A ( n = 5 mice per group) ( d ), sham- or DMM-operated Zfp36l1 +/− and WT mice ( e ; n = 6 mice per group), or sham- or DMM-operated mice subjected to IA injection with Ad-shC or Ad-shZFP36L1 ( f ; n = 5 mice per group). Means ± s.e.m. with one-way ANOVA ( a – c ) and two-tailed t -test ( d – f ). * P

Techniques Used: Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Quantitation Assay, Mouse Assay, IA, Injection, Two Tailed Test

Upregulation of ZFP36L1 in chondrocytes stimulated with OA-associated catabolic regulators. a Microarray analysis of AU-rich element (ARE)-binding proteins in chondrocytes treated with IL-1β or infected with Ad-HIF-2α or Ad-ZIP8. b , c qRT-PCR analyses of ZFP36 family members in chondrocytes treated with IL-1β or infected with 800 MOI of control virus (Ad-C) or the indicated MOIs of Ad-HIF-2α or Ad-ZIP8 ( n = 3 or 5). d Representative images of Alcian blue staining and ZFP36L1 immunostaining in undamaged and damaged regions of OA cartilage from the same patient ( n = 7 patients). e Representative images of Safranin-O staining and ZFP36L1 immunostaining in mouse OA cartilage caused by DMM surgery ( n = 5 mice per group) or by IA injection of Ad-HIF-2α or Ad-ZIP8 ( n = 5 mice per group). Means ± s.e.m. with one-way ANOVA (* P
Figure Legend Snippet: Upregulation of ZFP36L1 in chondrocytes stimulated with OA-associated catabolic regulators. a Microarray analysis of AU-rich element (ARE)-binding proteins in chondrocytes treated with IL-1β or infected with Ad-HIF-2α or Ad-ZIP8. b , c qRT-PCR analyses of ZFP36 family members in chondrocytes treated with IL-1β or infected with 800 MOI of control virus (Ad-C) or the indicated MOIs of Ad-HIF-2α or Ad-ZIP8 ( n = 3 or 5). d Representative images of Alcian blue staining and ZFP36L1 immunostaining in undamaged and damaged regions of OA cartilage from the same patient ( n = 7 patients). e Representative images of Safranin-O staining and ZFP36L1 immunostaining in mouse OA cartilage caused by DMM surgery ( n = 5 mice per group) or by IA injection of Ad-HIF-2α or Ad-ZIP8 ( n = 5 mice per group). Means ± s.e.m. with one-way ANOVA (* P

Techniques Used: Microarray, Binding Assay, Infection, Quantitative RT-PCR, Staining, Immunostaining, Mouse Assay, IA, Injection

ZFP36L1 targets HSP70 family members in chondrocytes. a Microarray analysis of chondrocytes infected with 400 MOI of Ad-shZFP36L1 to knock down ZFP36L1 (3 replicates). b , c qRT-PCR ( b ) and RT-PCR and western blot analyses ( c ) of ZFP36L1, HSPA1A, HSPA1B, and HSP70 in chondrocytes infected with Ad-C (800 MOI) or the indicated MOIs of Ad-shZFP36L1 or Ad-ZFP36L1 for 36 h ( n = 4). Representative images are presented ( c ). d RNA binding of ZFP36L1 in chondrocytes infected with Ad-C or Ad-ZFP36L1. The binding of ZFP36L1 to the 3′-UTR of HSPA1A was determined by RT-PCR of immunoprecipitates obtained using anti-ZFP36L1 or IgG. Representative images are presented from five biologically independent samples. e mRNA decay assay. Chondrocytes were infected with Ad-HSPA1A with or without Ad- ZFP36L1 for 12 h, and then exposed to Actinomycin D (1 μg ml − 1 ) for the indicated time periods. The mRNA levels of HSPA1A were quantified by qRT-PCR analysis ( n = 9). Representative western blot images of HSP70 and ZFP36L1 proteins. Means ± s.e.m. with one-way ANOVA (* P
Figure Legend Snippet: ZFP36L1 targets HSP70 family members in chondrocytes. a Microarray analysis of chondrocytes infected with 400 MOI of Ad-shZFP36L1 to knock down ZFP36L1 (3 replicates). b , c qRT-PCR ( b ) and RT-PCR and western blot analyses ( c ) of ZFP36L1, HSPA1A, HSPA1B, and HSP70 in chondrocytes infected with Ad-C (800 MOI) or the indicated MOIs of Ad-shZFP36L1 or Ad-ZFP36L1 for 36 h ( n = 4). Representative images are presented ( c ). d RNA binding of ZFP36L1 in chondrocytes infected with Ad-C or Ad-ZFP36L1. The binding of ZFP36L1 to the 3′-UTR of HSPA1A was determined by RT-PCR of immunoprecipitates obtained using anti-ZFP36L1 or IgG. Representative images are presented from five biologically independent samples. e mRNA decay assay. Chondrocytes were infected with Ad-HSPA1A with or without Ad- ZFP36L1 for 12 h, and then exposed to Actinomycin D (1 μg ml − 1 ) for the indicated time periods. The mRNA levels of HSPA1A were quantified by qRT-PCR analysis ( n = 9). Representative western blot images of HSP70 and ZFP36L1 proteins. Means ± s.e.m. with one-way ANOVA (* P

Techniques Used: Microarray, Infection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, RNA Binding Assay, Binding Assay, Mrna Decay Assay

9) Product Images from "In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good."

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2005840

Protospacer and 5’-triphosphate determine the intensity of the gRNA-mediated IFNβ response. (A) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with equal amounts of gRNAs containing different 20-nucleotide protospacers. gRNAs were ordered by decreasing levels of IFNB1 activation. gRNA1 refers to the gRNA that has been used in all previous experiments. (B) qRT-PCR analysis of ISG15 transcript levels in primary HSPCs nucleofected with equal amounts of gRNA 1, 3, 6, 8, and 11 from panel A. Average values of two biological replicates +/−SD are shown. (C) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with synthetic (“syn”), IVT, and phosphatase-treated IVT gRNAs (gRNA1). (D) Viability of human primary HSPCs 24 h postnucleofection with no RNP and Cas9 or dCas9 RNPs. dCas9 or Cas9 were complexed with synthetic (“syn”) or IVT gRNA targeting the HBB gene. Viability was determined by trypan blue exclusion test. (E) qRT-PCR analysis of ISG15 and DDX58 (RIG-I) transcript levels in human primary HSPCs 16 h postnucleofection. dCas9 or Cas9 were complexed with synthetic or IVT gRNA targeting the HBB gene, respectively. Ct values were normalized against Ct of mock-nucleofected cells. Average values of two biological replicates +/−SD are shown. (F) Viability of human primary HSPCs 16 h posttransfection with RNPs. RNPs consisted of dCas9 complexed with synthetic, IVT, or CIP-treated IVT gRNAs targeting a noncoding intron of JAK2 (left panel) or Cas9 complexed with gRNAs targeting exon 1 of HBB (right panel). Viability was determined by trypan blue exclusion test. (G) Editing outcomes in HSPCs 48 h after nucleofection with RNPs targeting the HBB locus. Indel frequencies were determined by amplicon NGS. Statistical significances were calculated by unpaired t test (* p
Figure Legend Snippet: Protospacer and 5’-triphosphate determine the intensity of the gRNA-mediated IFNβ response. (A) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with equal amounts of gRNAs containing different 20-nucleotide protospacers. gRNAs were ordered by decreasing levels of IFNB1 activation. gRNA1 refers to the gRNA that has been used in all previous experiments. (B) qRT-PCR analysis of ISG15 transcript levels in primary HSPCs nucleofected with equal amounts of gRNA 1, 3, 6, 8, and 11 from panel A. Average values of two biological replicates +/−SD are shown. (C) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with synthetic (“syn”), IVT, and phosphatase-treated IVT gRNAs (gRNA1). (D) Viability of human primary HSPCs 24 h postnucleofection with no RNP and Cas9 or dCas9 RNPs. dCas9 or Cas9 were complexed with synthetic (“syn”) or IVT gRNA targeting the HBB gene. Viability was determined by trypan blue exclusion test. (E) qRT-PCR analysis of ISG15 and DDX58 (RIG-I) transcript levels in human primary HSPCs 16 h postnucleofection. dCas9 or Cas9 were complexed with synthetic or IVT gRNA targeting the HBB gene, respectively. Ct values were normalized against Ct of mock-nucleofected cells. Average values of two biological replicates +/−SD are shown. (F) Viability of human primary HSPCs 16 h posttransfection with RNPs. RNPs consisted of dCas9 complexed with synthetic, IVT, or CIP-treated IVT gRNAs targeting a noncoding intron of JAK2 (left panel) or Cas9 complexed with gRNAs targeting exon 1 of HBB (right panel). Viability was determined by trypan blue exclusion test. (G) Editing outcomes in HSPCs 48 h after nucleofection with RNPs targeting the HBB locus. Indel frequencies were determined by amplicon NGS. Statistical significances were calculated by unpaired t test (* p

Techniques Used: Quantitative RT-PCR, Transfection, Activation Assay, Amplification, Next-Generation Sequencing

Transfection of IVT gRNAs into HEK293 cells triggers a type I interferon response. (A) qRT-PCR analysis of IFNB1 and ISG15 transcript levels in HEK293 cells transfected with increasing amounts of gRNA with and without Cas9 protein. In the samples with Cas9, gRNAs were complexed with constant amounts (100 pmol, 100 nM final concentration) of Cas9 protein. Cells were harvested for RNA extraction 30 h after transfection using CRISPRMAX transfection reagent. Ct values were normalized to Ct values of mock-transfected HEK293 cells to determine fold activation. (B) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with equimolar amounts (50 nM) of IVT gRNA, SeV DI RNA, or HCV PAMP, respectively. (C) qRT-PCR analysis of IFNB1 and ISG15 transcript levels in HEK293 cells over a 48-h time course after transfection with 50 nM via lipofection (Lipofectamine2000 or RNAiMAX) or nucleofection, respectively. For all panels, average values of 3 biological replicates +/−SD are shown. The underlying data for this figure can be found in S1 Data . Cas9, CRISPR-associated 9; Ct, cycle threshold; gRNA, guide RNA; HCV, hepatitis C virus; HEK 293, human embryonic kidney 293; IFNB1 , interferon beta 1 ; IVT, in vitro–transcribed; PAMP, pathogen-associated molecular pattern; qRT-PCR, quantitative real-time PCR; SeV DI, Sendai virus defective interfering.
Figure Legend Snippet: Transfection of IVT gRNAs into HEK293 cells triggers a type I interferon response. (A) qRT-PCR analysis of IFNB1 and ISG15 transcript levels in HEK293 cells transfected with increasing amounts of gRNA with and without Cas9 protein. In the samples with Cas9, gRNAs were complexed with constant amounts (100 pmol, 100 nM final concentration) of Cas9 protein. Cells were harvested for RNA extraction 30 h after transfection using CRISPRMAX transfection reagent. Ct values were normalized to Ct values of mock-transfected HEK293 cells to determine fold activation. (B) qRT-PCR analysis of IFNB1 transcript levels in HEK293 cells transfected with equimolar amounts (50 nM) of IVT gRNA, SeV DI RNA, or HCV PAMP, respectively. (C) qRT-PCR analysis of IFNB1 and ISG15 transcript levels in HEK293 cells over a 48-h time course after transfection with 50 nM via lipofection (Lipofectamine2000 or RNAiMAX) or nucleofection, respectively. For all panels, average values of 3 biological replicates +/−SD are shown. The underlying data for this figure can be found in S1 Data . Cas9, CRISPR-associated 9; Ct, cycle threshold; gRNA, guide RNA; HCV, hepatitis C virus; HEK 293, human embryonic kidney 293; IFNB1 , interferon beta 1 ; IVT, in vitro–transcribed; PAMP, pathogen-associated molecular pattern; qRT-PCR, quantitative real-time PCR; SeV DI, Sendai virus defective interfering.

Techniques Used: Transfection, Quantitative RT-PCR, Concentration Assay, RNA Extraction, Activation Assay, CRISPR, In Vitro, Real-time Polymerase Chain Reaction

IVT gRNAs are recognized via the RIG-I pathway. (A) qRT-PCR analysis of increase in IFNB1 transcript levels (left) and transcript levels of the two main cytosolic RIG-I-like receptors ( DDX58 and IFIH1 ) after introduction of IVT gRNA. Cell lines were ordered by responsiveness to gRNA-mediated induction of IFNB1 transcript levels. Cells were harvested for RNA extraction 30 h after transfection. Ct values were normalized to Ct values of mock-transfected cells for each cell line to determine fold activation. IFNB1 levels for K562 cells were too low to be determined (n.d.). (B) Western blot analysis for RIG-I and MDA5 expression of mock-transfected and gRNA-transfected HEK293 cells after 48 h. (C) qRT-PCR analysis of IFNB1 transcript levels in HEK293 RIG-I (left), MDA5 (middle), and MAVS (right) KO cells. Shown are three biological replicates of three clonal populations of RIG-I, MDA5, or MAVS KO cells, respectively. IFNB1 levels for RIG-I KO clone #5 were too low to be determined (n.d.). For panels A and C, cells were harvested for RNA extraction 30 h after transfection using RNAiMAX transfection reagent. Average values of three biological replicates +/−SD are shown. Statistical significances were calculated by unpaired t test (* p
Figure Legend Snippet: IVT gRNAs are recognized via the RIG-I pathway. (A) qRT-PCR analysis of increase in IFNB1 transcript levels (left) and transcript levels of the two main cytosolic RIG-I-like receptors ( DDX58 and IFIH1 ) after introduction of IVT gRNA. Cell lines were ordered by responsiveness to gRNA-mediated induction of IFNB1 transcript levels. Cells were harvested for RNA extraction 30 h after transfection. Ct values were normalized to Ct values of mock-transfected cells for each cell line to determine fold activation. IFNB1 levels for K562 cells were too low to be determined (n.d.). (B) Western blot analysis for RIG-I and MDA5 expression of mock-transfected and gRNA-transfected HEK293 cells after 48 h. (C) qRT-PCR analysis of IFNB1 transcript levels in HEK293 RIG-I (left), MDA5 (middle), and MAVS (right) KO cells. Shown are three biological replicates of three clonal populations of RIG-I, MDA5, or MAVS KO cells, respectively. IFNB1 levels for RIG-I KO clone #5 were too low to be determined (n.d.). For panels A and C, cells were harvested for RNA extraction 30 h after transfection using RNAiMAX transfection reagent. Average values of three biological replicates +/−SD are shown. Statistical significances were calculated by unpaired t test (* p

Techniques Used: Quantitative RT-PCR, RNA Extraction, Transfection, Activation Assay, Western Blot, Expressing

10) Product Images from "An investigation of nutrient-dependent mRNA translation in Drosophila larvae"

Article Title: An investigation of nutrient-dependent mRNA translation in Drosophila larvae

Journal: Biology Open

doi: 10.1242/bio.20149407

Translational control of genes whose total mRNA levels are decreased by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.
Figure Legend Snippet: Translational control of genes whose total mRNA levels are decreased by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; green bars, starved larvae.

Techniques Used: Quantitative RT-PCR

Translational control of genes whose total mRNA levels are increased by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; red bars, starved larvae.
Figure Legend Snippet: Translational control of genes whose total mRNA levels are increased by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B–G) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; red bars, starved larvae.

Techniques Used: Quantitative RT-PCR

Translational control of genes whose total mRNA levels are unchanged by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B) Schematic showing the twelve polysome fractions used for qRT-PCR analysis. The traces indicate representative polysome profiles from fed (black trace) and 18 hr starved (red trace) larvae. The twelve fractions processed for RNA extraction and qRT-PCR analysis are indicated. (C–H) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; blue bars, starved larvae.
Figure Legend Snippet: Translational control of genes whose total mRNA levels are unchanged by starvation. (A) Total mRNA levels of each of the six genes were measured by qRT-PCR in fed vs. 18 hr starved larvae. Data are presented as mean ± SEM. (B) Schematic showing the twelve polysome fractions used for qRT-PCR analysis. The traces indicate representative polysome profiles from fed (black trace) and 18 hr starved (red trace) larvae. The twelve fractions processed for RNA extraction and qRT-PCR analysis are indicated. (C–H) qRT-PCR analysis of each of the six selected genes. Each data point in the figure shows the mean (± SEM) % of total mRNA in each of the twelve fractions. Grey bars, fed larvae; blue bars, starved larvae.

Techniques Used: Quantitative RT-PCR, RNA Extraction

11) Product Images from "Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil"

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S205334

5-Fu sensitivity of cervical cancer cells is mediated by the E6/E7-promoted glycolysis. ( A ) CaSki and ( B ) SiHa cells were transfected with control or E6/E7 overexpression vector for 48 hrs, cells were treated with or without Akt inhibitor for 16 hrs. Western blot was performed to check the phosphorylation of Akt and Glut-1, HK2, and LDHA protein expressions. ( C ) CaSki and ( D ) SiHa cells were treated with the same protocol and the mRNAs of Glut-1, HK2, and LDHA were measured by qRT-PCR. ( E ) CaSki and ( F ) SiHa cells were treated with the same protocol and the glucose uptake and lactate production were detected. * p
Figure Legend Snippet: 5-Fu sensitivity of cervical cancer cells is mediated by the E6/E7-promoted glycolysis. ( A ) CaSki and ( B ) SiHa cells were transfected with control or E6/E7 overexpression vector for 48 hrs, cells were treated with or without Akt inhibitor for 16 hrs. Western blot was performed to check the phosphorylation of Akt and Glut-1, HK2, and LDHA protein expressions. ( C ) CaSki and ( D ) SiHa cells were treated with the same protocol and the mRNAs of Glut-1, HK2, and LDHA were measured by qRT-PCR. ( E ) CaSki and ( F ) SiHa cells were treated with the same protocol and the glucose uptake and lactate production were detected. * p

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

Elevated cellular glycolysis in 5-Fu-resistant cervical cancer cells. ( A ) Glucose uptake and lactate production were compared in CaSki parental- and 5-Fu-resistant cells. ( B ) Real-time comparison of extracellular acidification rate (ECAR) between CaSki parental- and 5-Fu-resistant cells. ( C ) Protein or ( D ) mRNA expressions of glycolysis enzymes, Glut1, HK2, and LDHA from CaSki parental- and 5-Fu-resistant cells were measured by Western blot or qRT-PCR. β-actin was a loading control. Results are shown as mean±SEM. * p
Figure Legend Snippet: Elevated cellular glycolysis in 5-Fu-resistant cervical cancer cells. ( A ) Glucose uptake and lactate production were compared in CaSki parental- and 5-Fu-resistant cells. ( B ) Real-time comparison of extracellular acidification rate (ECAR) between CaSki parental- and 5-Fu-resistant cells. ( C ) Protein or ( D ) mRNA expressions of glycolysis enzymes, Glut1, HK2, and LDHA from CaSki parental- and 5-Fu-resistant cells were measured by Western blot or qRT-PCR. β-actin was a loading control. Results are shown as mean±SEM. * p

Techniques Used: Western Blot, Quantitative RT-PCR

The combination of E6/E7 shRNA and 5-Fu suppresses growth of 5-Fu-resistant cervical cancer xenografts in vivo. ( A ) Mice tumor growth in CsSki 5-Fu-resistant cells xenografts after intravenous administration of 5-Fu and intratumor administration of E6/E7 shRNA. Mice were grouped as: control treatment, E6/E7 shRNA injection alone, 5-Fu alone, or the combination of E6/E7 shRNA and 5-Fu. Tumor volume was measured every 3 days until day 21 when mice with tumors were euthanized. ( B, C ) The representative mice tumor from the above xenograft experiments. The relative mRNA expressions of ( D ) Glut1, ( E ) HK2, and ( F ) LDHA from tumors of the above mice were measured by qRT-PCR. Results are shown as mean±SEM.
Figure Legend Snippet: The combination of E6/E7 shRNA and 5-Fu suppresses growth of 5-Fu-resistant cervical cancer xenografts in vivo. ( A ) Mice tumor growth in CsSki 5-Fu-resistant cells xenografts after intravenous administration of 5-Fu and intratumor administration of E6/E7 shRNA. Mice were grouped as: control treatment, E6/E7 shRNA injection alone, 5-Fu alone, or the combination of E6/E7 shRNA and 5-Fu. Tumor volume was measured every 3 days until day 21 when mice with tumors were euthanized. ( B, C ) The representative mice tumor from the above xenograft experiments. The relative mRNA expressions of ( D ) Glut1, ( E ) HK2, and ( F ) LDHA from tumors of the above mice were measured by qRT-PCR. Results are shown as mean±SEM.

Techniques Used: shRNA, In Vivo, Mouse Assay, Injection, Quantitative RT-PCR

12) Product Images from "Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots"

Article Title: Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004809

OsSERK2 and XB24 are involved in EFR signaling in rice. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (C) and (D) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Data points or bars depict average relative light production ± SE of at least six biological replicates. Defense gene expression of PR10b (E) and Os04g10010 (F) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (G) and (H) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Data points or bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: OsSERK2 and XB24 are involved in EFR signaling in rice. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (C) and (D) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Data points or bars depict average relative light production ± SE of at least six biological replicates. Defense gene expression of PR10b (E) and Os04g10010 (F) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (G) and (H) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Data points or bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

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

EFR and EFR::XA21 recognize the elf18 sequence derived from Xoo EF-Tu. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli or elf18 Xoo at a concentration of 500 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. Fully mature leaves of Ubi :: EFR :: GFP -9-11-12 (C) and Ubi :: EFR :: XA21 :: GFP -3-6-7 (D) lines were treated with 1 μM elf18 E . coli for the indicated time. Upper panel anti-p42/44 MAP kinase western blot on total protein extracts, lower panel CBB stain of membrane as loading control. (E) Total ROS production over 3 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: EFR and EFR::XA21 recognize the elf18 sequence derived from Xoo EF-Tu. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli or elf18 Xoo at a concentration of 500 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. Fully mature leaves of Ubi :: EFR :: GFP -9-11-12 (C) and Ubi :: EFR :: XA21 :: GFP -3-6-7 (D) lines were treated with 1 μM elf18 E . coli for the indicated time. Upper panel anti-p42/44 MAP kinase western blot on total protein extracts, lower panel CBB stain of membrane as loading control. (E) Total ROS production over 3 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

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

Transgenic expression of EFR and EFR::XA21 in rice leads to elf18 E . coli responsiveness. (A) Relative expression level of EFR and EFR :: XA21 in three independent PCR positive transgenic lines for each immune receptor. Expression was measured by qRT-PCR using primers annealing to the EFR ectodomain. Bars depict average expression level relative to actin expression ± SE of three technical replicates. This experiment was repeated at least three times with similar results. (B) Protein level of EFR and EFR::XA21 using an anti-GFP antibody detecting the C-terminal GFP fusion protein. Upper panel anti-GFP western blot, lower panel CBB stain of membrane as loading control. See S4 Fig for full western blot. Defense gene expression of PR10b (C) and Os04g10010 (D) in response to elf18 E . coli (500 nM) in mature leaves of the three independent Ubi :: EFR :: GFP and Ubi :: EFR :: XA21 :: GFP lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. This experiment was repeated twice with similar results. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: Transgenic expression of EFR and EFR::XA21 in rice leads to elf18 E . coli responsiveness. (A) Relative expression level of EFR and EFR :: XA21 in three independent PCR positive transgenic lines for each immune receptor. Expression was measured by qRT-PCR using primers annealing to the EFR ectodomain. Bars depict average expression level relative to actin expression ± SE of three technical replicates. This experiment was repeated at least three times with similar results. (B) Protein level of EFR and EFR::XA21 using an anti-GFP antibody detecting the C-terminal GFP fusion protein. Upper panel anti-GFP western blot, lower panel CBB stain of membrane as loading control. See S4 Fig for full western blot. Defense gene expression of PR10b (C) and Os04g10010 (D) in response to elf18 E . coli (500 nM) in mature leaves of the three independent Ubi :: EFR :: GFP and Ubi :: EFR :: XA21 :: GFP lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. This experiment was repeated twice with similar results. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

Techniques Used: Transgenic Assay, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining

13) Product Images from "Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots"

Article Title: Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004809

OsSERK2 and XB24 are involved in EFR signaling in rice. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (C) and (D) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Data points or bars depict average relative light production ± SE of at least six biological replicates. Defense gene expression of PR10b (E) and Os04g10010 (F) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (G) and (H) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Data points or bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: OsSERK2 and XB24 are involved in EFR signaling in rice. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (C) and (D) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (67 and 71) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and OsSerk2RNAi- X-B-4-2. Data points or bars depict average relative light production ± SE of at least six biological replicates. Defense gene expression of PR10b (E) and Os04g10010 (F) in response to elf18 E . coli at a concentration of 500 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. (G) and (H) temporal and total ROS production over 4 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Ubi :: EFR :: GFP -9-11-12 and double transgenic F2 (14 and 18) plants from two independent crosses between Ubi :: EFR :: GFP -9-11 and XB24 overexpressing (OE) line A109-6-5-1. Data points or bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

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

EFR and EFR::XA21 recognize the elf18 sequence derived from Xoo EF-Tu. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli or elf18 Xoo at a concentration of 500 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. Fully mature leaves of Ubi :: EFR :: GFP -9-11-12 (C) and Ubi :: EFR :: XA21 :: GFP -3-6-7 (D) lines were treated with 1 μM elf18 E . coli for the indicated time. Upper panel anti-p42/44 MAP kinase western blot on total protein extracts, lower panel CBB stain of membrane as loading control. (E) Total ROS production over 3 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: EFR and EFR::XA21 recognize the elf18 sequence derived from Xoo EF-Tu. Defense gene expression of PR10b (A) and Os04g10010 (B) in response to elf18 E . coli or elf18 Xoo at a concentration of 500 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. Fully mature leaves of Ubi :: EFR :: GFP -9-11-12 (C) and Ubi :: EFR :: XA21 :: GFP -3-6-7 (D) lines were treated with 1 μM elf18 E . coli for the indicated time. Upper panel anti-p42/44 MAP kinase western blot on total protein extracts, lower panel CBB stain of membrane as loading control. (E) Total ROS production over 3 hours in response to elf18 E . coli or elf18 Xoo at a concentration of 100 nM in mature leaves of Kitaake, Ubi :: EFR :: GFP -9-11-12 and Ubi :: EFR :: XA21 :: GFP -3-6-7 lines. Bars depict average relative light production ± SE of at least six biological replicates. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

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

Transgenic expression of EFR and EFR::XA21 in rice leads to elf18 E . coli responsiveness. (A) Relative expression level of EFR and EFR :: XA21 in three independent PCR positive transgenic lines for each immune receptor. Expression was measured by qRT-PCR using primers annealing to the EFR ectodomain. Bars depict average expression level relative to actin expression ± SE of three technical replicates. This experiment was repeated at least three times with similar results. (B) Protein level of EFR and EFR::XA21 using an anti-GFP antibody detecting the C-terminal GFP fusion protein. Upper panel anti-GFP western blot, lower panel CBB stain of membrane as loading control. See S4 Fig for full western blot. Defense gene expression of PR10b (C) and Os04g10010 (D) in response to elf18 E . coli (500 nM) in mature leaves of the three independent Ubi :: EFR :: GFP and Ubi :: EFR :: XA21 :: GFP lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. This experiment was repeated twice with similar results. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p
Figure Legend Snippet: Transgenic expression of EFR and EFR::XA21 in rice leads to elf18 E . coli responsiveness. (A) Relative expression level of EFR and EFR :: XA21 in three independent PCR positive transgenic lines for each immune receptor. Expression was measured by qRT-PCR using primers annealing to the EFR ectodomain. Bars depict average expression level relative to actin expression ± SE of three technical replicates. This experiment was repeated at least three times with similar results. (B) Protein level of EFR and EFR::XA21 using an anti-GFP antibody detecting the C-terminal GFP fusion protein. Upper panel anti-GFP western blot, lower panel CBB stain of membrane as loading control. See S4 Fig for full western blot. Defense gene expression of PR10b (C) and Os04g10010 (D) in response to elf18 E . coli (500 nM) in mature leaves of the three independent Ubi :: EFR :: GFP and Ubi :: EFR :: XA21 :: GFP lines. Expression levels were measured by qRT-PCR and normalized to actin reference gene expression. Data shown is normalized to the Kitaake mock treated (2 hour) sample. Bars depict average expression level ± SE of three technical replicates. This experiment was repeated twice with similar results. Statistical analysis was performed using the Tukey-Kramer HSD test. Different letters indicate significant differences (p

Techniques Used: Transgenic Assay, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining

14) Product Images from "BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses"

Article Title: BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122892

YAP1 targets CYR61 and connective tissue growth factor (CTGF) expression and secretion are regulated by BMP9 and endoglin. All cells were plated on fibronectin. ( A-B ) Quantitative real-time RT-PCR (qRT-PCR) of ( A ) CTGF and ( B ) CYR61 following BMP9 treatment and endoglin suppression using shRNA. ( C ) Immunofluorescence confocal microscopy analysis of CTGF in response to BMP9 treatment and endoglin suppression. ( D ) qRT-PCR of CTGF (gray) and CYR61 (black) mRNA from wild type and eng +/- mouse lung mRNA preparations. ( E ) qRT-PCR of CTGF following BMP9 treatment and Alk1 suppression using shRNA. ( F-G ) qRT-PCR of ( F ) CTGF and ( G ) CYR61 following BMP9 treatment and YAP1 suppression using shRNA. Human cyclophilin primers were used for normalization.
Figure Legend Snippet: YAP1 targets CYR61 and connective tissue growth factor (CTGF) expression and secretion are regulated by BMP9 and endoglin. All cells were plated on fibronectin. ( A-B ) Quantitative real-time RT-PCR (qRT-PCR) of ( A ) CTGF and ( B ) CYR61 following BMP9 treatment and endoglin suppression using shRNA. ( C ) Immunofluorescence confocal microscopy analysis of CTGF in response to BMP9 treatment and endoglin suppression. ( D ) qRT-PCR of CTGF (gray) and CYR61 (black) mRNA from wild type and eng +/- mouse lung mRNA preparations. ( E ) qRT-PCR of CTGF following BMP9 treatment and Alk1 suppression using shRNA. ( F-G ) qRT-PCR of ( F ) CTGF and ( G ) CYR61 following BMP9 treatment and YAP1 suppression using shRNA. Human cyclophilin primers were used for normalization.

Techniques Used: Expressing, Quantitative RT-PCR, shRNA, Immunofluorescence, Confocal Microscopy

Inflammatory cytokines are regulated by BMP9 and endoglin expression and signaling. (A) CCL2 and CCR2 levels of expression in HUVEC following treatment with TGFβ or BMP9, as compared to control (NT). (B) qRT-PCR analysis of time course of BMP9-dependent changes in mRNA levels for CCL2, CCR2, and CCL5. (C) Relative to control lentivirus (NT), qRT-PCR indicates shRNA for endoglin or ALK1 (e, a, respectively) rescues BMP9-dependent repression of CCL2 expression. (D) CCL2 and CCR2 expression in WT (n = 3) and eng +/- (n = 3) mouse lung mRNA preparations. *, p
Figure Legend Snippet: Inflammatory cytokines are regulated by BMP9 and endoglin expression and signaling. (A) CCL2 and CCR2 levels of expression in HUVEC following treatment with TGFβ or BMP9, as compared to control (NT). (B) qRT-PCR analysis of time course of BMP9-dependent changes in mRNA levels for CCL2, CCR2, and CCL5. (C) Relative to control lentivirus (NT), qRT-PCR indicates shRNA for endoglin or ALK1 (e, a, respectively) rescues BMP9-dependent repression of CCL2 expression. (D) CCL2 and CCR2 expression in WT (n = 3) and eng +/- (n = 3) mouse lung mRNA preparations. *, p

Techniques Used: Expressing, Quantitative RT-PCR, shRNA

15) Product Images from "Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways "

Article Title: Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways

Journal:

doi: 10.1152/physiolgenomics.90276.2008

Relative expression of CXCR4 mRNA, detected with qRT-PCR in blood of pregnant heifers carrying control, TI, or PI fetuses. Data are presented as means ± SE. ** P
Figure Legend Snippet: Relative expression of CXCR4 mRNA, detected with qRT-PCR in blood of pregnant heifers carrying control, TI, or PI fetuses. Data are presented as means ± SE. ** P

Techniques Used: Expressing, Quantitative RT-PCR

Relative expression of mRNA for cytosolic dsRNA sensors: RIG-I ( A ) and MDA-5 ( B ), and for ISGs: OAS-1 ( C ) and IFIT2 ( D ) in blood of heifers carrying control, TI, or PI fetuses, detected with qRT-PCR. Data are presented as means ± SE. *
Figure Legend Snippet: Relative expression of mRNA for cytosolic dsRNA sensors: RIG-I ( A ) and MDA-5 ( B ), and for ISGs: OAS-1 ( C ) and IFIT2 ( D ) in blood of heifers carrying control, TI, or PI fetuses, detected with qRT-PCR. Data are presented as means ± SE. *

Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

Experimental design. Heifers ( n = 6 per group) were inoculated with BVDV on day 75 (PI group) and day 175 (TI group) of gestation. Control group was kept uninfected throughout the experiment. Black stars show days of viremia detected by qRT-PCR.
Figure Legend Snippet: Experimental design. Heifers ( n = 6 per group) were inoculated with BVDV on day 75 (PI group) and day 175 (TI group) of gestation. Control group was kept uninfected throughout the experiment. Black stars show days of viremia detected by qRT-PCR.

Techniques Used: Quantitative RT-PCR

16) Product Images from "BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress"

Article Title: BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.278

A human genomic region including rs3814113 regulates BNC2 expression. ( a ) Genomic view of the 6.6 kb, surrounding rs3814113, with annotated human mRNAs from GenBank, according to UCSC genome browser. Positions for guides 6 and 9 (guide RNAs used for CRISPR (clustered regularly interspaced short palindromic repeat) deletion of the region), for Screen 6F and Screen 7F (primers used for PCR screening of KO), and for AK 5+3 (amplicon used to detect AK024561 transcript) are indicated. ( b ) Agarose gel of PCR with AK 5+3 primers to detect AK024561 in human samples: top panels, 14 normal human tissues and 3 PBMCs from healthy donors; bottom panels, 11 serous ovarian cancers (10 high grade, 1 low grade=LG) and 8 non-malignant control samples (4 fallopian tubes, 1 round ligament=RL, 3 ovaries). gDNA=genomic DNA used as PCR positive control; RT-PCR on non-retrotranscribed RNA, to exclude genomic contamination; GAPDH and U6 =housekeeping genes used for normalization. ( c ) Representative agarose gel of PCR with Screen 6F+Screen 7R primer pair showing the deletion of the 5 kb region surrounding rs3814113. ( d ) qRT-PCR analysis of BNC2 and CNTLN expression in 293FT KO cell clones. Points for WT and KO samples represent normalized expression data from four distinct WT and five distinct KO clones. The values for each clone are the average of two biologically independent experiments. Three distinct primer pairs (encompassing BNC2's exon 1-2, exon 2-2a and exon 6) were used to detect BNC2 expression levels. t -Test assuming unequal variances was used for statistical analysis. Scatter dot plot bars indicate mean±S.E. * P
Figure Legend Snippet: A human genomic region including rs3814113 regulates BNC2 expression. ( a ) Genomic view of the 6.6 kb, surrounding rs3814113, with annotated human mRNAs from GenBank, according to UCSC genome browser. Positions for guides 6 and 9 (guide RNAs used for CRISPR (clustered regularly interspaced short palindromic repeat) deletion of the region), for Screen 6F and Screen 7F (primers used for PCR screening of KO), and for AK 5+3 (amplicon used to detect AK024561 transcript) are indicated. ( b ) Agarose gel of PCR with AK 5+3 primers to detect AK024561 in human samples: top panels, 14 normal human tissues and 3 PBMCs from healthy donors; bottom panels, 11 serous ovarian cancers (10 high grade, 1 low grade=LG) and 8 non-malignant control samples (4 fallopian tubes, 1 round ligament=RL, 3 ovaries). gDNA=genomic DNA used as PCR positive control; RT-PCR on non-retrotranscribed RNA, to exclude genomic contamination; GAPDH and U6 =housekeeping genes used for normalization. ( c ) Representative agarose gel of PCR with Screen 6F+Screen 7R primer pair showing the deletion of the 5 kb region surrounding rs3814113. ( d ) qRT-PCR analysis of BNC2 and CNTLN expression in 293FT KO cell clones. Points for WT and KO samples represent normalized expression data from four distinct WT and five distinct KO clones. The values for each clone are the average of two biologically independent experiments. Three distinct primer pairs (encompassing BNC2's exon 1-2, exon 2-2a and exon 6) were used to detect BNC2 expression levels. t -Test assuming unequal variances was used for statistical analysis. Scatter dot plot bars indicate mean±S.E. * P

Techniques Used: Expressing, CRISPR, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Positive Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Clone Assay

Oxidative stress reduces BNC2 expression in vitro and in vivo . ( a ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 and COV504 cells treated with increasing amounts of H 2 O 2 (0.06, 0.125 and 0.25 mM) at 3 or 24 h post-treatment. Total H2AX was used as loading control. Phospho-H2AX (pH2AX) was detected to check effectiveness of H 2 O 2 treatment. ( b ) qRT-PCR analysis of BNC2 expression in COV318 and OVCAR4 at 5 h after H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( c ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H 2 O 2 and collected 1, 6 and 24 h post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H 2 O 2 treatment. ( d ) qRT-PCR analysis of BNC2 expression in COV318 at 1, 6 and 24 h after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( e ) ChIP for H3K4me3 enrichment (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 treated with 0.06 mM H 2 O 2 at different time points. Bars indicate mean±S.E. from two independent experiments. ( f ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H 2 O 2 and collected 1 week post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H 2 O 2 treatment. ( g ) qRT-PCR analysis of BNC2 expression in COV318 at 1 week after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( h ) ChIP for H3K4me3 enrichment (left panel) and H3K27me3 enrichment (right panel) (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 at 1 week after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( i ) Left panel: Western blot detection of BNC2 protein levels in NP40-insoluble extracts from prepubertal mice oviducts treated or not with hCG 16 h earlier. COV318 was used as positive control for BNC2 expression. Right panel: Densitometric analysis of BNC2 western blot bands in oviducts from hCG treated ( n =10) and untreated ( n =10) prepubertal mice as described above. Scatter dot plot bars indicate mean±S.E. from two independent experiments, including the one presented in the left panel. * P
Figure Legend Snippet: Oxidative stress reduces BNC2 expression in vitro and in vivo . ( a ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 and COV504 cells treated with increasing amounts of H 2 O 2 (0.06, 0.125 and 0.25 mM) at 3 or 24 h post-treatment. Total H2AX was used as loading control. Phospho-H2AX (pH2AX) was detected to check effectiveness of H 2 O 2 treatment. ( b ) qRT-PCR analysis of BNC2 expression in COV318 and OVCAR4 at 5 h after H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( c ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H 2 O 2 and collected 1, 6 and 24 h post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H 2 O 2 treatment. ( d ) qRT-PCR analysis of BNC2 expression in COV318 at 1, 6 and 24 h after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( e ) ChIP for H3K4me3 enrichment (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 treated with 0.06 mM H 2 O 2 at different time points. Bars indicate mean±S.E. from two independent experiments. ( f ) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H 2 O 2 and collected 1 week post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H 2 O 2 treatment. ( g ) qRT-PCR analysis of BNC2 expression in COV318 at 1 week after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( h ) ChIP for H3K4me3 enrichment (left panel) and H3K27me3 enrichment (right panel) (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 at 1 week after 0.06 mM H 2 O 2 treatment. Bars indicate mean±S.E. from two independent experiments. ( i ) Left panel: Western blot detection of BNC2 protein levels in NP40-insoluble extracts from prepubertal mice oviducts treated or not with hCG 16 h earlier. COV318 was used as positive control for BNC2 expression. Right panel: Densitometric analysis of BNC2 western blot bands in oviducts from hCG treated ( n =10) and untreated ( n =10) prepubertal mice as described above. Scatter dot plot bars indicate mean±S.E. from two independent experiments, including the one presented in the left panel. * P

Techniques Used: Expressing, In Vitro, In Vivo, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation, Mouse Assay, Positive Control

BNC2 expression and genetic regulation. ( a ) qRT-PCR analysis of BNC2 expression in a panel of 16 ovarian cancer cell lines, grouped in likely and unlikely HGSOC. ( b ) Western blot detection of BNC2 shows several isoforms in NP40-insoluble protein extracts from seven likely HGSOC cell lines. H2AX total was used as loading control. ( c ) ChIP for histone mark enrichment (measured by qRT-PCR) in the putative promoter/enhancer genomic regions comprising BNC2 , CNTLN and the intergenic region in between, according to UCSC ( Supplementary Figure S3 ). H3K4me3 (top panels) and H3K27Ac (bottom panels)=histone H3 trymethylated on lysine 4 and acetylated on lysine 27, respectively. BNC2 intron 2 (Intr 2) was set as 1
Figure Legend Snippet: BNC2 expression and genetic regulation. ( a ) qRT-PCR analysis of BNC2 expression in a panel of 16 ovarian cancer cell lines, grouped in likely and unlikely HGSOC. ( b ) Western blot detection of BNC2 shows several isoforms in NP40-insoluble protein extracts from seven likely HGSOC cell lines. H2AX total was used as loading control. ( c ) ChIP for histone mark enrichment (measured by qRT-PCR) in the putative promoter/enhancer genomic regions comprising BNC2 , CNTLN and the intergenic region in between, according to UCSC ( Supplementary Figure S3 ). H3K4me3 (top panels) and H3K27Ac (bottom panels)=histone H3 trymethylated on lysine 4 and acetylated on lysine 27, respectively. BNC2 intron 2 (Intr 2) was set as 1

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

17) Product Images from "The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1"

Article Title: The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1

Journal: Molecular Microbiology

doi: 10.1111/mmi.13256

Transcript levels of GlcNAc catabolic cluster genes are strongly increased during growth on GlcNAc . A. Gene expression analysis (semi‐quantitative RT‐PCR ) of GlcNAc cluster genes in T . atroviride and T . reesei after growth on 1% glucose (G) or 1% GlcNAc (N) for 16 h, 24 h and 38 h. As reference gene tef1 was used. B. Quantitative gene expression analysis ( qRT‐PCR ) of the GlcNAc cluster genes in T . reesei . Samples were normalised to tef1 expression levels and compared with the time point ‘glucose, 16 h’, which was set as 1 for the respective genes that were studied. The standard deviation of the mean expression values from at least two independent biological replicates is shown; P values are
Figure Legend Snippet: Transcript levels of GlcNAc catabolic cluster genes are strongly increased during growth on GlcNAc . A. Gene expression analysis (semi‐quantitative RT‐PCR ) of GlcNAc cluster genes in T . atroviride and T . reesei after growth on 1% glucose (G) or 1% GlcNAc (N) for 16 h, 24 h and 38 h. As reference gene tef1 was used. B. Quantitative gene expression analysis ( qRT‐PCR ) of the GlcNAc cluster genes in T . reesei . Samples were normalised to tef1 expression levels and compared with the time point ‘glucose, 16 h’, which was set as 1 for the respective genes that were studied. The standard deviation of the mean expression values from at least two independent biological replicates is shown; P values are

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

The transcription factor RON 1 is the key activator of the GlcNAc catabolic gene cluster. Fungi were grown on MA medium containing 1% glycerol as carbon source for 24 h and then shifted to minimal medium containing 1% GlcNAc ( N ) or 1% glucose ( G ) and cultivated for another 24 h. Gene expression levels of all GlcNAc cluster genes, ngt1 as well as nag1 and nag2 were evaluated by qRT ‐ PCR at 4, 8 and 24 h after the medium shift, normalised using tef1 as reference gene, and compared with the respective gene expression levels of the parental strain on glucose (control) which was set as 1. The 4, 8 and 24 h values were combined since expression of all three time points was very similar. Statistically significant differences ( P values
Figure Legend Snippet: The transcription factor RON 1 is the key activator of the GlcNAc catabolic gene cluster. Fungi were grown on MA medium containing 1% glycerol as carbon source for 24 h and then shifted to minimal medium containing 1% GlcNAc ( N ) or 1% glucose ( G ) and cultivated for another 24 h. Gene expression levels of all GlcNAc cluster genes, ngt1 as well as nag1 and nag2 were evaluated by qRT ‐ PCR at 4, 8 and 24 h after the medium shift, normalised using tef1 as reference gene, and compared with the respective gene expression levels of the parental strain on glucose (control) which was set as 1. The 4, 8 and 24 h values were combined since expression of all three time points was very similar. Statistically significant differences ( P values

Techniques Used: Expressing, Quantitative RT-PCR

CSP 2 is not involved in the regulation of GlcNAc genes during vegetative growth or under carbon starvation conditions. A. Growth of the T . reesei parental strain QM 9414 Δ tku70 ( WT ) and two csp2 knockout strains (Δ csp2/5 and Δ csp2/23 ) after 7 days on agar plates with MA medium with glucose and PDA . B. qRT‐PCR of the parental strain ( WT ), and two Δ ron1 and Δ csp2 strains. Mycelium was pre‐cultivated for 16 h in MA medium containing 1% glucose, and biomass samples were taken at 0, 5 and 15 h after a shift to MA medium lacking added carbon sources. All measured values were normalised to tef1 expression and compared with the time point [ WT , 0 h], which was set at 1. Bars indicate the SEM (standard error of the mean). ‘a’, ‘b’ and ‘c’ indicate significance at P
Figure Legend Snippet: CSP 2 is not involved in the regulation of GlcNAc genes during vegetative growth or under carbon starvation conditions. A. Growth of the T . reesei parental strain QM 9414 Δ tku70 ( WT ) and two csp2 knockout strains (Δ csp2/5 and Δ csp2/23 ) after 7 days on agar plates with MA medium with glucose and PDA . B. qRT‐PCR of the parental strain ( WT ), and two Δ ron1 and Δ csp2 strains. Mycelium was pre‐cultivated for 16 h in MA medium containing 1% glucose, and biomass samples were taken at 0, 5 and 15 h after a shift to MA medium lacking added carbon sources. All measured values were normalised to tef1 expression and compared with the time point [ WT , 0 h], which was set at 1. Bars indicate the SEM (standard error of the mean). ‘a’, ‘b’ and ‘c’ indicate significance at P

Techniques Used: Knock-Out, Quantitative RT-PCR, Expressing

18) Product Images from "SOX30 is a key regulator of desmosomal gene suppressing tumor growth and metastasis in lung adenocarcinoma"

Article Title: SOX30 is a key regulator of desmosomal gene suppressing tumor growth and metastasis in lung adenocarcinoma

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0778-3

Inhibition of SOX30 promotes tumorigenesis of lung cancer with urethane treatment. a The mRNA expression of DSP, JUP and DSC3 in lung tissues of SOX30-knockout mice were measured by qRT-PCR. b The protein expression of DSP, JUP and DSC3 in lung tissues of SOX30-knockout mice were detected by WB. c Lung tissues were processed and stained with H E for detection of tumor foci. d Tumor extracts were analyzed by WB. ACTIN was used as a loading control. e Schematic diagram of the mechanisms of SOX30 mediated suppression of ADC cell proliferation and metastasis based on our study
Figure Legend Snippet: Inhibition of SOX30 promotes tumorigenesis of lung cancer with urethane treatment. a The mRNA expression of DSP, JUP and DSC3 in lung tissues of SOX30-knockout mice were measured by qRT-PCR. b The protein expression of DSP, JUP and DSC3 in lung tissues of SOX30-knockout mice were detected by WB. c Lung tissues were processed and stained with H E for detection of tumor foci. d Tumor extracts were analyzed by WB. ACTIN was used as a loading control. e Schematic diagram of the mechanisms of SOX30 mediated suppression of ADC cell proliferation and metastasis based on our study

Techniques Used: Inhibition, Expressing, Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot, Staining

SOX30 is positive correlated with desmosomal gene expression in ADC, but not in SCC. a Heatmaps for correlations between SOX30 and desmosomal genes in the TCGA lung adenocarcinoma RNAseq (IlluminaHiSeq; n = 571) data set. b Heatmaps for correlations between SOX30 and desmosomal genes in the TCGA lung squamous carcinoma RNAseq (IlluminaHiSeq; n = 553) data set. Correlation coefficient R and P -values were calculated by a Spearman correlation analysis. c qRT-PCR analysis of desmosomal gene expression in A549 and LTEP-a-2 cells transiently transfected with the vector control or SOX30 expression vector. d qRT-PCR analysis of desmosomal gene expression in H520 and H226 cells transiently transfected with the vector control or SOX30 expression vector. ACTIN was used as an internal control. e The protein levels of DSP, and JUP and DSC3 were monitored by WB after SOX30 overexpression in A549 and LTEP-a-2 cells. f The protein levels of DSP, and JUP and DSC3 were monitored by WB after SOX30 overexpression in H520 and H226 cells. g The protein levels of SOX30, DSP, JUP and DSC3 were further monitored by IHC in human ADC tissues. h The protein levels of SOX30, DSP, JUP and DSC3 were further monitored by IHC in human SCC tissues. Scale bar represents 50 mm
Figure Legend Snippet: SOX30 is positive correlated with desmosomal gene expression in ADC, but not in SCC. a Heatmaps for correlations between SOX30 and desmosomal genes in the TCGA lung adenocarcinoma RNAseq (IlluminaHiSeq; n = 571) data set. b Heatmaps for correlations between SOX30 and desmosomal genes in the TCGA lung squamous carcinoma RNAseq (IlluminaHiSeq; n = 553) data set. Correlation coefficient R and P -values were calculated by a Spearman correlation analysis. c qRT-PCR analysis of desmosomal gene expression in A549 and LTEP-a-2 cells transiently transfected with the vector control or SOX30 expression vector. d qRT-PCR analysis of desmosomal gene expression in H520 and H226 cells transiently transfected with the vector control or SOX30 expression vector. ACTIN was used as an internal control. e The protein levels of DSP, and JUP and DSC3 were monitored by WB after SOX30 overexpression in A549 and LTEP-a-2 cells. f The protein levels of DSP, and JUP and DSC3 were monitored by WB after SOX30 overexpression in H520 and H226 cells. g The protein levels of SOX30, DSP, JUP and DSC3 were further monitored by IHC in human ADC tissues. h The protein levels of SOX30, DSP, JUP and DSC3 were further monitored by IHC in human SCC tissues. Scale bar represents 50 mm

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

19) Product Images from "Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder"

Article Title: Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder

Journal: Nature Communications

doi: 10.1038/s41467-017-00932-7

Downregulation of NRF2 induced by luteolin. a Representative western blot of endogenous level of NRF2 in whole protein lysates of treated human primary fibroblast of patient 1. NRF2 p.T80K mutant cells were exposed to 50 µM luteolin or DMSO for 24 h. Full blots are shown in Supplementary Fig. 7 . b Quantitative analysis of western blot images illustrating the endogenous level of NRF2 relative normalized to ACTB and DMSO treated cells. Data are given as means ± SEM, n = 3 independent experiments. Statistical differences were obtained with unpaired Welch’s t -test: *** p ≤ 0.001. c qRT–PCR analysis of AKR1B10 and AKR1C1 gene expression in primary fibroblasts of patient 1 treated with 50 µM luteolin or DMSO for 24 h. Expression is normalized to that of GAPDH . % of mRNA is equal to 2 −∆∆CT and normalized relative to DMSO. Data are given as means ± SEM, n = 2 independent experiments
Figure Legend Snippet: Downregulation of NRF2 induced by luteolin. a Representative western blot of endogenous level of NRF2 in whole protein lysates of treated human primary fibroblast of patient 1. NRF2 p.T80K mutant cells were exposed to 50 µM luteolin or DMSO for 24 h. Full blots are shown in Supplementary Fig. 7 . b Quantitative analysis of western blot images illustrating the endogenous level of NRF2 relative normalized to ACTB and DMSO treated cells. Data are given as means ± SEM, n = 3 independent experiments. Statistical differences were obtained with unpaired Welch’s t -test: *** p ≤ 0.001. c qRT–PCR analysis of AKR1B10 and AKR1C1 gene expression in primary fibroblasts of patient 1 treated with 50 µM luteolin or DMSO for 24 h. Expression is normalized to that of GAPDH . % of mRNA is equal to 2 −∆∆CT and normalized relative to DMSO. Data are given as means ± SEM, n = 2 independent experiments

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

Increased stabilization and activation of mutant NRF2. a Representative western blot of endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 in protein lysates of human primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. Full blots are shown in Supplementary Fig. 6 . b Quantitative analysis of western blot images illustrating the endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 relative normalized to ACTB and NRF2 WT 1. c qRT–PCR analysis of NFE2L2, KEAP1 and target gene expression in primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. AKR1B10 and AKR1C1 are visualized on a separated X axis due to the high range. Expression is normalized to that of ACTB . % of mRNA is equal to 2 −∆∆CT and normalized relative to NRF2 WT 1. Redox calibration confirms full functionality of roGFP1 as well as identical response ranges for NRF2 WT 2 and NRF2 p.T80K fibroblast cells. d Response range calibration of an exemplary NRF2 WT 2 and NRF2 p.T80K fibroblast cell performed as a continuous recording of the roGFP1 ratio F 395 /F 470 within a ROI of cytoplasm of the cell, scale bar is 20 µM. Plotted traces represent full oxidation (R ox, induced by 5 mM H 2 O 2 , 5 min) and full reduction (R red , induced by 10 mM DTT, 5 min). After calibration the relative degrees of roGFP1 oxidation and corresponding roGFP1 redox potentials can be calculated. e Baseline redox conditions of NRF2 WT 2 and NRF2 p.T80K fibroblasts. Upper diagram shows the relative level of roGFP1 oxidation of NRF2 WT 2 and NRF2 p.T80K cells at rest (OxD roGFP1 , Eq. 1). Lower diagram represents corresponding steady-state roGFP1 redox potential (E roGFP1 , Eq. 2). b , c Data are given as means ± SEM, n ≥ 3 independent experiments. Data were analyzed by one-way analysis of variance with multiple comparisons: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. e Data are given as means ± SEM. Number of measured cells are given within the bar. Statistical differences were obtained with unpaired Welch’s t -test: *** p ≤ 0.001
Figure Legend Snippet: Increased stabilization and activation of mutant NRF2. a Representative western blot of endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 in protein lysates of human primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. Full blots are shown in Supplementary Fig. 6 . b Quantitative analysis of western blot images illustrating the endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 relative normalized to ACTB and NRF2 WT 1. c qRT–PCR analysis of NFE2L2, KEAP1 and target gene expression in primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. AKR1B10 and AKR1C1 are visualized on a separated X axis due to the high range. Expression is normalized to that of ACTB . % of mRNA is equal to 2 −∆∆CT and normalized relative to NRF2 WT 1. Redox calibration confirms full functionality of roGFP1 as well as identical response ranges for NRF2 WT 2 and NRF2 p.T80K fibroblast cells. d Response range calibration of an exemplary NRF2 WT 2 and NRF2 p.T80K fibroblast cell performed as a continuous recording of the roGFP1 ratio F 395 /F 470 within a ROI of cytoplasm of the cell, scale bar is 20 µM. Plotted traces represent full oxidation (R ox, induced by 5 mM H 2 O 2 , 5 min) and full reduction (R red , induced by 10 mM DTT, 5 min). After calibration the relative degrees of roGFP1 oxidation and corresponding roGFP1 redox potentials can be calculated. e Baseline redox conditions of NRF2 WT 2 and NRF2 p.T80K fibroblasts. Upper diagram shows the relative level of roGFP1 oxidation of NRF2 WT 2 and NRF2 p.T80K cells at rest (OxD roGFP1 , Eq. 1). Lower diagram represents corresponding steady-state roGFP1 redox potential (E roGFP1 , Eq. 2). b , c Data are given as means ± SEM, n ≥ 3 independent experiments. Data were analyzed by one-way analysis of variance with multiple comparisons: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. e Data are given as means ± SEM. Number of measured cells are given within the bar. Statistical differences were obtained with unpaired Welch’s t -test: *** p ≤ 0.001

Techniques Used: Activation Assay, Mutagenesis, Western Blot, Variant Assay, Quantitative RT-PCR, Expressing

20) Product Images from "OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3"

Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3

Journal: Nature Communications

doi: 10.1038/ncomms3519

Depletion of OTUB1 represses TGFβ-induced transcription. ( a ) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. ( b ) C2C12 cells stably expressing a SMAD3-dependent TGFβ-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1# 1. Cells were treated with or without 50 pM TGFβ for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. ( n =3). ( c ) HaCaT cells stably expressing shRNA against OTUB1 or transfected with control (−) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. ( d ) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). ( e ) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFβ for 1 h. TGFβ was then washed off and SB505124 (1 μM) added. RNA was isolated 45 min after TGFβ removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P
Figure Legend Snippet: Depletion of OTUB1 represses TGFβ-induced transcription. ( a ) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. ( b ) C2C12 cells stably expressing a SMAD3-dependent TGFβ-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1# 1. Cells were treated with or without 50 pM TGFβ for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. ( n =3). ( c ) HaCaT cells stably expressing shRNA against OTUB1 or transfected with control (−) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. ( d ) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). ( e ) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFβ for 1 h. TGFβ was then washed off and SB505124 (1 μM) added. RNA was isolated 45 min after TGFβ removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

Techniques Used: Transfection, SDS Page, Stable Transfection, Expressing, Luciferase, Construct, Lysis, Activity Assay, shRNA, Isolation, Quantitative RT-PCR

OTUB1 prevents SMAD3 ubiquitylation in vitro. ( a ) For in-cell polyubiquitylation of FLAG-SMAD2/3/4, vectors encoding FLAG-SMAD2/3/4 were co-transfected with HA-NEDD4L and HA-ubiquitin in HEK293 cells and treated with 50 pM TGFβ and 10 μM bortezomib for 3 h before lysis and FLAG-SMAD2/3/4 were immunoprecipitated. An in vitro DUB assay of in-cell polyubiquitylated FLAG-SMAD2/3/4 was performed with GST-OTUB1 and the indicated GST-OTUB1 mutants in DUB assay buffer for 1 h at 30 °C. The assay mix was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( b ) An in vitro ubiquitylation assay was performed with human recombinant SMAD2. SMAD2 was incubated with E1, E2, E3 and ubiquitin in ubiquitylation assay buffer for 1 h at 30 °C. Increasing concentrations of GST-OTUB1 and GST-OTUB1 C91S (8–60 ng μl −1 ) were added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) FLAG-SMAD2/3/4 IPs (from HEK293 cells expressing FLAG-SMAD2/3/4 treated with 50 pM TGFβ for 1 h before lysis) were ubiquitylated in vitro in ubiquitylation assay buffer for 1 h at 30 °C using E1, E2, E3 and ubiquitin. The indicated DUBs were added at the start of the ubiquitylation assay (time 0) and proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) HaCaT cells were stably transfected with vectors encoding siRNA-resistant silent mutations (rescue) of the indicated OTUB1 constructs. These cells were then transfected with control FoxO4 or OTUB1 siRNA for 48 h and treated with or without TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P
Figure Legend Snippet: OTUB1 prevents SMAD3 ubiquitylation in vitro. ( a ) For in-cell polyubiquitylation of FLAG-SMAD2/3/4, vectors encoding FLAG-SMAD2/3/4 were co-transfected with HA-NEDD4L and HA-ubiquitin in HEK293 cells and treated with 50 pM TGFβ and 10 μM bortezomib for 3 h before lysis and FLAG-SMAD2/3/4 were immunoprecipitated. An in vitro DUB assay of in-cell polyubiquitylated FLAG-SMAD2/3/4 was performed with GST-OTUB1 and the indicated GST-OTUB1 mutants in DUB assay buffer for 1 h at 30 °C. The assay mix was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( b ) An in vitro ubiquitylation assay was performed with human recombinant SMAD2. SMAD2 was incubated with E1, E2, E3 and ubiquitin in ubiquitylation assay buffer for 1 h at 30 °C. Increasing concentrations of GST-OTUB1 and GST-OTUB1 C91S (8–60 ng μl −1 ) were added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) FLAG-SMAD2/3/4 IPs (from HEK293 cells expressing FLAG-SMAD2/3/4 treated with 50 pM TGFβ for 1 h before lysis) were ubiquitylated in vitro in ubiquitylation assay buffer for 1 h at 30 °C using E1, E2, E3 and ubiquitin. The indicated DUBs were added at the start of the ubiquitylation assay (time 0) and proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) HaCaT cells were stably transfected with vectors encoding siRNA-resistant silent mutations (rescue) of the indicated OTUB1 constructs. These cells were then transfected with control FoxO4 or OTUB1 siRNA for 48 h and treated with or without TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

Techniques Used: In Vitro, Transfection, Lysis, Immunoprecipitation, SDS Page, Ubiquitin Assay, Recombinant, Incubation, Expressing, Stable Transfection, Construct, Isolation, Quantitative RT-PCR

21) Product Images from "Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus"

Article Title: Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2016.00090

Assessment of Scl1.3 expression. (A) Assessment of Scl1.3 production by M3-type GAS. Cell wall (CW) and culture supernatant (Sup) protein fractions prepared from exponential phase cultures of several M3-type strains were analyzed by western immunoblotting, using anti-Scl1 rabbit polyclonal antibody. Recombinant protein rScl1.3V, corresponding to the variable region of Scl1.3, was used as a positive control. Expected molecular masses: Scl1.3, 11.4 kDa; rScl1.3V, 8.3 kDa. M, PageRuler™ Plus Prestained Protein Ladder. (B) Detection of Scl1.3 on the surface of M3-type GAS. Flow cytometry analysis of several M3-type strains is shown using anti-Scl1 antibody described in part (A) (color-shaded histograms) or a secondary only control (2° sample, black outlined histogram). Median fluorescence intensities (MFI) are shown in parentheses for each strain. (C) Assessment of scl1 transcription. Fold-change of scl1 transcript levels are shown compared to scl1.3 transcription in M3-type strain MGAS315. qRT-PCR was performed on RNA obtained from exponential phase cultures. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (student's t -test). (D) Assessment of mga and emm transcription. Relative expression levels of mga and emm genes were compared between MGAS315 and four additional M3 strains or the M1 strain MGAS5005. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; ** P ≤ 0.01. (E) Assessment of M3-protein production by M3-type GAS. The same cell wall (CW) and culture supernatant (Sup) protein samples prepared from exponential phase cultures of M3-type strains (used in A ) were analyzed by western immunoblotting, using anti-M3 protein rabbit polyclonal antibody. Expected molecular mass: 65 kDa. M, PageRuler™ Plus Prestained Protein Ladder.
Figure Legend Snippet: Assessment of Scl1.3 expression. (A) Assessment of Scl1.3 production by M3-type GAS. Cell wall (CW) and culture supernatant (Sup) protein fractions prepared from exponential phase cultures of several M3-type strains were analyzed by western immunoblotting, using anti-Scl1 rabbit polyclonal antibody. Recombinant protein rScl1.3V, corresponding to the variable region of Scl1.3, was used as a positive control. Expected molecular masses: Scl1.3, 11.4 kDa; rScl1.3V, 8.3 kDa. M, PageRuler™ Plus Prestained Protein Ladder. (B) Detection of Scl1.3 on the surface of M3-type GAS. Flow cytometry analysis of several M3-type strains is shown using anti-Scl1 antibody described in part (A) (color-shaded histograms) or a secondary only control (2° sample, black outlined histogram). Median fluorescence intensities (MFI) are shown in parentheses for each strain. (C) Assessment of scl1 transcription. Fold-change of scl1 transcript levels are shown compared to scl1.3 transcription in M3-type strain MGAS315. qRT-PCR was performed on RNA obtained from exponential phase cultures. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (student's t -test). (D) Assessment of mga and emm transcription. Relative expression levels of mga and emm genes were compared between MGAS315 and four additional M3 strains or the M1 strain MGAS5005. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; ** P ≤ 0.01. (E) Assessment of M3-protein production by M3-type GAS. The same cell wall (CW) and culture supernatant (Sup) protein samples prepared from exponential phase cultures of M3-type strains (used in A ) were analyzed by western immunoblotting, using anti-M3 protein rabbit polyclonal antibody. Expected molecular mass: 65 kDa. M, PageRuler™ Plus Prestained Protein Ladder.

Techniques Used: Expressing, Western Blot, Recombinant, Positive Control, Flow Cytometry, Cytometry, Fluorescence, Quantitative RT-PCR

Characterization of the scl2.3 locus in M3-type GAS. (A) Assessment of Scl2.3 production by M3-type GAS. The same cell wall (CW) and culture supernatant (Sup) protein samples prepared from exponential phase cultur es of several M3-type strains (used in Figures 2A,E ) were analyzed by western immunoblotting, using anti-rScl2.3V rabbit polyclonal antibody. Recombinant protein rScl2.3V, corresponding to the variable region of Scl2.3 protein, was used as a positive control. Expected molecular masses based on MGAS315: Scl2.3, 52.5 kDa; rScl2.3V, 10.1 kDa. Aberrant migration of detected Scl2.3 variants is characteristic of Scl proteins. M, PageRuler™ Plus Prestained Protein Ladder. (B) Detection of Scl2.3 on the surface of M3-type GAS. Flow cytometry analysis of several M3-type strains is shown using anti-rScl2.3V rabbit polyclonal antibody (color-shaded histograms) or a secondary-only control (2° sample, black outlined histogram). Median fluorescence intensities (MFI) are shown in parentheses for each strain. (C) Assessment of scl2 transcription. Fold-change of scl2 transcription levels are shown compared to scl2.3 transcription in M3-type MGAS315. qRT-PCR was performed on reverse-transcribed RNA obtained from exponential phase cultures. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; * P ≤ 0.05, ** P ≤ 0.01 (student's t -test).
Figure Legend Snippet: Characterization of the scl2.3 locus in M3-type GAS. (A) Assessment of Scl2.3 production by M3-type GAS. The same cell wall (CW) and culture supernatant (Sup) protein samples prepared from exponential phase cultur es of several M3-type strains (used in Figures 2A,E ) were analyzed by western immunoblotting, using anti-rScl2.3V rabbit polyclonal antibody. Recombinant protein rScl2.3V, corresponding to the variable region of Scl2.3 protein, was used as a positive control. Expected molecular masses based on MGAS315: Scl2.3, 52.5 kDa; rScl2.3V, 10.1 kDa. Aberrant migration of detected Scl2.3 variants is characteristic of Scl proteins. M, PageRuler™ Plus Prestained Protein Ladder. (B) Detection of Scl2.3 on the surface of M3-type GAS. Flow cytometry analysis of several M3-type strains is shown using anti-rScl2.3V rabbit polyclonal antibody (color-shaded histograms) or a secondary-only control (2° sample, black outlined histogram). Median fluorescence intensities (MFI) are shown in parentheses for each strain. (C) Assessment of scl2 transcription. Fold-change of scl2 transcription levels are shown compared to scl2.3 transcription in M3-type MGAS315. qRT-PCR was performed on reverse-transcribed RNA obtained from exponential phase cultures. Results are shown from three independent experiments, each performed in triplicate wells. Standard errors and statistical analysis were computed from averaged ΔCt values for each biological replicate prior to normalization against the endogenous reference gene tufA ; * P ≤ 0.05, ** P ≤ 0.01 (student's t -test).

Techniques Used: Western Blot, Recombinant, Positive Control, Migration, Flow Cytometry, Cytometry, Fluorescence, Quantitative RT-PCR

22) Product Images from "Ethyl pyruvate reduces organic dust-induced airway inflammation by targeting HMGB1-RAGE signaling"

Article Title: Ethyl pyruvate reduces organic dust-induced airway inflammation by targeting HMGB1-RAGE signaling

Journal: Respiratory Research

doi: 10.1186/s12931-019-0992-3

ODE exposure modulates NF-κB subunit gene expression with time. qRT-PCR analysis on NF-κB sub unit genes was performed on control and ODE exposed cells at 6, 24 and 48 h (a-3). Compared controls, ODE-exposure induced a significant increase in nfkbp65 ( a ), nfkbp52 ( b ) and crel ( d ) at 6, 24 and 48 h (#, p
Figure Legend Snippet: ODE exposure modulates NF-κB subunit gene expression with time. qRT-PCR analysis on NF-κB sub unit genes was performed on control and ODE exposed cells at 6, 24 and 48 h (a-3). Compared controls, ODE-exposure induced a significant increase in nfkbp65 ( a ), nfkbp52 ( b ) and crel ( d ) at 6, 24 and 48 h (#, p

Techniques Used: Expressing, Quantitative RT-PCR

ODE exposure increases tlr2 and tlr4 gene expression with time. qRT-PCR analysis on tlr2 ( a ) and tlr4 ( b ) genes was performed on control and ODE exposed cells at 6, 24 and 48 h. Compared to controls, ODE exposure resulted in a significant increase in fold in the expression of both tlr2 and tlr4 (###, p
Figure Legend Snippet: ODE exposure increases tlr2 and tlr4 gene expression with time. qRT-PCR analysis on tlr2 ( a ) and tlr4 ( b ) genes was performed on control and ODE exposed cells at 6, 24 and 48 h. Compared to controls, ODE exposure resulted in a significant increase in fold in the expression of both tlr2 and tlr4 (###, p

Techniques Used: Expressing, Quantitative RT-PCR

23) Product Images from "Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium"

Article Title: Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium

Journal: PLoS ONE

doi: 10.1371/journal.pone.0125921

Retinyl-ester processing in dgat1 -/- and lrat -/- mouse eyes. (A) Levels of the DGAT1 mRNA by qRT-PCR in retina samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (B) Levels of the DGAT1 mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (C) Levels of the LRAT mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. Error bars for (A), (B), and (C) represent standard deviation of the mean for four (n = 4) samples of mRNA tested (p =
Figure Legend Snippet: Retinyl-ester processing in dgat1 -/- and lrat -/- mouse eyes. (A) Levels of the DGAT1 mRNA by qRT-PCR in retina samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (B) Levels of the DGAT1 mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (C) Levels of the LRAT mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. Error bars for (A), (B), and (C) represent standard deviation of the mean for four (n = 4) samples of mRNA tested (p =

Techniques Used: Quantitative RT-PCR, Mouse Assay, Standard Deviation

DGAT1 expression in retina and RPE. (A) Immunofluorescent analysis of DGAT1 (green) and CRALBP (red) in distal ocular sections from six-month-old BALB/c mice. Nuclei were counter-stained with DAPI (blue). The merged image shows overlapping expression of DGAT1 and CRALBP (yellow). Labels identifying retinal layers are shown to the right of the image. RPE, retinal pigment epithelium; IPM, interphotoreceptor matrix; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Müller-cell endfeet contact the vitreous within the GCL. Note expression of both DGAT1 and CRALBP in the apical microvilli of Müller cells, extending beyond the OLM into the IPM. (B) Light microscopy of twenty day old primary cultured bovine Müller-cells (scale bar = 100μm, 10X). (C) Expression of MFAT, DGAT1, CRALBP, GFAP, and DES1 mRNA’s by qRT-PCR on cDNA from primary-cultured bovine Müller-cell RNA. Levels were normalized to the actin mRNA. CRALBP, GFAP, MFAT and DES1 are positive controls for Müller-cell expression. Error bars show standard deviation of the mean for four (n = 4) independent experiments (p =
Figure Legend Snippet: DGAT1 expression in retina and RPE. (A) Immunofluorescent analysis of DGAT1 (green) and CRALBP (red) in distal ocular sections from six-month-old BALB/c mice. Nuclei were counter-stained with DAPI (blue). The merged image shows overlapping expression of DGAT1 and CRALBP (yellow). Labels identifying retinal layers are shown to the right of the image. RPE, retinal pigment epithelium; IPM, interphotoreceptor matrix; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Müller-cell endfeet contact the vitreous within the GCL. Note expression of both DGAT1 and CRALBP in the apical microvilli of Müller cells, extending beyond the OLM into the IPM. (B) Light microscopy of twenty day old primary cultured bovine Müller-cells (scale bar = 100μm, 10X). (C) Expression of MFAT, DGAT1, CRALBP, GFAP, and DES1 mRNA’s by qRT-PCR on cDNA from primary-cultured bovine Müller-cell RNA. Levels were normalized to the actin mRNA. CRALBP, GFAP, MFAT and DES1 are positive controls for Müller-cell expression. Error bars show standard deviation of the mean for four (n = 4) independent experiments (p =

Techniques Used: Expressing, Mouse Assay, Staining, Light Microscopy, Cell Culture, Quantitative RT-PCR, Standard Deviation

24) Product Images from "Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum"

Article Title: Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of Tribolium castaneum

Journal: PLoS ONE

doi: 10.1371/journal.pone.0199056

qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.
Figure Legend Snippet: qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq. Tc ELOVL, elongation of very long chain fatty acids protein 7; Tc MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; Tc HSP70, heat shock 70kDa protein; Tc MAPK, MAP kinase; Tc DUSP, Dual specificity MAP kinase phosphatase; Tc SD, superoxide dismutase, Cu-Zn family; Tc AG, alpha-glucosidase; Tc DACHS, DACHS, Hippo signling pathway; Tc FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.

Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Binding Assay, Polymerase Chain Reaction

Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.
Figure Legend Snippet: Gene expression pattern of glycolytic (A) and Krebs (B) cycle enzymes of Tribolium castaneum larvae in response to hypoxia. Total RNA was isolated from the larvae after 12 hours’ hypoxia treatment. qRT-PCR was used to illustrate gene expression. Tc HK, hexokinase; Tc PGI, phosphoglucose isomerase; Tc PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Tc FBP, fructose 1,6-bisphosphate aldolase; Tc TPI, triosephosphate isomerase; Tc GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tc PGK, phosphoglycerate kinase; Tc PGM, phosphoglycerate mutase; Tc ENO, Enolase; Tc PK, Pyruvate kinase; Tc LDH, L-lactate dehydrogenase; Tc PDC, pyruvate dehydrogenase; Tc ACO, aconitate hydratase, mitochondria; Tc IDH, isocitrate dehydrogenase; Tc SCSb, succinyl-CoA synthetase beta chain; Tc SDH, succinate dehydrogenase; Tc FH, fumarate hydratase; Tc MDH, malate dehydrogenase. Red color represents upregulate, green color represents downregulate and black color represents no change.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

25) Product Images from "Macroautophagy and Selective Mitophagy Ameliorate Chondrogenic Differentiation Potential in Adipose Stem Cells of Equine Metabolic Syndrome: New Findings in the Field of Progenitor Cells Differentiation"

Article Title: Macroautophagy and Selective Mitophagy Ameliorate Chondrogenic Differentiation Potential in Adipose Stem Cells of Equine Metabolic Syndrome: New Findings in the Field of Progenitor Cells Differentiation

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2016/3718468

Mitochondria condition and clearance in ASC CTRL and ASC EMS . Representative photographs showing the results of DAPI, MitoRed, and anti-LAMP2 stainings. Interestingly, although LAMP2 expression was increased in ASC EMS , clearance of mitochondria in those cells seems to be reduced as we observed decreased number of mitochondria fused with lysosomes in comparison to control group (a). Flow cytometry analysis confirmed increased expression of LAMP2 in ASC EMS (b). Mitochondria deterioration was confirmed with flow cytometry using JC1 test, as we observed decreased mitochondrial membrane potential in ASC EMS (c). Moreover, the antioxidative protection coming from mitochondrial MnSOD was reduced (d) as established with qRT-PCR. The mir-140 transported via MVs was increased in ASC EMS (e) which correlates with increased expression of MFN. The serum level of FGF21, characteristic of obesity and diabetes, was upregulated in ASC EMS (f). Results are expressed as mean ± SD. ∗ p
Figure Legend Snippet: Mitochondria condition and clearance in ASC CTRL and ASC EMS . Representative photographs showing the results of DAPI, MitoRed, and anti-LAMP2 stainings. Interestingly, although LAMP2 expression was increased in ASC EMS , clearance of mitochondria in those cells seems to be reduced as we observed decreased number of mitochondria fused with lysosomes in comparison to control group (a). Flow cytometry analysis confirmed increased expression of LAMP2 in ASC EMS (b). Mitochondria deterioration was confirmed with flow cytometry using JC1 test, as we observed decreased mitochondrial membrane potential in ASC EMS (c). Moreover, the antioxidative protection coming from mitochondrial MnSOD was reduced (d) as established with qRT-PCR. The mir-140 transported via MVs was increased in ASC EMS (e) which correlates with increased expression of MFN. The serum level of FGF21, characteristic of obesity and diabetes, was upregulated in ASC EMS (f). Results are expressed as mean ± SD. ∗ p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR

miRNA expression during chondrogenic differentiation. Using qRT-PCR miRNA in chondrocytes precursors was established after day 10. No differences in mir-140 (a) and mir-223 were observed (c). Only mir-146 expression was significantly reduced in ASC EMS group (b). Results are expressed as mean ± SD. ∗ p
Figure Legend Snippet: miRNA expression during chondrogenic differentiation. Using qRT-PCR miRNA in chondrocytes precursors was established after day 10. No differences in mir-140 (a) and mir-223 were observed (c). Only mir-146 expression was significantly reduced in ASC EMS group (b). Results are expressed as mean ± SD. ∗ p

Techniques Used: Expressing, Quantitative RT-PCR

26) Product Images from "Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.)"

Article Title: Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.)

Journal: BMC Plant Biology

doi: 10.1186/s12870-015-0512-7

qRT-PCR analysis of relative CaHsfs transcript levels in R9 plants exposed to various abiotic stresses. qRT-PCR data of HS as seen in Fig. 4 . R9: pepper thermotolerant line; L-HS: heat stress (40 °C for 2 h); R-NaCl and S-NaCl: salt stress (300 mM NaCl for 6 h) responsiveness of CaHsf genes in roots and stems, respectively; R-Mannitol and S-Mannitol: osmotic stress (5 % mannitol for 6 h) responsiveness of CaHsf genes in roots and stems, respectively. The expression levels under salt and osmotic stress treatments were relative to that of the samples treated with water. L-CaCl 2 , −Put, −ABA and -MeJA: CaCl 2 (15 mM for 6 h), putrescine (Put, 1.5 mM for 6 h), abscisic acid (ABA, 100 μM for 3 h) and methyl jasmonate (MeJA, 100 μM for 6 h) responsiveness of CaHsf genes in leaves, respectively. MeJA was dissolved in 10 % ethanol and other substances were dissolved in water; therefore, control seedlings were sprayed with 10 % ethanol (for the MeJA treatment) or water (for the CaCl 2 , Put and ABA treatments). The expression levels are relative to that of the control samples
Figure Legend Snippet: qRT-PCR analysis of relative CaHsfs transcript levels in R9 plants exposed to various abiotic stresses. qRT-PCR data of HS as seen in Fig. 4 . R9: pepper thermotolerant line; L-HS: heat stress (40 °C for 2 h); R-NaCl and S-NaCl: salt stress (300 mM NaCl for 6 h) responsiveness of CaHsf genes in roots and stems, respectively; R-Mannitol and S-Mannitol: osmotic stress (5 % mannitol for 6 h) responsiveness of CaHsf genes in roots and stems, respectively. The expression levels under salt and osmotic stress treatments were relative to that of the samples treated with water. L-CaCl 2 , −Put, −ABA and -MeJA: CaCl 2 (15 mM for 6 h), putrescine (Put, 1.5 mM for 6 h), abscisic acid (ABA, 100 μM for 3 h) and methyl jasmonate (MeJA, 100 μM for 6 h) responsiveness of CaHsf genes in leaves, respectively. MeJA was dissolved in 10 % ethanol and other substances were dissolved in water; therefore, control seedlings were sprayed with 10 % ethanol (for the MeJA treatment) or water (for the CaCl 2 , Put and ABA treatments). The expression levels are relative to that of the control samples

Techniques Used: Quantitative RT-PCR, Expressing

Relative gene expression levels of CaHsfs , analysed by qRT-PCR, in response to HS treatment in B6 and R9 leaves. HS treatment: 40 °C for 2 h; B6: pepper thermosensitive line; R9: pepper thermotolerant line. qRT-PCR data were normalized using the pepper ubiquitin-conjugating protein gene ( UBI-3 ) and are shown relative to 0 h. The relative expression levels were calculated using the -ΔΔCT method and then a heat map with HemI was created
Figure Legend Snippet: Relative gene expression levels of CaHsfs , analysed by qRT-PCR, in response to HS treatment in B6 and R9 leaves. HS treatment: 40 °C for 2 h; B6: pepper thermosensitive line; R9: pepper thermotolerant line. qRT-PCR data were normalized using the pepper ubiquitin-conjugating protein gene ( UBI-3 ) and are shown relative to 0 h. The relative expression levels were calculated using the -ΔΔCT method and then a heat map with HemI was created

Techniques Used: Expressing, Quantitative RT-PCR

27) Product Images from "Characterization of Apoptosis, Autophagy and Oxidative Stress in Pancreatic Islets Cells and Intestinal Epithelial Cells Isolated from Equine Metabolic Syndrome (EMS) Horses"

Article Title: Characterization of Apoptosis, Autophagy and Oxidative Stress in Pancreatic Islets Cells and Intestinal Epithelial Cells Isolated from Equine Metabolic Syndrome (EMS) Horses

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103068

Morphology and proliferation of PIs in culture. The cultures were monitored after 72 ( A ), 120 ( B ) and 168 ( C , D ) h in culture. Immunofluorescence staining for insulin ( E ) was quantified using ImageJ by the meaning of fluorescence intensity. The obtained data indicate decreased insulin secretion by PIs EMS ( F ). Proliferation of cells was established using BrdU assay which indicates DNA synthesis rate. In the control group we observed increased BrdU incorporation in comparison to EMS, where DNA synthesis remained at a similar level during propagation. Additionally, using qRT-PCR we established the expression of two genes involved in ER stress. The amount of both CHOP ( G ) and PERK ( H ) mRNA was increased in PIs EMS , but only CHOP showed a statistically significant difference ( p
Figure Legend Snippet: Morphology and proliferation of PIs in culture. The cultures were monitored after 72 ( A ), 120 ( B ) and 168 ( C , D ) h in culture. Immunofluorescence staining for insulin ( E ) was quantified using ImageJ by the meaning of fluorescence intensity. The obtained data indicate decreased insulin secretion by PIs EMS ( F ). Proliferation of cells was established using BrdU assay which indicates DNA synthesis rate. In the control group we observed increased BrdU incorporation in comparison to EMS, where DNA synthesis remained at a similar level during propagation. Additionally, using qRT-PCR we established the expression of two genes involved in ER stress. The amount of both CHOP ( G ) and PERK ( H ) mRNA was increased in PIs EMS , but only CHOP showed a statistically significant difference ( p

Techniques Used: Immunofluorescence, Staining, Fluorescence, BrdU Staining, DNA Synthesis, BrdU Incorporation Assay, Quantitative RT-PCR, Expressing

Autophagy in PIs. Using confocal microscopy, co-localization of LAMP2 and mitochondria was visualized in investigated cultures ( A ). Representative photographs indicate increased lysosome and mitolysosome formation in EMS groups. Moreover, using qRT-PCR, the expression of autophagy-related genes including LAMP2 ( B ), LC3 ( C ), Parkin ( D ) and Beclin ( E ) was evaluated. The obtained data confirmed increased autophagy in PIs EMS . Scale bars: confocal microscope: 5 µm. Results expressed as mean ± S.D. * p
Figure Legend Snippet: Autophagy in PIs. Using confocal microscopy, co-localization of LAMP2 and mitochondria was visualized in investigated cultures ( A ). Representative photographs indicate increased lysosome and mitolysosome formation in EMS groups. Moreover, using qRT-PCR, the expression of autophagy-related genes including LAMP2 ( B ), LC3 ( C ), Parkin ( D ) and Beclin ( E ) was evaluated. The obtained data confirmed increased autophagy in PIs EMS . Scale bars: confocal microscope: 5 µm. Results expressed as mean ± S.D. * p

Techniques Used: Confocal Microscopy, Quantitative RT-PCR, Expressing, Microscopy

28) Product Images from "Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism"

Article Title: Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0094988

The pUR4 fibronectin inhibitor promotes elevated expression of SM differentiation genes. SMC were incubated in Medium 231 supplemented with smooth muscle growth supplement. 30–60 min after seeding, pUR4 or III-11C were added as described in Methods . RNA was prepared from cells 5 days after seeding as described in Methods . qRT-PCR was performed in triplicate for each sample using SYBR green chemistry as described in Methods . The data was normalized to the levels of GAPDH in each sample. Data represent the average of 4 separate experiments. The relative levels of γ-actin, SM α-actin, calponin, and α8 integrin are shown; pUR4 treated samples were set equal to 1. The error bars represent the s.e.m. *p
Figure Legend Snippet: The pUR4 fibronectin inhibitor promotes elevated expression of SM differentiation genes. SMC were incubated in Medium 231 supplemented with smooth muscle growth supplement. 30–60 min after seeding, pUR4 or III-11C were added as described in Methods . RNA was prepared from cells 5 days after seeding as described in Methods . qRT-PCR was performed in triplicate for each sample using SYBR green chemistry as described in Methods . The data was normalized to the levels of GAPDH in each sample. Data represent the average of 4 separate experiments. The relative levels of γ-actin, SM α-actin, calponin, and α8 integrin are shown; pUR4 treated samples were set equal to 1. The error bars represent the s.e.m. *p

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, SYBR Green Assay

The pUR4 fibronectin inhibitor promotes elevated levels of SRF protein. mRNA or protein were isolated from cells 5 days after seeding as described in Methods . A) qRT-PCR was performed in triplicate for each sample using SYBR green chemistry as described in Methods . The data was normalized to the levels of GAPDH in each sample. Data represent the average of 4 separate experiments, and the error bars the s.e.m. The relative levels of SRF mRNA are shown; pUR4 treated samples were set equal to 1. B) Western blotting was performed using antibodies to SRF. C) SRF protein levels in 3 separate experiments were quantitated using an Odyssey Infrared Imager following normalization to the levels of tubulin in each sample. Normalized SRF levels in pUR4 treated cells were set equal to 1. The error bar represents the s.e.m. *p
Figure Legend Snippet: The pUR4 fibronectin inhibitor promotes elevated levels of SRF protein. mRNA or protein were isolated from cells 5 days after seeding as described in Methods . A) qRT-PCR was performed in triplicate for each sample using SYBR green chemistry as described in Methods . The data was normalized to the levels of GAPDH in each sample. Data represent the average of 4 separate experiments, and the error bars the s.e.m. The relative levels of SRF mRNA are shown; pUR4 treated samples were set equal to 1. B) Western blotting was performed using antibodies to SRF. C) SRF protein levels in 3 separate experiments were quantitated using an Odyssey Infrared Imager following normalization to the levels of tubulin in each sample. Normalized SRF levels in pUR4 treated cells were set equal to 1. The error bar represents the s.e.m. *p

Techniques Used: Isolation, Quantitative RT-PCR, SYBR Green Assay, Western Blot

29) Product Images from "Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA"

Article Title: Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA

Journal: Nature Communications

doi: 10.1038/s41467-018-08059-z

HMR’s TAD is required for the activation of thermoresponsive genes. a qRT-PCR analyses of the steady-state levels of warm-temperature-induced PIF4 direct target genes, YUC8 , IAA29 , and IAA19 , in 4-d-old Col-0, pif4-2 , hmr-5 , and hmr-22 seedlings grown under 27 °C in Rc, LD (R-LD), and SD (R-SD) conditions with 10 μmol m −2 s −1 R light. Samples were taken at 96 h after stratification for Rc, 104 h (or ZT 8) for LD, and 94 h (or ZT 22) for SD. Error bars represent SD of three replicates. Different letters denote statistically significant differences in transcript levels (ANOVA, Tukey’s HSD, P
Figure Legend Snippet: HMR’s TAD is required for the activation of thermoresponsive genes. a qRT-PCR analyses of the steady-state levels of warm-temperature-induced PIF4 direct target genes, YUC8 , IAA29 , and IAA19 , in 4-d-old Col-0, pif4-2 , hmr-5 , and hmr-22 seedlings grown under 27 °C in Rc, LD (R-LD), and SD (R-SD) conditions with 10 μmol m −2 s −1 R light. Samples were taken at 96 h after stratification for Rc, 104 h (or ZT 8) for LD, and 94 h (or ZT 22) for SD. Error bars represent SD of three replicates. Different letters denote statistically significant differences in transcript levels (ANOVA, Tukey’s HSD, P

Techniques Used: Activation Assay, Quantitative RT-PCR

HMR interacts with PIF4 and facilitates thermoresponsive PIF4 accumulation . a qRT-PCR analyses of the steady-state transcript levels of PIF4 in 4-d-old Col-0, pif4-2 , hmr-5 , and hmr-22 seedlings grown at 27 °C in Rc, LD (R-LD), and SD (R-SD) conditions with 10 μmol m −2 s −1 R light. Samples were taken at 96 h after stratification for Rc, 104 h (or ZT 8) for LD, and 94 h (or ZT 22) for SD. Error bars represent SD of three biological replicates. Different letters denote statistically significant differences in PIF4 transcript levels (ANOVA, Tukey’s HSD, P
Figure Legend Snippet: HMR interacts with PIF4 and facilitates thermoresponsive PIF4 accumulation . a qRT-PCR analyses of the steady-state transcript levels of PIF4 in 4-d-old Col-0, pif4-2 , hmr-5 , and hmr-22 seedlings grown at 27 °C in Rc, LD (R-LD), and SD (R-SD) conditions with 10 μmol m −2 s −1 R light. Samples were taken at 96 h after stratification for Rc, 104 h (or ZT 8) for LD, and 94 h (or ZT 22) for SD. Error bars represent SD of three biological replicates. Different letters denote statistically significant differences in PIF4 transcript levels (ANOVA, Tukey’s HSD, P

Techniques Used: Quantitative RT-PCR

30) Product Images from "Small molecule NSC59984 restores p53 pathway signaling and anti-tumor effects against colorectal cancer via p73 activation and degradation of mutant p53"

Article Title: Small molecule NSC59984 restores p53 pathway signaling and anti-tumor effects against colorectal cancer via p73 activation and degradation of mutant p53

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-13-1079

NSC59984 induces mutant p53 degradation via MDM2-mediated ubiquitination. A. Mutant p53 protein levels in SW480 cells treated with 10µM of MG132 and NSC59984 for 16 hr. B. Mutant p53 protein levels in SW480 cells treated with 5µM of nutlin-3 and 25µM of NSC59984 for 16 hr. C. Ubiquitination (Ub) of mutant p53 in cells treated with NSC59984 and MG132. Cells were transfected with HA-Ub for 48 hr, followed by treatment with 25µM of NSC59984 and MG132 for 16 hr. Cell lysates were subjected to immunoprecipitation. D. Mutant p53 mRNA level in SW480 cells treated with NSC59984 for 3hr and 16 hr, mRNA was quantified by qRT-PCR. Data were normalized to GAPDH and plotted relative to cells treated with the DMSO control. Data are expressed as mean ± SD.
Figure Legend Snippet: NSC59984 induces mutant p53 degradation via MDM2-mediated ubiquitination. A. Mutant p53 protein levels in SW480 cells treated with 10µM of MG132 and NSC59984 for 16 hr. B. Mutant p53 protein levels in SW480 cells treated with 5µM of nutlin-3 and 25µM of NSC59984 for 16 hr. C. Ubiquitination (Ub) of mutant p53 in cells treated with NSC59984 and MG132. Cells were transfected with HA-Ub for 48 hr, followed by treatment with 25µM of NSC59984 and MG132 for 16 hr. Cell lysates were subjected to immunoprecipitation. D. Mutant p53 mRNA level in SW480 cells treated with NSC59984 for 3hr and 16 hr, mRNA was quantified by qRT-PCR. Data were normalized to GAPDH and plotted relative to cells treated with the DMSO control. Data are expressed as mean ± SD.

Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, Quantitative RT-PCR

NSC59984 restores p53-responsive transcriptional activity in mutant p53- expressing tumor cells. A. Structure of NSC59984. B. Imaging bioluminescence assay of p53-responsive transcriptional activity in cells at 24 hr after NSC59984 treatment. Data are representative of triplicate wells. C. The fold-increase of p53 responsive bioluminescence (B). D. mRNA levels of p21, Puma and Noxa in cells at 3 hr after NSC59984 treatment. mRNA levels were quantified by qRT-PCR. Data were normalized to GAPDH expression and plotted relative to cells treated with DMSO as control. Data are expressed as mean ± SD, *p
Figure Legend Snippet: NSC59984 restores p53-responsive transcriptional activity in mutant p53- expressing tumor cells. A. Structure of NSC59984. B. Imaging bioluminescence assay of p53-responsive transcriptional activity in cells at 24 hr after NSC59984 treatment. Data are representative of triplicate wells. C. The fold-increase of p53 responsive bioluminescence (B). D. mRNA levels of p21, Puma and Noxa in cells at 3 hr after NSC59984 treatment. mRNA levels were quantified by qRT-PCR. Data were normalized to GAPDH expression and plotted relative to cells treated with DMSO as control. Data are expressed as mean ± SD, *p

Techniques Used: Activity Assay, Mutagenesis, Expressing, Imaging, ATP Bioluminescent Assay, Quantitative RT-PCR

31) Product Images from "Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts"

Article Title: Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0139464

Sip4 is involved in expression of carnitine shuttle genes in K . lactis . (A) Schematic overview of metabolic pathways and key genes essential for the utilization of non-fermentable carbon source ethanol in yeast as found in the yeast genome database [ 33 ]. (B) RNA levels of genes related to the carnitine shuttle in sip4 Δ, cat8 Δ and sip4 Δ cat8 Δ mutants relative to congenic wild-type strains were determined by qRT-PCR in S . cerevisiae (left panel) and K . lactis (right panel). Cultures were shifted from 2% glucose to 3% ethanol medium for 2 hours at an OD 600 of 0.8 to 1.0. Gene expression levels were normalized to the reference gene HEM2 and quantified relative to wild-type levels (set to 1.0; dashed line) Data points and error bars represent mean values ± standard deviations obtained with three independent biological samples each measured in technical triplicates. Asterisks indicate statistically significant differences compared to wild-type ( t -test; * P
Figure Legend Snippet: Sip4 is involved in expression of carnitine shuttle genes in K . lactis . (A) Schematic overview of metabolic pathways and key genes essential for the utilization of non-fermentable carbon source ethanol in yeast as found in the yeast genome database [ 33 ]. (B) RNA levels of genes related to the carnitine shuttle in sip4 Δ, cat8 Δ and sip4 Δ cat8 Δ mutants relative to congenic wild-type strains were determined by qRT-PCR in S . cerevisiae (left panel) and K . lactis (right panel). Cultures were shifted from 2% glucose to 3% ethanol medium for 2 hours at an OD 600 of 0.8 to 1.0. Gene expression levels were normalized to the reference gene HEM2 and quantified relative to wild-type levels (set to 1.0; dashed line) Data points and error bars represent mean values ± standard deviations obtained with three independent biological samples each measured in technical triplicates. Asterisks indicate statistically significant differences compared to wild-type ( t -test; * P

Techniques Used: Expressing, Quantitative RT-PCR

KlSIP4-6HA and KlCAT8-6HA RNA and protein levels after a shift to ethanol. (A and B) Time course of KlSIP4-6HA (A) and KlCAT8-6HA (B) gene expression. Wild-type cells expressing KlSip4-(HA) 6 (JA6/S4HA) or KlCat8-(HA) 6 (JA6/C8HA) were grown in glucose and shifted to ethanol medium at time zero. Samples were taken at the indicated time points, RNA was isolated and qRT-PCR was performed in triplicates. Fold changes relative to the time 0 sample were calculated by the 2 −∆∆CT method, normalized to the reference gene KlHEM2 . (C and D) Time course of protein levels. In parallel to the RNA preparations (panel A and B) total protein extracts were prepared, separated by SDS-PAGE and analyzed by Western blotting using anti-HA antibody (top panel). Nop1, detected with an anti-Nop1 antibody served as loading control (bottom panel). The position of KlSip4-(HA) 6 (89.1 kDa), KlCat8-(HA) 6 (166.4 kDa) and KlNop1 (34.8 kDa) are indicated by arrows.
Figure Legend Snippet: KlSIP4-6HA and KlCAT8-6HA RNA and protein levels after a shift to ethanol. (A and B) Time course of KlSIP4-6HA (A) and KlCAT8-6HA (B) gene expression. Wild-type cells expressing KlSip4-(HA) 6 (JA6/S4HA) or KlCat8-(HA) 6 (JA6/C8HA) were grown in glucose and shifted to ethanol medium at time zero. Samples were taken at the indicated time points, RNA was isolated and qRT-PCR was performed in triplicates. Fold changes relative to the time 0 sample were calculated by the 2 −∆∆CT method, normalized to the reference gene KlHEM2 . (C and D) Time course of protein levels. In parallel to the RNA preparations (panel A and B) total protein extracts were prepared, separated by SDS-PAGE and analyzed by Western blotting using anti-HA antibody (top panel). Nop1, detected with an anti-Nop1 antibody served as loading control (bottom panel). The position of KlSip4-(HA) 6 (89.1 kDa), KlCat8-(HA) 6 (166.4 kDa) and KlNop1 (34.8 kDa) are indicated by arrows.

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

32) Product Images from "Stat5b Regulates Sexually Dimorphic Gene Expression in Zebrafish Liver"

Article Title: Stat5b Regulates Sexually Dimorphic Gene Expression in Zebrafish Liver

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00676

Verification of the transcriptome data by qRT-PCR. (A) Profile of Illumina sequencing value for selected genes with normalized expression data (FPKM value). (B,C) Relative expression of selected genes by qRT-PCR. Values are indicated as means ± standard deviation (SD). The data was assessed using a Student’s t -test. A probability P
Figure Legend Snippet: Verification of the transcriptome data by qRT-PCR. (A) Profile of Illumina sequencing value for selected genes with normalized expression data (FPKM value). (B,C) Relative expression of selected genes by qRT-PCR. Values are indicated as means ± standard deviation (SD). The data was assessed using a Student’s t -test. A probability P

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

Identification of candidate genes correlated with growth traits. (A) Venn diagram of the candidate DEGs among three comparisons: WFM vs. WM (purple circle), SFM vs. SM (green circle) and growth traits-related candidate genes in female blue module (orange circle). (B) Venn diagram of the candidate DEGs among three comparisons: WFM vs. WM (purple circle), SFM vs. SM (green circle) and growth traits-related candidate genes in male turquoise module (orange circle). (C) qRT-PCR verification of several candidate genes related to growth traits, including esr1 , vtg2 , clo1a1a , greb1, and igf2b . Ef1a was used for the internal reference. (D) Two-way ANOVAs of mRNA expression levels of esr1 , vtg2 , clo1a1a , greb1, and igf2b . Values are indicated as means ± standard deviation (SD). All the experiments were conducted in triplicate. The data was assessed using a Student’s t -test and two-way ANOVA. Asterisk denoted significant differences and a probability P
Figure Legend Snippet: Identification of candidate genes correlated with growth traits. (A) Venn diagram of the candidate DEGs among three comparisons: WFM vs. WM (purple circle), SFM vs. SM (green circle) and growth traits-related candidate genes in female blue module (orange circle). (B) Venn diagram of the candidate DEGs among three comparisons: WFM vs. WM (purple circle), SFM vs. SM (green circle) and growth traits-related candidate genes in male turquoise module (orange circle). (C) qRT-PCR verification of several candidate genes related to growth traits, including esr1 , vtg2 , clo1a1a , greb1, and igf2b . Ef1a was used for the internal reference. (D) Two-way ANOVAs of mRNA expression levels of esr1 , vtg2 , clo1a1a , greb1, and igf2b . Values are indicated as means ± standard deviation (SD). All the experiments were conducted in triplicate. The data was assessed using a Student’s t -test and two-way ANOVA. Asterisk denoted significant differences and a probability P

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

33) Product Images from "RNA interference-mediated knockdown of voltage-gated sodium channel (MpNav) gene causes mortality in peach-potato aphid, Myzus persicae"

Article Title: RNA interference-mediated knockdown of voltage-gated sodium channel (MpNav) gene causes mortality in peach-potato aphid, Myzus persicae

Journal: Scientific Reports

doi: 10.1038/s41598-019-41832-8

Stage-specific expression pattern of the M . persicae gene MpNa v H1 in the whole insect body analyzed by qRT-PCR. The letters above the bars show significant differences (least significant difference in one-way analysis of variance, P
Figure Legend Snippet: Stage-specific expression pattern of the M . persicae gene MpNa v H1 in the whole insect body analyzed by qRT-PCR. The letters above the bars show significant differences (least significant difference in one-way analysis of variance, P

Techniques Used: Expressing, Quantitative RT-PCR

Dietary delivery of MpNa v dsRNA alters MpNa v expression in M . persicae . Relative abundances of MpNa v H1 gene transcripts, as determined by qRT-PCR of cDNA made from total RNA isolated from whole body 1 to 7 days after the indicated dietary treatments of M . persicae . Data represent the mean ± SEM of three independent experiments. MpNa v mRNA was normalized with Actin as an endogenous control. Treatment was compared with controls using ANOVA (Tukey Kramer multiple comparison, p
Figure Legend Snippet: Dietary delivery of MpNa v dsRNA alters MpNa v expression in M . persicae . Relative abundances of MpNa v H1 gene transcripts, as determined by qRT-PCR of cDNA made from total RNA isolated from whole body 1 to 7 days after the indicated dietary treatments of M . persicae . Data represent the mean ± SEM of three independent experiments. MpNa v mRNA was normalized with Actin as an endogenous control. Treatment was compared with controls using ANOVA (Tukey Kramer multiple comparison, p

Techniques Used: Expressing, Quantitative RT-PCR, Isolation

34) Product Images from "The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells"

Article Title: The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013445

( A ) MRNA microarray validation with qRT-PCR analysis in grouped RISC-IP U-87 astrocytoma and primary astrocytes samples. Grouped RISC-IP data were compared to the grouped global mRNA from U-87 astrocytoma and primary astrocytes samples. Eight mRNAs were selected from the grouped mRNA microarray dataset and examined by qRT-PCR. Fold change from the mRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all mRNA expression values were normalized to the beta-actin endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Importantly, the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of up-regulation and down-regulation can be compared. ( B ) MRNAs in RISC compared to the global cellular milieu in U-87 astrocytoma cells. MRNA expression in U-87 astrocytoma cells were normalized to primary astrocytes mRNA expression. All mRNAs had a fold change > 2.5 and were significantly expressed (p
Figure Legend Snippet: ( A ) MRNA microarray validation with qRT-PCR analysis in grouped RISC-IP U-87 astrocytoma and primary astrocytes samples. Grouped RISC-IP data were compared to the grouped global mRNA from U-87 astrocytoma and primary astrocytes samples. Eight mRNAs were selected from the grouped mRNA microarray dataset and examined by qRT-PCR. Fold change from the mRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all mRNA expression values were normalized to the beta-actin endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Importantly, the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of up-regulation and down-regulation can be compared. ( B ) MRNAs in RISC compared to the global cellular milieu in U-87 astrocytoma cells. MRNA expression in U-87 astrocytoma cells were normalized to primary astrocytes mRNA expression. All mRNAs had a fold change > 2.5 and were significantly expressed (p

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

MiRNA microarray validation with qRT-PCR analysis for (A) global miRNA and (B) RISC-specific miRNA. Nine random miRNAs were selected from the miRNA microarray datasets and examined by qRT-PCR. Fold change from the miRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all miRNA expression values were normalized to the RNU6B endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Note: only the general trend of up-regulation and down-regulation can be compared but the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods.
Figure Legend Snippet: MiRNA microarray validation with qRT-PCR analysis for (A) global miRNA and (B) RISC-specific miRNA. Nine random miRNAs were selected from the miRNA microarray datasets and examined by qRT-PCR. Fold change from the miRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all miRNA expression values were normalized to the RNU6B endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Note: only the general trend of up-regulation and down-regulation can be compared but the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods.

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

35) Product Images from "Identification of mTOR inhibitor-resistant genes in cutaneous squamous cell carcinoma"

Article Title: Identification of mTOR inhibitor-resistant genes in cutaneous squamous cell carcinoma

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S174966

CCND1 overexpression induces everolimus resistance in HSC-1 cells. Notes: ( A ) qRT-PCR analysis of exogenous CCND1 expression. ( B ) Cell viability in control and CCND1-overexpressing cells following treatment with 100 nM of everolimus. Abbreviation: qRT-PCR, quantitative real-time PCR.
Figure Legend Snippet: CCND1 overexpression induces everolimus resistance in HSC-1 cells. Notes: ( A ) qRT-PCR analysis of exogenous CCND1 expression. ( B ) Cell viability in control and CCND1-overexpressing cells following treatment with 100 nM of everolimus. Abbreviation: qRT-PCR, quantitative real-time PCR.

Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

IPA of the genes associated with everolimus resistance. Notes: ( A ) The functional pathway analysis network using IPA. ( B ) Validation of the DEG expression levels significantly involved in the pathway network using qRT-PCR analysis (* P
Figure Legend Snippet: IPA of the genes associated with everolimus resistance. Notes: ( A ) The functional pathway analysis network using IPA. ( B ) Validation of the DEG expression levels significantly involved in the pathway network using qRT-PCR analysis (* P

Techniques Used: Indirect Immunoperoxidase Assay, Functional Assay, Expressing, Quantitative RT-PCR

36) Product Images from "Dexamethasone Induces Cross-Linked Actin Networks in Trabecular Meshwork Cells Through Noncanonical Wnt Signaling"

Article Title: Dexamethasone Induces Cross-Linked Actin Networks in Trabecular Meshwork Cells Through Noncanonical Wnt Signaling

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.13-12447

Degenerate PCR and qRT-PCR
Figure Legend Snippet: Degenerate PCR and qRT-PCR

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR

Promoter B of Wnt5a is activated in TM cells and SMAD agonist BMP4 induces CLAN formation in TM cells. ( A ) The qRT-PCR analysis of DEX-treated TM cells. Promoter B specific transcript is upregulated by DEX treatment. Bmp4 also is upregulated, while Foxc1
Figure Legend Snippet: Promoter B of Wnt5a is activated in TM cells and SMAD agonist BMP4 induces CLAN formation in TM cells. ( A ) The qRT-PCR analysis of DEX-treated TM cells. Promoter B specific transcript is upregulated by DEX treatment. Bmp4 also is upregulated, while Foxc1

Techniques Used: Quantitative RT-PCR

37) Product Images from "Physiological and transcriptomic analyses reveal a response mechanism to cold stress in Santalum album L. leaves"

Article Title: Physiological and transcriptomic analyses reveal a response mechanism to cold stress in Santalum album L. leaves

Journal: Scientific Reports

doi: 10.1038/srep42165

Expression patterns of cold-inducible genes shown by qRT-PCR following cold stress (4 °C) treatment. Three measurements were averaged from the results of three replicated experiments and statistically treated using a t -test. * P ≤ 0.05 > 0.005; ** P ≤ 0.005
Figure Legend Snippet: Expression patterns of cold-inducible genes shown by qRT-PCR following cold stress (4 °C) treatment. Three measurements were averaged from the results of three replicated experiments and statistically treated using a t -test. * P ≤ 0.05 > 0.005; ** P ≤ 0.005

Techniques Used: Expressing, Quantitative RT-PCR

Differentially expressed genes involved in terpenoid biosynthesis in response to cold stress. ( a ) Heat map of changes of transcript abundance for eight genes. ( b ) qRT-PCR determination of transcript levels of these genes in leaves and roots following cold treatment (4 °C). Three measurements were averaged from the results of three replicated experiments and statistically treated using a t -test. * P ≤ 0.05 > 0.005; ** P ≤ 0.005
Figure Legend Snippet: Differentially expressed genes involved in terpenoid biosynthesis in response to cold stress. ( a ) Heat map of changes of transcript abundance for eight genes. ( b ) qRT-PCR determination of transcript levels of these genes in leaves and roots following cold treatment (4 °C). Three measurements were averaged from the results of three replicated experiments and statistically treated using a t -test. * P ≤ 0.05 > 0.005; ** P ≤ 0.005

Techniques Used: Quantitative RT-PCR

38) Product Images from "Selective Activation of Striatal NGF-TrkA/p75NTR/MAPK Intracellular Signaling in Rats That Show Suppression of Methamphetamine Intake 30 Days following Drug Abstinence"

Article Title: Selective Activation of Striatal NGF-TrkA/p75NTR/MAPK Intracellular Signaling in Rats That Show Suppression of Methamphetamine Intake 30 Days following Drug Abstinence

Journal: International Journal of Neuropsychopharmacology

doi: 10.1093/ijnp/pyx105

Distinct neurotrophin, neuropeptide, and immediate early genes (IEGs) expression in the dorsal striatum of shock-resistant (SR) and shock-sensitive (SS) rats. (a) Independent qRT-PCR analyses for neurotrophin mRNA levels in the dorsal striatum with increased expression of Bdnf in both SR and SS rats, decrease TrkA levels in SS rats, and increased Gfra2 levels also in SS rats. (b) Differential expression neuropeptide-related genes and receptors in the dorsal striatum, with SS rats having increased mRNA levels for crhr1 , crhbp , and unc2 compared with saline controls and SR rats. (c) Altered mRNA levels of IEGs in the dorsal striatum of SR and SS rats. Both SR and SS rats had increased expression of c-fos mRNA levels compared with saline controls, while only SR rats had increased egr1 and egr2 mRNA levels compared with saline and SS rats. Key to statistics: * P
Figure Legend Snippet: Distinct neurotrophin, neuropeptide, and immediate early genes (IEGs) expression in the dorsal striatum of shock-resistant (SR) and shock-sensitive (SS) rats. (a) Independent qRT-PCR analyses for neurotrophin mRNA levels in the dorsal striatum with increased expression of Bdnf in both SR and SS rats, decrease TrkA levels in SS rats, and increased Gfra2 levels also in SS rats. (b) Differential expression neuropeptide-related genes and receptors in the dorsal striatum, with SS rats having increased mRNA levels for crhr1 , crhbp , and unc2 compared with saline controls and SR rats. (c) Altered mRNA levels of IEGs in the dorsal striatum of SR and SS rats. Both SR and SS rats had increased expression of c-fos mRNA levels compared with saline controls, while only SR rats had increased egr1 and egr2 mRNA levels compared with saline and SS rats. Key to statistics: * P

Techniques Used: Expressing, Quantitative RT-PCR

Transcriptional alterations 30 days after the punishment phase of methamphetamine (METH) self-administration (SA) in the dorsal striatum of shock-resistant (SR) and shock-sensitive (SS) rats. (a) The graph shows neurotrophic- and neuropeptide-associated genes that were differentially expressed between SR and SS rats as determined by RT 2 Profiler PCR Arrays. (b) The figure shows transcriptional responses obtained by Profiler Arrays (“x” axis) and individual qRT-PCRs (“y” axis) for upregulated genes in the dorsal striatum of SR rats (in grey circles) and SS rats (in black circles). Data are presented as fold-changes relative to saline control rats. A significant correlation was observed between the 2 analyses ( P = .001). Key to statistics: # P
Figure Legend Snippet: Transcriptional alterations 30 days after the punishment phase of methamphetamine (METH) self-administration (SA) in the dorsal striatum of shock-resistant (SR) and shock-sensitive (SS) rats. (a) The graph shows neurotrophic- and neuropeptide-associated genes that were differentially expressed between SR and SS rats as determined by RT 2 Profiler PCR Arrays. (b) The figure shows transcriptional responses obtained by Profiler Arrays (“x” axis) and individual qRT-PCRs (“y” axis) for upregulated genes in the dorsal striatum of SR rats (in grey circles) and SS rats (in black circles). Data are presented as fold-changes relative to saline control rats. A significant correlation was observed between the 2 analyses ( P = .001). Key to statistics: # P

Techniques Used: Polymerase Chain Reaction

39) Product Images from "hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia"

Article Title: hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia

Journal: Oncogenesis

doi: 10.1038/oncsis.2016.72

NEIL1 was downregulated upon concurrent suppression of MTH1 and MYH. ( a ) Expression of DNA glycosylases were analyzed after 96 h of Dox treatment using qRT–PCR and the results are shown in a heat-map diagram. ( b ) Detailed NEIL1 expression levels are represented in a bar chart. Data are presented as mean±s.e.m. from three independent experiments in triplicate. P- values were obtained using one-way analysis of variance (ANOVA). * P
Figure Legend Snippet: NEIL1 was downregulated upon concurrent suppression of MTH1 and MYH. ( a ) Expression of DNA glycosylases were analyzed after 96 h of Dox treatment using qRT–PCR and the results are shown in a heat-map diagram. ( b ) Detailed NEIL1 expression levels are represented in a bar chart. Data are presented as mean±s.e.m. from three independent experiments in triplicate. P- values were obtained using one-way analysis of variance (ANOVA). * P

Techniques Used: Expressing, Quantitative RT-PCR

Simultaneous suppression of MTH1 and MYH was efficiently achieved using a two-vector system. ( a ) Steps of cell line establishment are illustrated here. Top 15% of cells expressing GFP and RFP670 were sorted to improve the knockdown efficiency. Expression levels of MYH and MTH1 were analyzed after 96 h of treatment with Dox in shRNA set1 ( b ) and shRNA set2 ( c ) using qRT–PCR analysis. Data were normalized to NT-shRNA expressing cells and presented as mean±s.e.m. from three independent experiments in triplicate. To investigate the knockdown efficiency at protein level, western blot analysis was performed after 96 h of Dox treatment of cells with shRNA set1 ( d ) and shRNA set2 ( e ). Data were double normalized with GAPDH and NT-shRNA control samples and expressed as percentage. The graphs represent mean±s.e.m. from three independent experiments. P- values were calculated using one-way analysis of variance (ANOVA). * P
Figure Legend Snippet: Simultaneous suppression of MTH1 and MYH was efficiently achieved using a two-vector system. ( a ) Steps of cell line establishment are illustrated here. Top 15% of cells expressing GFP and RFP670 were sorted to improve the knockdown efficiency. Expression levels of MYH and MTH1 were analyzed after 96 h of treatment with Dox in shRNA set1 ( b ) and shRNA set2 ( c ) using qRT–PCR analysis. Data were normalized to NT-shRNA expressing cells and presented as mean±s.e.m. from three independent experiments in triplicate. To investigate the knockdown efficiency at protein level, western blot analysis was performed after 96 h of Dox treatment of cells with shRNA set1 ( d ) and shRNA set2 ( e ). Data were double normalized with GAPDH and NT-shRNA control samples and expressed as percentage. The graphs represent mean±s.e.m. from three independent experiments. P- values were calculated using one-way analysis of variance (ANOVA). * P

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

40) Product Images from "Opposite Roles of Wnt7a and Sfrp1 in Modulating Proper Development of Neural Progenitors in the Mouse Cerebral Cortex"

Article Title: Opposite Roles of Wnt7a and Sfrp1 in Modulating Proper Development of Neural Progenitors in the Mouse Cerebral Cortex

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00247

Wnt7a and Sfrp1 are co-expressed in neural progenitors and show opposite expression trends. (A) In coronal sections of mouse E12.5 cerebral cortices, Wnt7a , Sfrp1 , Pax6 , Ngn2 , and Hes5 were expressed in the ventricular zone (arrowheads). Conversely, Wnt7b was expressed in newborn neurons. Red boxes show high power views. (B) qRT-PCR analysis of Wnt7a and Sfrp1 expression levels at different embryonic stages (E12.5, E13.5, E14.5, E15.5, and E17.5). All comparisons were made with that of values at E12.5. Values of histogram represent mean ± SEM, and each dot represents a data point in each biology repeat ( n = 3, ∗ P
Figure Legend Snippet: Wnt7a and Sfrp1 are co-expressed in neural progenitors and show opposite expression trends. (A) In coronal sections of mouse E12.5 cerebral cortices, Wnt7a , Sfrp1 , Pax6 , Ngn2 , and Hes5 were expressed in the ventricular zone (arrowheads). Conversely, Wnt7b was expressed in newborn neurons. Red boxes show high power views. (B) qRT-PCR analysis of Wnt7a and Sfrp1 expression levels at different embryonic stages (E12.5, E13.5, E14.5, E15.5, and E17.5). All comparisons were made with that of values at E12.5. Values of histogram represent mean ± SEM, and each dot represents a data point in each biology repeat ( n = 3, ∗ P

Techniques Used: Expressing, Quantitative RT-PCR

41) Product Images from "Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and stimulating osteoblast activity in mouse"

Article Title: Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and stimulating osteoblast activity in mouse

Journal: Development (Cambridge, England)

doi: 10.1242/dev.126227

Bmp-mTORC1 signaling induces expression of protein anabolism genes. (A) Western blot of protein extracts from long bones at P33. Examples are shown for two mice with each genotype. (B) qRT-PCR with RNA isolated from calvaria at P33. (C) qRT-PCR with RNA isolated from Bmpr1a f/f newborn calvarial cells infected with Ad-Cre or Ad-Gfp virus followed by vehicle or BMP2 treatment for 96 h. (D) Western blot in ST2 cells following 1 h treatment with vehicle (Veh) or BMP2. Phosphorylated ribosomal protein S6 (P-S6) is normalized to S6. Quantification (average with s.d.) is shown for three independent experiments. (E) qRT-PCR with RNA from ST2 cells treated with BMP2 or vehicle for 72 h with or without chemical inhibitors. * P
Figure Legend Snippet: Bmp-mTORC1 signaling induces expression of protein anabolism genes. (A) Western blot of protein extracts from long bones at P33. Examples are shown for two mice with each genotype. (B) qRT-PCR with RNA isolated from calvaria at P33. (C) qRT-PCR with RNA isolated from Bmpr1a f/f newborn calvarial cells infected with Ad-Cre or Ad-Gfp virus followed by vehicle or BMP2 treatment for 96 h. (D) Western blot in ST2 cells following 1 h treatment with vehicle (Veh) or BMP2. Phosphorylated ribosomal protein S6 (P-S6) is normalized to S6. Quantification (average with s.d.) is shown for three independent experiments. (E) qRT-PCR with RNA from ST2 cells treated with BMP2 or vehicle for 72 h with or without chemical inhibitors. * P

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

42) Product Images from "Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya"

Article Title: Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2016.00855

Expression pattern validation of 21 selected DEGs by qRT-PCR in transgenic SunUp relative to the donor control Sunset . The transcriptional level of candidate genes was examined by real-time PCR with three biological replicates. EIF was used as an internal control. RNA-Seq data were highly consistent with qRT-PCR results ( r = 0.9247). The Y -axis indicates the fold change of transcript abundance in SU-NP relative to the control SS-NP.
Figure Legend Snippet: Expression pattern validation of 21 selected DEGs by qRT-PCR in transgenic SunUp relative to the donor control Sunset . The transcriptional level of candidate genes was examined by real-time PCR with three biological replicates. EIF was used as an internal control. RNA-Seq data were highly consistent with qRT-PCR results ( r = 0.9247). The Y -axis indicates the fold change of transcript abundance in SU-NP relative to the control SS-NP.

Techniques Used: Expressing, Quantitative RT-PCR, Transgenic Assay, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

43) Product Images from "Antagonistic Regulation, Yet Synergistic Defense: Effect of Bergapten and Protease Inhibitor on Development of Cowpea Bruchid Callosobruchus maculatus"

Article Title: Antagonistic Regulation, Yet Synergistic Defense: Effect of Bergapten and Protease Inhibitor on Development of Cowpea Bruchid Callosobruchus maculatus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041877

scN attenuates transcriptional changes induced by bergapten. Selected bergapten- and scN-coregulated genes involved in polysaccharide or protein degradation ( CmGH5 , CmCatL s, CmCatB ), detoxification ( CmCYP6G1 s, CmGST , CmPOD ), defense ( CmDrsL1-1 ), development ( CmJHE7 ) and transport ( CmSUT1 ) were subjected to qPT-PCR analyses. Total RNA was extracted from midgut of the 4 th instar larvae feeding on artificial diet containing 1,000 ppm scN, 250 ppm bergapten or 1,000 ppm scN + 250 ppm bergapten, respectively. Insects feeding on diet without bergapten or scN served as the control. Reverse transcription and qRT-PCR reactions were performed as described in Material and Methods . Transcript fold induction derived from qRT-PCR is shown as bar graphs. The lower panel shows microarray results of the corresponding genes. “+”, “−”: up- or down-regulation when subjected to scN or bergapten treatment. CmGH5 , Glycoside hydrolase; CmCatLa and CmCatLb , cathepsin L-like proteases; CmCatB , cathepsin B-like protease; CmCYP6G1-2 and CmCYP6G1-3 , Cytochrome P450s; CmGST , Glutathione S-transferase; CmDrsL1-1 , Drosomycin-like I; CmJHE7 , Juvenile hormone esterase; CmSUT1 , Sugar transporter 1; CmPOD , Peroxidase precursor.
Figure Legend Snippet: scN attenuates transcriptional changes induced by bergapten. Selected bergapten- and scN-coregulated genes involved in polysaccharide or protein degradation ( CmGH5 , CmCatL s, CmCatB ), detoxification ( CmCYP6G1 s, CmGST , CmPOD ), defense ( CmDrsL1-1 ), development ( CmJHE7 ) and transport ( CmSUT1 ) were subjected to qPT-PCR analyses. Total RNA was extracted from midgut of the 4 th instar larvae feeding on artificial diet containing 1,000 ppm scN, 250 ppm bergapten or 1,000 ppm scN + 250 ppm bergapten, respectively. Insects feeding on diet without bergapten or scN served as the control. Reverse transcription and qRT-PCR reactions were performed as described in Material and Methods . Transcript fold induction derived from qRT-PCR is shown as bar graphs. The lower panel shows microarray results of the corresponding genes. “+”, “−”: up- or down-regulation when subjected to scN or bergapten treatment. CmGH5 , Glycoside hydrolase; CmCatLa and CmCatLb , cathepsin L-like proteases; CmCatB , cathepsin B-like protease; CmCYP6G1-2 and CmCYP6G1-3 , Cytochrome P450s; CmGST , Glutathione S-transferase; CmDrsL1-1 , Drosomycin-like I; CmJHE7 , Juvenile hormone esterase; CmSUT1 , Sugar transporter 1; CmPOD , Peroxidase precursor.

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Microarray

44) Product Images from "Transcriptomic profiles reveal the genome-wide responses of the harmful dinoflagellate Cochlodinium polykrikoides when exposed to the algicide copper sulfate"

Article Title: Transcriptomic profiles reveal the genome-wide responses of the harmful dinoflagellate Cochlodinium polykrikoides when exposed to the algicide copper sulfate

Journal: BMC Genomics

doi: 10.1186/s12864-015-2341-3

qRT-PCR validation of 13 DEGs identified by RNA-Seq. The relative expression levels of 12 h, 24 h, and 48 h CuSO 4 treated sample were presented. The housekeeping gene α-tubulin ( TUA ) was used as an internal control for qRT-PCR normalization. The relative expression level of control was considered as 1, and the control samples were not shown in the figure
Figure Legend Snippet: qRT-PCR validation of 13 DEGs identified by RNA-Seq. The relative expression levels of 12 h, 24 h, and 48 h CuSO 4 treated sample were presented. The housekeeping gene α-tubulin ( TUA ) was used as an internal control for qRT-PCR normalization. The relative expression level of control was considered as 1, and the control samples were not shown in the figure

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

45) Product Images from "Identification of 2′,4′-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus"

Article Title: Identification of 2′,4′-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23061492

Identification and assessment of antivirulence 2′,4′- DHC. ( A ) rtxA1 gene expression analysis of 10 secondary antivirulence hits. A fresh culture of wild-type (WT) V. vulnificus was incubated to grow with and without various compounds at the mentioned concentrations (µM) until OD 600 1.8–2.0 and total RNA was isolated to prepare cDNA for qRT-PCR analysis. Gene expression level was normalized with the expression level of gyrB. The relative expression level against the non-treated WT control sample is displayed; ( B ) chemical structure of 2′,4′-DHC .
Figure Legend Snippet: Identification and assessment of antivirulence 2′,4′- DHC. ( A ) rtxA1 gene expression analysis of 10 secondary antivirulence hits. A fresh culture of wild-type (WT) V. vulnificus was incubated to grow with and without various compounds at the mentioned concentrations (µM) until OD 600 1.8–2.0 and total RNA was isolated to prepare cDNA for qRT-PCR analysis. Gene expression level was normalized with the expression level of gyrB. The relative expression level against the non-treated WT control sample is displayed; ( B ) chemical structure of 2′,4′-DHC .

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

46) Product Images from "Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants"

Article Title: Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Journal: PLoS ONE

doi: 10.1371/journal.pone.0153494

Expression profiles of TaEXPA2 in different tissues of wheat plants in response to abiotic stresses and signaling molecules, as detected by qRT-PCR. (A) Expression of TaEXPA2 in different organs/tissues of wheat. (B-D) Total RNA was isolated from 2-week-old wheat seedling leaves that were collected after exposure to 20% PEG and 150 mM NaCl (B), 2 mM ABA, 50 mM GA (C), 10 mM MeJA, and 10 mM SA (D). Wheat seedlings incubated in sterile water (CK) were used as controls. The α -tubulin gene was used as an internal reference. The experiments were repeated at least three times.
Figure Legend Snippet: Expression profiles of TaEXPA2 in different tissues of wheat plants in response to abiotic stresses and signaling molecules, as detected by qRT-PCR. (A) Expression of TaEXPA2 in different organs/tissues of wheat. (B-D) Total RNA was isolated from 2-week-old wheat seedling leaves that were collected after exposure to 20% PEG and 150 mM NaCl (B), 2 mM ABA, 50 mM GA (C), 10 mM MeJA, and 10 mM SA (D). Wheat seedlings incubated in sterile water (CK) were used as controls. The α -tubulin gene was used as an internal reference. The experiments were repeated at least three times.

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

47) Product Images from "Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota"

Article Title: Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota

Journal: Microbiome

doi: 10.1186/s40168-017-0357-4

Effect of inulin and scFOS on LPS-induced murine endotoxemia. a Diagram illustrating the animal protocol employed to induce murine endotoxemia. b Hematoxylin and eosin staining of the terminal ileum from mice gavaged with prebiotics (staining representative of at least five individual animals). c Body weights of animals throughout the duration of the study protocol ( n = 8/group). d – g RNA extracted from terminal ileum were measured for inflammatory cytokines and chemokines using qRT-PCR ( n = 8/group). h , i Terminal ileal sections were lysed and immunoblotted for MAPK phosphorylation ( n = 4/group). Bars represent means ± SEM, * P
Figure Legend Snippet: Effect of inulin and scFOS on LPS-induced murine endotoxemia. a Diagram illustrating the animal protocol employed to induce murine endotoxemia. b Hematoxylin and eosin staining of the terminal ileum from mice gavaged with prebiotics (staining representative of at least five individual animals). c Body weights of animals throughout the duration of the study protocol ( n = 8/group). d – g RNA extracted from terminal ileum were measured for inflammatory cytokines and chemokines using qRT-PCR ( n = 8/group). h , i Terminal ileal sections were lysed and immunoblotted for MAPK phosphorylation ( n = 4/group). Bars represent means ± SEM, * P

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

48) Product Images from "Characterization and Expression Profiling of Neuropeptides and G-Protein-Coupled Receptors (GPCRs) for Neuropeptides in the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae)"

Article Title: Characterization and Expression Profiling of Neuropeptides and G-Protein-Coupled Receptors (GPCRs) for Neuropeptides in the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae)

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123912

qRT-PCR results of neuropeptides throughout the D. citri life cycle. The y -axis represents the relative expression level and the x -axis the life cycle. The standard error is represented by the error bar and significant differences are represented by the different letters ( p
Figure Legend Snippet: qRT-PCR results of neuropeptides throughout the D. citri life cycle. The y -axis represents the relative expression level and the x -axis the life cycle. The standard error is represented by the error bar and significant differences are represented by the different letters ( p

Techniques Used: Quantitative RT-PCR, Expressing

49) Product Images from "Comparative Transcriptomics Provides Insights into Reticulate and Adaptive Evolution of a Butterfly Radiation"

Article Title: Comparative Transcriptomics Provides Insights into Reticulate and Adaptive Evolution of a Butterfly Radiation

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evz202

—Tissue- and sex-specific expression of four genes in eight butterfly species. For each gene, we construct a maximum likelihood phylogeny based on its conserved cluster, and the scale bar represents the percentage of substitutions per site. The branches highlighted in red, if any, indicate a significant pattern of positive selection, and the numbers in brackets are significant sites identified using CodeML branch-site model. Each heat map grid stands for relative expression base on a normalized gender- and tissue-specific qRT-PCR result with n = 3.
Figure Legend Snippet: —Tissue- and sex-specific expression of four genes in eight butterfly species. For each gene, we construct a maximum likelihood phylogeny based on its conserved cluster, and the scale bar represents the percentage of substitutions per site. The branches highlighted in red, if any, indicate a significant pattern of positive selection, and the numbers in brackets are significant sites identified using CodeML branch-site model. Each heat map grid stands for relative expression base on a normalized gender- and tissue-specific qRT-PCR result with n = 3.

Techniques Used: Expressing, Construct, Selection, Quantitative RT-PCR

50) Product Images from "Allelic diversity in the transcriptomes of contrasting rust-infected genotypes of Lathyrus sativus, a lasting resource for smart breeding"

Article Title: Allelic diversity in the transcriptomes of contrasting rust-infected genotypes of Lathyrus sativus, a lasting resource for smart breeding

Journal: BMC Plant Biology

doi: 10.1186/s12870-014-0376-2

Correlation between RNA-seq and qRT-PCR. The relative expression levels obtained by RNA-seq using DEGseq and by qRT-PCR using the ΔΔCt method. Pearson’s correlation coefficient (R) between relative expression levels is shown above the trendline.
Figure Legend Snippet: Correlation between RNA-seq and qRT-PCR. The relative expression levels obtained by RNA-seq using DEGseq and by qRT-PCR using the ΔΔCt method. Pearson’s correlation coefficient (R) between relative expression levels is shown above the trendline.

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

51) Product Images from "Gut Antibody Deficiency in a Mouse Model of CVID Results in Spontaneous Development of a Gluten-Sensitive Enteropathy"

Article Title: Gut Antibody Deficiency in a Mouse Model of CVID Results in Spontaneous Development of a Gluten-Sensitive Enteropathy

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02484

Immune Involvement in Intestinal Malabsorption in CD19 −/− . (A) DE gene transcripts in CD19 −/− mice associated with innate immunity. (B) qRT-PCR of Cpa3 mRNA expression. (C) Representative flow cytometry plot of activated peritoneal mast cells with relative abundance shown. (D) DE gene transcripts in CD19 −/− mice associated with adaptive immunity. (E) Representative anti-CD3 immunohistochemistry staining of WT and CD19 −/− ileal sections. (F) The relative and absolute abundance of CD4 and CD8 T cells isolated from the gut mucosa (lamina propria) of WT and CD19 −/− are provided. (A,D) Quasi-likelihood ratio test to identify significantly DE genes. (B,C,F) Student's t -test; ns, non-significant, * = p
Figure Legend Snippet: Immune Involvement in Intestinal Malabsorption in CD19 −/− . (A) DE gene transcripts in CD19 −/− mice associated with innate immunity. (B) qRT-PCR of Cpa3 mRNA expression. (C) Representative flow cytometry plot of activated peritoneal mast cells with relative abundance shown. (D) DE gene transcripts in CD19 −/− mice associated with adaptive immunity. (E) Representative anti-CD3 immunohistochemistry staining of WT and CD19 −/− ileal sections. (F) The relative and absolute abundance of CD4 and CD8 T cells isolated from the gut mucosa (lamina propria) of WT and CD19 −/− are provided. (A,D) Quasi-likelihood ratio test to identify significantly DE genes. (B,C,F) Student's t -test; ns, non-significant, * = p

Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Immunohistochemistry, Staining, Isolation

52) Product Images from "Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation and less remyelination following cuprizone exposure"

Article Title: Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation and less remyelination following cuprizone exposure

Journal: Glia

doi: 10.1002/glia.23214

DKO mice have increased pro-inflammatory cytokine expression at 6-weeks cuprizone treatment and 1-week recovery relative to WT mice. A 2-mm section of corpora callosa was isolated at the midline from DKO (gray thatched bars) and WT (black bars) mice following perfusion with PBS. The isolated region was homogenized in Trizol. Extracted RNA was reverse transcribed and qRT-PCR was performed. All genes were normalized to HPRT expression with naïve (0-weeks) WT mice set as the reference. Differences in expression are shown as 2−ddCt. Data is presented as mean±SEM and analyzed in GraphPad Prism by student’s t-test, p≤0.05 as significant.
Figure Legend Snippet: DKO mice have increased pro-inflammatory cytokine expression at 6-weeks cuprizone treatment and 1-week recovery relative to WT mice. A 2-mm section of corpora callosa was isolated at the midline from DKO (gray thatched bars) and WT (black bars) mice following perfusion with PBS. The isolated region was homogenized in Trizol. Extracted RNA was reverse transcribed and qRT-PCR was performed. All genes were normalized to HPRT expression with naïve (0-weeks) WT mice set as the reference. Differences in expression are shown as 2−ddCt. Data is presented as mean±SEM and analyzed in GraphPad Prism by student’s t-test, p≤0.05 as significant.

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

53) Product Images from "Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence"

Article Title: Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Journal: Scientific Reports

doi: 10.1038/s41598-017-11892-9

Validation of the microarray data by qRT-PCR. mRNA levels of the indicated genes quantified by qRT-PCR in: ( A ) The P . aeruginosa PAO1 strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN, relative to the same strain grown in LB (grey bars), in comparison with microarray data for the same genes (white bars); ( B ) The P . aeruginosa PAO1 strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN (white bars), with 1 mM MgSO 4 (light-grey bars), or with 27 µM PAßN plus 1 mM MgSO 4 (dark-grey bars) relative to the same strain grown in LB; ( C ) The P . aeruginosa PAO1-KP strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN (white bars), with 1 mM MgSO 4 (light-grey bars), or with 27 µM PAßN plus 1 mM MgSO 4 (dark-grey bars), and the P . aeruginosa PAO1-KP ∆efflux strain grown to an A 600 of 2.5 in LB (black bars), relative to the PAO-KP strain grown in LB. The average of two independent analyses performed on three technical replicates is shown with SD.
Figure Legend Snippet: Validation of the microarray data by qRT-PCR. mRNA levels of the indicated genes quantified by qRT-PCR in: ( A ) The P . aeruginosa PAO1 strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN, relative to the same strain grown in LB (grey bars), in comparison with microarray data for the same genes (white bars); ( B ) The P . aeruginosa PAO1 strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN (white bars), with 1 mM MgSO 4 (light-grey bars), or with 27 µM PAßN plus 1 mM MgSO 4 (dark-grey bars) relative to the same strain grown in LB; ( C ) The P . aeruginosa PAO1-KP strain grown to an A 600 of 2.5 in LB supplemented with 27 µM PAßN (white bars), with 1 mM MgSO 4 (light-grey bars), or with 27 µM PAßN plus 1 mM MgSO 4 (dark-grey bars), and the P . aeruginosa PAO1-KP ∆efflux strain grown to an A 600 of 2.5 in LB (black bars), relative to the PAO-KP strain grown in LB. The average of two independent analyses performed on three technical replicates is shown with SD.

Techniques Used: Microarray, Quantitative RT-PCR

54) Product Images from "A systemic increase in the recombination frequency upon local infection of Arabidopsis thaliana plants with oilseed rape mosaic virus depends on plant age, the initial inoculum concentration and the time for virus replication"

Article Title: A systemic increase in the recombination frequency upon local infection of Arabidopsis thaliana plants with oilseed rape mosaic virus depends on plant age, the initial inoculum concentration and the time for virus replication

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2013.00061

Detecting coat protein mRNA . Plants were infected with 100 ng of ORMV, both infected and non-infected leaves were taken for the analysis at different time points (from 0 to 5 days). Four independent plants were used per each experimental time point. mRNA was isolated, converted to cDNA and used to amplify coat protein RNA via qRT-PCR. ORMV was converted to cDNA and used as a negative control in the amplification (no amplification was found in this sample). Negative control sample contained no gDNA/cDNA. Y axis shows arbitrary units of cDNA amplified using primers against gene coding for coat protein. X axis shows time in hours after infection. Data are shown in arbitrary units (with SD). Asterisks show significant difference from negative control ( P
Figure Legend Snippet: Detecting coat protein mRNA . Plants were infected with 100 ng of ORMV, both infected and non-infected leaves were taken for the analysis at different time points (from 0 to 5 days). Four independent plants were used per each experimental time point. mRNA was isolated, converted to cDNA and used to amplify coat protein RNA via qRT-PCR. ORMV was converted to cDNA and used as a negative control in the amplification (no amplification was found in this sample). Negative control sample contained no gDNA/cDNA. Y axis shows arbitrary units of cDNA amplified using primers against gene coding for coat protein. X axis shows time in hours after infection. Data are shown in arbitrary units (with SD). Asterisks show significant difference from negative control ( P

Techniques Used: Infection, Isolation, Quantitative RT-PCR, Negative Control, Amplification

55) Product Images from "Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus"

Article Title: Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18050887

Validation of selected differentially expressed miRNAs by qRT-PCR in the leaf (L), flower bud (F), branching site at bolting (Br1), and branching site at early flowering (Br2) in 6098B (solid line) and Purler (dotted line), performed on the same tissue samples used for verifying gene expression. Data represent means (three biological replicates) ± standard deviation.
Figure Legend Snippet: Validation of selected differentially expressed miRNAs by qRT-PCR in the leaf (L), flower bud (F), branching site at bolting (Br1), and branching site at early flowering (Br2) in 6098B (solid line) and Purler (dotted line), performed on the same tissue samples used for verifying gene expression. Data represent means (three biological replicates) ± standard deviation.

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

56) Product Images from "SUV39H1 downregulation induces deheterochromatinization of satellite regions and senescence after exposure to ionizing radiation"

Article Title: SUV39H1 downregulation induces deheterochromatinization of satellite regions and senescence after exposure to ionizing radiation

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00411

Altered p53 activation and binding to the SUV39H1 promoter after irradiation. (A) Representative Western blots images and quantification of band intensities of the protein levels normalized to PD 38 0 Gy. Averages from three biological replicates ± SD . (B) ChIP-qRT-PCR target amplification normalized to 10% of the amplification from input DNA fragments. Averages from three biological replicates ± SD . * p
Figure Legend Snippet: Altered p53 activation and binding to the SUV39H1 promoter after irradiation. (A) Representative Western blots images and quantification of band intensities of the protein levels normalized to PD 38 0 Gy. Averages from three biological replicates ± SD . (B) ChIP-qRT-PCR target amplification normalized to 10% of the amplification from input DNA fragments. Averages from three biological replicates ± SD . * p

Techniques Used: Activation Assay, Binding Assay, Irradiation, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR, Amplification

57) Product Images from "Postnatal exposure to trichloroethylene alters glutathione redox homeostasis, methylation potential, and neurotrophin expression in the mouse hippocampus"

Article Title: Postnatal exposure to trichloroethylene alters glutathione redox homeostasis, methylation potential, and neurotrophin expression in the mouse hippocampus

Journal: Neurotoxicology

doi: 10.1016/j.neuro.2012.02.017

TCE exposure decreases neurotrophic factor gene expression in hippocampus. qRT-PCR was performed using hippocampus samples from 6 mice in each treatment group collected at PND42. *Statistically different from results obtained from control mice for each
Figure Legend Snippet: TCE exposure decreases neurotrophic factor gene expression in hippocampus. qRT-PCR was performed using hippocampus samples from 6 mice in each treatment group collected at PND42. *Statistically different from results obtained from control mice for each

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

58) Product Images from "The Expression of Sphingosine Kinase-1 in Head and Neck Carcinoma"

Article Title: The Expression of Sphingosine Kinase-1 in Head and Neck Carcinoma

Journal: Cells, Tissues, Organs

doi: 10.1159/000318173

SPHK1 mRNA expression in OSCC patients. Total RNA was prepared from cancerous or non-malignant epithelial tissues obtained by LCM of samples belonging to 4 OSCC patients, reverse-transcribed and subjected to qRT-PCR using oligonucleotide primers specific
Figure Legend Snippet: SPHK1 mRNA expression in OSCC patients. Total RNA was prepared from cancerous or non-malignant epithelial tissues obtained by LCM of samples belonging to 4 OSCC patients, reverse-transcribed and subjected to qRT-PCR using oligonucleotide primers specific

Techniques Used: Expressing, Laser Capture Microdissection, Quantitative RT-PCR

59) Product Images from "Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women"

Article Title: Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women

Journal: Molecular Human Reproduction

doi: 10.1093/molehr/gav038

NALCN contributes to the Gd 3+ -sensitive leak current in hMSMCs. ( A ) qRT–PCR analysis of NALCN mRNA levels after transduction with shRNAs. Bars represent the mean values ± SEM. Levels were normalized to SDHA and TOP1 and compared with scrambled
Figure Legend Snippet: NALCN contributes to the Gd 3+ -sensitive leak current in hMSMCs. ( A ) qRT–PCR analysis of NALCN mRNA levels after transduction with shRNAs. Bars represent the mean values ± SEM. Levels were normalized to SDHA and TOP1 and compared with scrambled

Techniques Used: Quantitative RT-PCR, Transduction

60) Product Images from "Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration"

Article Title: Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration

Journal: Journal of Cell Science

doi: 10.1242/jcs.076331

Biological activity of axolotl Prod1. ( A ) Axolotl AL1 cells were transfected with NP1, axolotl Prod1 (AP1), NP1ΔC or GFPA, serum starved for 72 hours and analysed by qRT-PCR. The results are means ± s.e.m. of three independent experiments
Figure Legend Snippet: Biological activity of axolotl Prod1. ( A ) Axolotl AL1 cells were transfected with NP1, axolotl Prod1 (AP1), NP1ΔC or GFPA, serum starved for 72 hours and analysed by qRT-PCR. The results are means ± s.e.m. of three independent experiments

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

61) Product Images from "Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration"

Article Title: Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration

Journal: Journal of Cell Science

doi: 10.1242/jcs.076331

Biological activity of axolotl Prod1. ( A ) Axolotl AL1 cells were transfected with NP1, axolotl Prod1 (AP1), NP1ΔC or GFPA, serum starved for 72 hours and analysed by qRT-PCR. The results are means ± s.e.m. of three independent experiments
Figure Legend Snippet: Biological activity of axolotl Prod1. ( A ) Axolotl AL1 cells were transfected with NP1, axolotl Prod1 (AP1), NP1ΔC or GFPA, serum starved for 72 hours and analysed by qRT-PCR. The results are means ± s.e.m. of three independent experiments

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

62) Product Images from "Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein"

Article Title: Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008468

Validation of selected RNAi knockdowns using qRT-PCR. RNA was extracted from cultures at 24 hours following induction, and the level of mRNA corresponding to the knockdown target compared to uninduced controls. Data were normalized against tubulin mRNA. See methods for experimental details. ORFs are designated by their geneDB accessions.
Figure Legend Snippet: Validation of selected RNAi knockdowns using qRT-PCR. RNA was extracted from cultures at 24 hours following induction, and the level of mRNA corresponding to the knockdown target compared to uninduced controls. Data were normalized against tubulin mRNA. See methods for experimental details. ORFs are designated by their geneDB accessions.

Techniques Used: Quantitative RT-PCR

63) Product Images from "Evaluation of a Novel Non-Destructive Catch and Release Technology for Harvesting Autologous Adult Stem Cells"

Article Title: Evaluation of a Novel Non-Destructive Catch and Release Technology for Harvesting Autologous Adult Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053933

qRT-PCR analysis of CD90 fold change relative to a control capture containing no antibody after normalisation to a beta-actin control. Error bars represent 1 standard deviation from the mean, n = 3 (technical replicates).
Figure Legend Snippet: qRT-PCR analysis of CD90 fold change relative to a control capture containing no antibody after normalisation to a beta-actin control. Error bars represent 1 standard deviation from the mean, n = 3 (technical replicates).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Related Articles

Real-time Polymerase Chain Reaction:

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Article Snippet: .. All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates. .. Amount of viral load in specimens were interpolated from standard curve using Prism Graphpad software.

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Article Snippet: Quantitative real time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler (Bio-Rad). .. For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. qRT-PCR primer pairs used were as follows: Os04g10010 -Q1/-Q2(5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’) for Os04g10010 gene, PR10b -Q1/-Q2 (5’- GTCGCGGTGTCGGTGGAGAG-3’, 5’- ACGGCGTCGATGAATCCGGC-3’) for PR10b , EFR_ecto -Q1/-Q2 (5’- TGCATCTTTGCTCAAGCCAGGT-3’, 5’-GCGGCCACATGTGACTCCAA-3’) for EFR_ectodomain , Actin -Q1/-Q2 (5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’) for the reference gene actin. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec.

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Article Snippet: .. A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad). .. Expression levels of the mRNA were calculated using the comparative Ct method.

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil
Article Snippet: For cDNA synthesis, reverse transcription of 1 μg RNA was carried out by the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. .. All of the reactions were performed in triplicate in an ABI PRISM 7300 real-time PCR system.

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Article Snippet: .. The expression was normalized using soybean ACTIN as internal control. qRT-PCR reactions were performed in 96-well plates (iQ5 Real Time PCR System; Bio-Rad) for all tissues tested, and data were analyzed according to methods described previously ( ). .. Histochemical Staining for Starch The leaves of 4-week-old Arabidopsis plants were subjected for starch-iodine staining.

RNA Extraction:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: Paragraph title: RNA extraction, viral load and gene expression analyses ... All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates.

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Article Title: BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses
Article Snippet: Paragraph title: RNA Extraction and Real Time Quantitative RT-PCR Analysis ... For these experiments, cells plated on fibronectin were harvested and washed, and their total RNA was isolated using RNeasy plus (QIAGEN). qRT-PCR reactions were run in triplicate on a BioRad iQ5 system.

Amplification:

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish
Article Snippet: All qRT-PCR reactions were performed in 20 μl reactions containing 10 μl 2× SYBR green master mix (Bio-Rad), 0.5 μl (10 μM) of each primer, 1 μl reverse transcriptase RT synthetic cDNA template, and 8 μl double distilled water. .. Specificity of amplification for each reaction was analyzed by dissociation curves using CFX manager software (Bio-Rad).

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.
Article Snippet: .. For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see ), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). ..

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean
Article Snippet: The thermal cycler settings were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 55°C for 20 s. This cycle was followed by a melting curve analysis ranging from 50 to 95°C, with temperature increasing steps of 0.5°C every 10 s. Melting curves for each amplicon were observed carefully to confirm the specificity of the primers used. .. All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

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Article Snippet: .. For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. qRT-PCR primer pairs used were as follows: Os04g10010 -Q1/-Q2(5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’) for Os04g10010 gene, PR10b -Q1/-Q2 (5’- GTCGCGGTGTCGGTGGAGAG-3’, 5’- ACGGCGTCGATGAATCCGGC-3’) for PR10b , EFR_ecto -Q1/-Q2 (5’- TGCATCTTTGCTCAAGCCAGGT-3’, 5’-GCGGCCACATGTGACTCCAA-3’) for EFR_ectodomain , Actin -Q1/-Q2 (5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’) for the reference gene actin. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec. .. The expression levels of Os04g10010 , PR10b and EFR-ectodomain were normalized to the actin gene expression level.

Article Title: Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways
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Synthesized:

Article Title: Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development
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Isolation:

Article Title: BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses
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Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology
Article Snippet: qRT-PCR RNA was isolated from cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. .. A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad).

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil
Article Snippet: Paragraph title: Total RNA isolation and qRT-PCR ... For cDNA synthesis, reverse transcription of 1 μg RNA was carried out by the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols.

Article Title: Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development
Article Snippet: Quantitative RT-PCR (qRT-PCR) Analysis of Gene Expression The total RNAs from tissues of soybean plants and Arabidopsis leaves were isolated following the protocol provided with RNA isolation kit supplied by Biotech, Beijing, China. .. The expression was normalized using soybean ACTIN as internal control. qRT-PCR reactions were performed in 96-well plates (iQ5 Real Time PCR System; Bio-Rad) for all tissues tested, and data were analyzed according to methods described previously ( ).

Size-exclusion Chromatography:

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish
Article Snippet: All qRT-PCR reactions were performed in 20 μl reactions containing 10 μl 2× SYBR green master mix (Bio-Rad), 0.5 μl (10 μM) of each primer, 1 μl reverse transcriptase RT synthetic cDNA template, and 8 μl double distilled water. .. Samples were run with the following program parameters: 95° for 1 min, followed by 39 cycles of 95° for 15 sec, 60° for 30 sec, 76° for 5 sec, 79° for 5 sec, and 81° for 5 sec, ending with the melt curve 65–95°, with 0.5°/sec with each temperature lasting for 0.5 sec.

Article Title: Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots
Article Snippet: .. For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. qRT-PCR primer pairs used were as follows: Os04g10010 -Q1/-Q2(5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’) for Os04g10010 gene, PR10b -Q1/-Q2 (5’- GTCGCGGTGTCGGTGGAGAG-3’, 5’- ACGGCGTCGATGAATCCGGC-3’) for PR10b , EFR_ecto -Q1/-Q2 (5’- TGCATCTTTGCTCAAGCCAGGT-3’, 5’-GCGGCCACATGTGACTCCAA-3’) for EFR_ectodomain , Actin -Q1/-Q2 (5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’) for the reference gene actin. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec. .. The expression levels of Os04g10010 , PR10b and EFR-ectodomain were normalized to the actin gene expression level.

Quantitative RT-PCR:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: .. Total RNA was reverse transcribed, and the resulting cDNA was PCR-amplified using the PCR primers and experimental conditions summarized in Supplementary Table . qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq. ..

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: .. All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates. .. Amount of viral load in specimens were interpolated from standard curve using Prism Graphpad software.

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish
Article Snippet: .. All qRT-PCR reactions were performed in 20 μl reactions containing 10 μl 2× SYBR green master mix (Bio-Rad), 0.5 μl (10 μM) of each primer, 1 μl reverse transcriptase RT synthetic cDNA template, and 8 μl double distilled water. .. Samples were run with the following program parameters: 95° for 1 min, followed by 39 cycles of 95° for 15 sec, 60° for 30 sec, 76° for 5 sec, 79° for 5 sec, and 81° for 5 sec, ending with the melt curve 65–95°, with 0.5°/sec with each temperature lasting for 0.5 sec.

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.
Article Snippet: .. For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see ), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). ..

Article Title: An investigation of nutrient-dependent mRNA translation in Drosophila larvae
Article Snippet: .. The cDNA was used as a template to perform qRT-PCR reactions (BioRad Laboratories, MyIQ PCR machine using SyBr Green PCR mix) using specific primer pairs (sequences available upon request). ..

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean
Article Snippet: .. All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed. ..

Article Title: BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses
Article Snippet: .. For these experiments, cells plated on fibronectin were harvested and washed, and their total RNA was isolated using RNeasy plus (QIAGEN). qRT-PCR reactions were run in triplicate on a BioRad iQ5 system. .. Primers used for quantitative RT-PCR and PCR analyses are listed below.

Article Title: Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs
Article Snippet: .. qRT-PCR The validation of gene expression results was done by qRT-PCR. qRT-PCR reactions were set up using the SsoFast™ EvaGreen® Supermix (BioRad) and analyzed according to SSoFast guidelines with annealing temperatures as specified for specific primer pairs (Table ). ..

Article Title: Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots
Article Snippet: .. For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. qRT-PCR primer pairs used were as follows: Os04g10010 -Q1/-Q2(5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’) for Os04g10010 gene, PR10b -Q1/-Q2 (5’- GTCGCGGTGTCGGTGGAGAG-3’, 5’- ACGGCGTCGATGAATCCGGC-3’) for PR10b , EFR_ecto -Q1/-Q2 (5’- TGCATCTTTGCTCAAGCCAGGT-3’, 5’-GCGGCCACATGTGACTCCAA-3’) for EFR_ectodomain , Actin -Q1/-Q2 (5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’) for the reference gene actin. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec. .. The expression levels of Os04g10010 , PR10b and EFR-ectodomain were normalized to the actin gene expression level.

Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology
Article Snippet: .. A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad). .. Expression levels of the mRNA were calculated using the comparative Ct method.

Article Title: Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways
Article Snippet: .. qRT-PCR reactions were performed with IQ SYBR green supermix (Bio-Rad) on the LightCycler480 (Roche, Basel, Switzerland) using the 384-well plate format. qRT-PCR reaction, performed in duplicate, contained 2 μl of cDNA, 5 μl of Supermix, and 1.5 μl of 7.5 nM solution of each primer. .. The qRT-PCR reaction was conducted at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 15 s. Upon completion of qRT-PCR amplification, melting curve analysis was performed to evaluate the quality of amplification as described previously ( ).

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil
Article Snippet: .. For cDNA synthesis, reverse transcription of 1 μg RNA was carried out by the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. ..

Article Title: Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development
Article Snippet: .. The expression was normalized using soybean ACTIN as internal control. qRT-PCR reactions were performed in 96-well plates (iQ5 Real Time PCR System; Bio-Rad) for all tissues tested, and data were analyzed according to methods described previously ( ). .. Histochemical Staining for Starch The leaves of 4-week-old Arabidopsis plants were subjected for starch-iodine staining.

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: .. The immunoprecipitated RNA was reverse transcribed, and the resulting cDNA was PCR-amplified. qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq (TaKaRa Bio). ..

Immunoprecipitation:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: .. The immunoprecipitated RNA was reverse transcribed, and the resulting cDNA was PCR-amplified. qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq (TaKaRa Bio). ..

SYBR Green Assay:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: Viral load and gene expression qRT-PCR were performed with 10 ng of cDNA/well using SsoAdvanced Universal Probe Supermix (BIO-RAD) or iQ SYBR Green Supermix (BIO-RAD), respectively. .. All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates.

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish
Article Snippet: .. All qRT-PCR reactions were performed in 20 μl reactions containing 10 μl 2× SYBR green master mix (Bio-Rad), 0.5 μl (10 μM) of each primer, 1 μl reverse transcriptase RT synthetic cDNA template, and 8 μl double distilled water. .. Samples were run with the following program parameters: 95° for 1 min, followed by 39 cycles of 95° for 15 sec, 60° for 30 sec, 76° for 5 sec, 79° for 5 sec, and 81° for 5 sec, ending with the melt curve 65–95°, with 0.5°/sec with each temperature lasting for 0.5 sec.

Article Title: An investigation of nutrient-dependent mRNA translation in Drosophila larvae
Article Snippet: .. The cDNA was used as a template to perform qRT-PCR reactions (BioRad Laboratories, MyIQ PCR machine using SyBr Green PCR mix) using specific primer pairs (sequences available upon request). ..

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean
Article Snippet: Briefly, each reaction (15 µl) contained 7.5 µl of SYBR Green, 100 nM forward primer, 100 nM universal primer and 2 µl diluted cDNA. .. All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology
Article Snippet: .. A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad). .. Expression levels of the mRNA were calculated using the comparative Ct method.

Article Title: Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways
Article Snippet: .. qRT-PCR reactions were performed with IQ SYBR green supermix (Bio-Rad) on the LightCycler480 (Roche, Basel, Switzerland) using the 384-well plate format. qRT-PCR reaction, performed in duplicate, contained 2 μl of cDNA, 5 μl of Supermix, and 1.5 μl of 7.5 nM solution of each primer. .. The qRT-PCR reaction was conducted at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 15 s. Upon completion of qRT-PCR amplification, melting curve analysis was performed to evaluate the quality of amplification as described previously ( ).

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil
Article Snippet: .. For cDNA synthesis, reverse transcription of 1 μg RNA was carried out by the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. ..

Activation Assay:

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.
Article Snippet: For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see ), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). .. To determine “fold activation” of genes, ΔCt values for target genes were then normalized against ΔCt values for the same target gene for mock-treated cells (ΔΔCt).

Incubation:

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean
Article Snippet: The reaction mix was then incubated in a 96 well plate and analyzed using iQ5 Real-Time PCR Detection System and iQ5 Optical System Software (Bio-Rad). .. All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

Polymerase Chain Reaction:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: .. Total RNA was reverse transcribed, and the resulting cDNA was PCR-amplified using the PCR primers and experimental conditions summarized in Supplementary Table . qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq. ..

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.
Article Snippet: .. For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see ), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). ..

Article Title: An investigation of nutrient-dependent mRNA translation in Drosophila larvae
Article Snippet: .. The cDNA was used as a template to perform qRT-PCR reactions (BioRad Laboratories, MyIQ PCR machine using SyBr Green PCR mix) using specific primer pairs (sequences available upon request). ..

Article Title: BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses
Article Snippet: For these experiments, cells plated on fibronectin were harvested and washed, and their total RNA was isolated using RNeasy plus (QIAGEN). qRT-PCR reactions were run in triplicate on a BioRad iQ5 system. .. Primers used for quantitative RT-PCR and PCR analyses are listed below.

Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology
Article Snippet: .. A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad). .. Expression levels of the mRNA were calculated using the comparative Ct method.

Article Title: Inhibiting the HPV16 oncogene-mediated glycolysis sensitizes human cervical carcinoma cells to 5-fluorouracil
Article Snippet: .. For cDNA synthesis, reverse transcription of 1 μg RNA was carried out by the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. ..

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: .. The immunoprecipitated RNA was reverse transcribed, and the resulting cDNA was PCR-amplified. qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq (TaKaRa Bio). ..

Infection:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: Briefly, 70% confluent mouse articular chondrocytes were infected with Ad-ZFP36L1 and Ad-HSPA1A at the indicated MOIs for 24 h. Cell lysates were immunoprecipitated with anti-ZFP36L1 (3 μg, 101AP; FabGennix International) or control IgG (1 μg, CS200621; EMD Milli pore Corp.) antibody. .. The immunoprecipitated RNA was reverse transcribed, and the resulting cDNA was PCR-amplified. qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq (TaKaRa Bio).

Expressing:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: Paragraph title: RNA extraction, viral load and gene expression analyses ... All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates.

Article Title: Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs
Article Snippet: .. qRT-PCR The validation of gene expression results was done by qRT-PCR. qRT-PCR reactions were set up using the SsoFast™ EvaGreen® Supermix (BioRad) and analyzed according to SSoFast guidelines with annealing temperatures as specified for specific primer pairs (Table ). ..

Article Title: Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense ResponsesThe Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots
Article Snippet: For qRT-PCR reactions, the Bio-Rad SsoFast EvaGreen Supermix was used. qRT-PCR primer pairs used were as follows: Os04g10010 -Q1/-Q2(5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’) for Os04g10010 gene, PR10b -Q1/-Q2 (5’- GTCGCGGTGTCGGTGGAGAG-3’, 5’- ACGGCGTCGATGAATCCGGC-3’) for PR10b , EFR_ecto -Q1/-Q2 (5’- TGCATCTTTGCTCAAGCCAGGT-3’, 5’-GCGGCCACATGTGACTCCAA-3’) for EFR_ectodomain , Actin -Q1/-Q2 (5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’) for the reference gene actin. qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec. .. The expression levels of Os04g10010 , PR10b and EFR-ectodomain were normalized to the actin gene expression level.

Article Title: Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology
Article Snippet: A total of 1 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis kit (Bio-Rad) according to the supplier’s protocol and was used as templates for the quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were performed in a 20-μl mixture containing cDNA, primers, and 1× SYBR GREEN PCR Master mix (Bio-Rad). .. Expression levels of the mRNA were calculated using the comparative Ct method.

Article Title: Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways
Article Snippet: qRT-PCR reactions were performed with IQ SYBR green supermix (Bio-Rad) on the LightCycler480 (Roche, Basel, Switzerland) using the 384-well plate format. qRT-PCR reaction, performed in duplicate, contained 2 μl of cDNA, 5 μl of Supermix, and 1.5 μl of 7.5 nM solution of each primer. .. The qRT-PCR results were analyzed with the Comparative Ct (ΔΔCt ) method, and data were presented as a relative expression.

Article Title: Soybean LEC2 Regulates Subsets of Genes Involved in Controlling the Biosynthesis and Catabolism of Seed Storage Substances and Seed Development
Article Snippet: .. The expression was normalized using soybean ACTIN as internal control. qRT-PCR reactions were performed in 96-well plates (iQ5 Real Time PCR System; Bio-Rad) for all tissues tested, and data were analyzed according to methods described previously ( ). .. Histochemical Staining for Starch The leaves of 4-week-old Arabidopsis plants were subjected for starch-iodine staining.

Reverse Transcription Polymerase Chain Reaction:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: Paragraph title: RT-PCR and qRT-PCR ... Total RNA was reverse transcribed, and the resulting cDNA was PCR-amplified using the PCR primers and experimental conditions summarized in Supplementary Table . qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq.

RNA Binding Assay:

Article Title: RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family
Article Snippet: The RNA-binding protein immunoprecipitation assay was performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. .. The immunoprecipitated RNA was reverse transcribed, and the resulting cDNA was PCR-amplified. qRT-PCR reactions were performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq (TaKaRa Bio).

Software:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates. .. Amount of viral load in specimens were interpolated from standard curve using Prism Graphpad software.

Article Title: Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish
Article Snippet: All qRT-PCR reactions were performed in 20 μl reactions containing 10 μl 2× SYBR green master mix (Bio-Rad), 0.5 μl (10 μM) of each primer, 1 μl reverse transcriptase RT synthetic cDNA template, and 8 μl double distilled water. .. Specificity of amplification for each reaction was analyzed by dissociation curves using CFX manager software (Bio-Rad).

Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean
Article Snippet: The reaction mix was then incubated in a 96 well plate and analyzed using iQ5 Real-Time PCR Detection System and iQ5 Optical System Software (Bio-Rad). .. All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

Plasmid Preparation:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: Standard curve (101 to 108 NS1 copies/μl) was generated using serial dilutions of plasmid expressing MR766 NS1 protein. .. All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates.

Generated:

Article Title: Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy
Article Snippet: Standard curve (101 to 108 NS1 copies/μl) was generated using serial dilutions of plasmid expressing MR766 NS1 protein. .. All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System on 96-well plates.

Article Title: In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathwaySometimes You’re the Scooper, and Sometimes You Get Scooped; How to Turn Both into Something Good.
Article Snippet: .. For qRT-PCR reactions, a total of 20 ng of cDNA was used as a template and combined with primers (see ), and EvaGreen Supermix (Biorad) and amplicons were generated using standard PCR amplification protocols for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems). ..

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  • 90
    Bio-Rad aurum total rna mini kit
    The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) <t>RNA</t> levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase <t>PCR</t> (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).
    Aurum Total Rna Mini Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) <t>RNA</t> levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase <t>PCR</t> (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).
    Iq Sybr Green Supermix, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 3038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) <t>RNA</t> levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase <t>PCR</t> (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).
    Ssoadvanced Universal Sybr Green Supermix, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 1105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) <t>RNA</t> levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase <t>PCR</t> (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).
    Miniopticon Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) RNA levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase PCR (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).

    Journal: Nucleic Acids Research

    Article Title: Regulation of telomere metabolism by the RNA processing protein Xrn1

    doi: 10.1093/nar/gkx072

    Figure Lengend Snippet: The lack of Xrn1 impairs telomere elongation. ( A ) Schematic representation of the HO-induced short telomere system. The ADH4 locus on chromosome VII was replaced with a fragment consisting of the ADE2 gene and 81 bp of TG telomeric sequences (zigzag lines) flanking the recognition site for the HO endonuclease. The centromere is shown as a circle. S, SpeI. ( B ) Elongation of the HO-induced telomere. Cell cultures carrying the system described in A and exponentially growing in raffinose were shifted to galactose at time zero to induce HO expression. SpeI-digested genomic DNA was subjected to Southern blot analysis using an ADE2 fragment as a probe. A bracket points out new telomere repeats added to the TG telomeric sequences. The band of about 1.6kb (INT) represents the endogenous ade2-101 gene. ( C–E ) XhoI-cut genomic DNA from exponentially growing cells was subjected to Southern blot analysis using a radiolabeled poly(GT) probe. The bar in (D) points out two independent xrn1-E176G cell cultures. ( F ) RNA levels of CDC13-EST1 from cells in (E) were evaluated by quantitative reverse transcriptase PCR (qRT-PCR). Quantities were normalized to ACT1 RNA levels and compared to that of wild - type cells that was set up to 1. The mean values ±s.d. are represented ( n = 3).

    Article Snippet: qRT-PCR Total RNA was extracted from cells using the Bio-Rad Aurum total RNA mini kit.

    Techniques: Expressing, Southern Blot, Polymerase Chain Reaction, Quantitative RT-PCR