Structured Review

TaKaRa mrna
Constructs of <t>DNA</t> and <t>mRNA</t> used in this study. (A) Construct of the model dsDNA. (B) Constructs of mRNA and DNA-linker having hydrazide group and the complex in the ligation reaction. T7φ2.5, T7φ2.5 promoter; clump, GC-rich sequence for hybridization to the DNA-linker; ε, T7 gene 10 translation enhancer; SD, Shine-Dalgarno sequence; ATG or AUG, initiation codon; TAG or UAG, amber stop codon; GFPN, N-terminus 10 amino acids of Green Fluorescent Protein gene; 6x His, Histidine hexamer tag; FLAG, FLAG epitope tag, TAA or UAA, ocher stop codon; RTP, hybridization region for RT-primer prRT-; FITC, fluorescein isothiocyanate labeled to deoxythymidine.
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1) Product Images from "An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering"

Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

Journal: International Journal of Biological Sciences

doi:

Constructs of DNA and mRNA used in this study. (A) Construct of the model dsDNA. (B) Constructs of mRNA and DNA-linker having hydrazide group and the complex in the ligation reaction. T7φ2.5, T7φ2.5 promoter; clump, GC-rich sequence for hybridization to the DNA-linker; ε, T7 gene 10 translation enhancer; SD, Shine-Dalgarno sequence; ATG or AUG, initiation codon; TAG or UAG, amber stop codon; GFPN, N-terminus 10 amino acids of Green Fluorescent Protein gene; 6x His, Histidine hexamer tag; FLAG, FLAG epitope tag, TAA or UAA, ocher stop codon; RTP, hybridization region for RT-primer prRT-; FITC, fluorescein isothiocyanate labeled to deoxythymidine.
Figure Legend Snippet: Constructs of DNA and mRNA used in this study. (A) Construct of the model dsDNA. (B) Constructs of mRNA and DNA-linker having hydrazide group and the complex in the ligation reaction. T7φ2.5, T7φ2.5 promoter; clump, GC-rich sequence for hybridization to the DNA-linker; ε, T7 gene 10 translation enhancer; SD, Shine-Dalgarno sequence; ATG or AUG, initiation codon; TAG or UAG, amber stop codon; GFPN, N-terminus 10 amino acids of Green Fluorescent Protein gene; 6x His, Histidine hexamer tag; FLAG, FLAG epitope tag, TAA or UAA, ocher stop codon; RTP, hybridization region for RT-primer prRT-; FITC, fluorescein isothiocyanate labeled to deoxythymidine.

Techniques Used: Construct, Ligation, Sequencing, Hybridization, FLAG-tag, Labeling

Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
Figure Legend Snippet: Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

Techniques Used: Nucleic Acid Electrophoresis, Ligation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Fluorescence, SYBR Green Assay, Negative Control

Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.
Figure Legend Snippet: Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

Techniques Used: Modification, Reverse Transcription Polymerase Chain Reaction

Results of the selective enrichment experiment. (A) Result of RT-PCR after single-round Ni-NTA screening of the mRNA-peptide fusion displaying 6x His-tag sequence. Left panel, RT-PCR of the screened samples translated with or without methionine. Right panel, RT-PCR of the screened samples translated using mRNA with or without modification of 4-acethyl-phenylalanyl tRNA Asn CUA before translation. Lane M: DNA ladder, Lane PC: positive control using mRNA before translation as template of RT-PCR. (B) Results of selective enrichment experiment for binary mixture: the FLAG-tag displaying fusion molecule and the 6x His-tag displaying fusion molecule. Initial molar ratio of the former to the latter is 87:13 for left panel and 17:83 for right panel. RT-PCR product at each screening round was analyzed by electrophoresis. Left panel; screening with Ni-NTA column. Right panel; screening with Anti-FLAG antibody column. (C) Results of selective enrichment experiment using PURESYSTEM for binary mixture: the FLAG-tag displaying fusion molecule and the 6x His-tag displaying fusion molecule. Initial molar ratio of the former to the latter is 76:24 for left panel and 6:94 for right panel. RT-PCR product at each screening round was analyzed by electrophoresis. Left panel; screening with Ni-NTA column. Right panel; screening with Anti-FLAG antibody column.
Figure Legend Snippet: Results of the selective enrichment experiment. (A) Result of RT-PCR after single-round Ni-NTA screening of the mRNA-peptide fusion displaying 6x His-tag sequence. Left panel, RT-PCR of the screened samples translated with or without methionine. Right panel, RT-PCR of the screened samples translated using mRNA with or without modification of 4-acethyl-phenylalanyl tRNA Asn CUA before translation. Lane M: DNA ladder, Lane PC: positive control using mRNA before translation as template of RT-PCR. (B) Results of selective enrichment experiment for binary mixture: the FLAG-tag displaying fusion molecule and the 6x His-tag displaying fusion molecule. Initial molar ratio of the former to the latter is 87:13 for left panel and 17:83 for right panel. RT-PCR product at each screening round was analyzed by electrophoresis. Left panel; screening with Ni-NTA column. Right panel; screening with Anti-FLAG antibody column. (C) Results of selective enrichment experiment using PURESYSTEM for binary mixture: the FLAG-tag displaying fusion molecule and the 6x His-tag displaying fusion molecule. Initial molar ratio of the former to the latter is 76:24 for left panel and 6:94 for right panel. RT-PCR product at each screening round was analyzed by electrophoresis. Left panel; screening with Ni-NTA column. Right panel; screening with Anti-FLAG antibody column.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Modification, Positive Control, FLAG-tag, Electrophoresis

2) Product Images from "miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6"

Article Title: miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2014.63

In vivo validation of the relationship between miR-98 and interleukin-6 (IL-6) production. ( a ) Metastatic tumor size of both the miRNA-negative control (miR-NC) and miR-98 groups over time. Data are presented as mean±s.d. ( n =3 in each group). *A statistically significant difference between the miR-98 and miR-NC groups, P ⩽0.018. ( b ) Representative gross image of lung metastases at day 35. ( c ) Kaplan–Meier survival curve, the dots in the figure indicate the censored cases. Log-rank tests indicate a significant difference between the groups, P =0.076. ( d ) Relative serum IL-6 levels after mice were treated with miRNA-NC or miR-98; n =3 for each group. ( e ) Western blot analysis of total Stat3 and p64 and phosphorylated Stat3 and p65 after 35 days treatment with miR-98 or miRNA-NC in tumor samples at day 35. ( f ) Relative lin28B mRNA levels in tumor samples at day 35 after mice were treated with miRNA-NC or miR-98. Data are presented as mean±s.d. ( n =3 in each group).
Figure Legend Snippet: In vivo validation of the relationship between miR-98 and interleukin-6 (IL-6) production. ( a ) Metastatic tumor size of both the miRNA-negative control (miR-NC) and miR-98 groups over time. Data are presented as mean±s.d. ( n =3 in each group). *A statistically significant difference between the miR-98 and miR-NC groups, P ⩽0.018. ( b ) Representative gross image of lung metastases at day 35. ( c ) Kaplan–Meier survival curve, the dots in the figure indicate the censored cases. Log-rank tests indicate a significant difference between the groups, P =0.076. ( d ) Relative serum IL-6 levels after mice were treated with miRNA-NC or miR-98; n =3 for each group. ( e ) Western blot analysis of total Stat3 and p64 and phosphorylated Stat3 and p65 after 35 days treatment with miR-98 or miRNA-NC in tumor samples at day 35. ( f ) Relative lin28B mRNA levels in tumor samples at day 35 after mice were treated with miRNA-NC or miR-98. Data are presented as mean±s.d. ( n =3 in each group).

Techniques Used: In Vivo, Negative Control, Mouse Assay, Western Blot

3) Product Images from "A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice"

Article Title: A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice

Journal: Scientific Reports

doi: 10.1038/srep32155

Vaccination against CTGF reduced the expression of profibrogenic factors in the fibrotic livers. Western blotting detection of TGF-β1 ( a ), PDGF-B ( b ), TIMP-1 ( c ) and RQ-PCR evaluation of CTGF mRNA ( d ) revealed that vaccination against CTGF decreased the expressions of TGF-β1, PDGF-B, TIMP-1 and CTGF in CCl 4 -induced fibrotic mouse livers. Western blotting evaluation of the hepatic tissue p-Smad2/Smad2 ratio revealed that the vaccination inhibited Smad2 phosphorylation ( e ). Following Western blotting, bands were quantified with Image-J software. Relative protein abundance in each sample was normalized to that of β-actin. The RQ-PCR results were represented as the means ± SEM of three independent experiments, each of which was performed in triplicate reactions. Error bars indicate SEM. TGF-β1, transforming growth factor β1; PDGF-B, platelet-derived growth factor B; TIMP-1, tissue inhibitor of metalloproteinase-1; RQ-PCR, Real-time quantitative reverse transcriptase polymerase chain reaction.
Figure Legend Snippet: Vaccination against CTGF reduced the expression of profibrogenic factors in the fibrotic livers. Western blotting detection of TGF-β1 ( a ), PDGF-B ( b ), TIMP-1 ( c ) and RQ-PCR evaluation of CTGF mRNA ( d ) revealed that vaccination against CTGF decreased the expressions of TGF-β1, PDGF-B, TIMP-1 and CTGF in CCl 4 -induced fibrotic mouse livers. Western blotting evaluation of the hepatic tissue p-Smad2/Smad2 ratio revealed that the vaccination inhibited Smad2 phosphorylation ( e ). Following Western blotting, bands were quantified with Image-J software. Relative protein abundance in each sample was normalized to that of β-actin. The RQ-PCR results were represented as the means ± SEM of three independent experiments, each of which was performed in triplicate reactions. Error bars indicate SEM. TGF-β1, transforming growth factor β1; PDGF-B, platelet-derived growth factor B; TIMP-1, tissue inhibitor of metalloproteinase-1; RQ-PCR, Real-time quantitative reverse transcriptase polymerase chain reaction.

Techniques Used: Expressing, Western Blot, Polymerase Chain Reaction, Software, Derivative Assay

4) Product Images from "Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus"

Article Title: Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001286

xNorrin induces anterior CNS formation in ventralized embryos. (A–D) xNorrin mRNA induces anterior neural tissues, while Wnt11 mRNA restores only a partial dorsal axis (without anterior structures) in UV-irradiated embryos. (A) A wild-type embryo (stage 33); (B) an embryo UV-irradiated (50 µJ) at the vegetal pole; (C) a UV-irradiated embryo injected with Wnt11 mRNA (500 pg) into one cell at the four-cell stage (arrowhead: partial dorsal axis); (D) a UV-irradiated embryo injected with xNorrin mRNA (500 pg) as in (C) (arrow: head). (E) Summary of (A–D). Fraction of the population within each group is indicated. (F) Histological analysis of stage 40 embryos. Arrowhead: muscle; arrows: brain and eye. (G–J) Whole-mount in situ hybridizations to Sox3 . (G) A wild-type (WT) embryo (100%, n = 65); (H) a UV-treated embryo (4% Sox3 positive, n = 70); (I) a UV+ Wnt11 (500 pg) rescued embryo (45% Sox3 positive, n = 77); (J) a UV+ xNorrin (500 pg) rescued embryo (83% Sox3 positive, n = 69). All embryos are shown with the anterior pole to the left. Arrowhead: posterior neural structure; arrow: anterior neural structure. (K) Neural marker expression detected by RT-PCR. xNorrin induced expression of anterior neural and pan-neural markers ( En2 , otx2 , Xpax6 , NCAM , and Sox2 ) in UV-irradiated embryos. Wnt11 induced only the hindbrain marker Krox20 in UV-irradiated embryos.
Figure Legend Snippet: xNorrin induces anterior CNS formation in ventralized embryos. (A–D) xNorrin mRNA induces anterior neural tissues, while Wnt11 mRNA restores only a partial dorsal axis (without anterior structures) in UV-irradiated embryos. (A) A wild-type embryo (stage 33); (B) an embryo UV-irradiated (50 µJ) at the vegetal pole; (C) a UV-irradiated embryo injected with Wnt11 mRNA (500 pg) into one cell at the four-cell stage (arrowhead: partial dorsal axis); (D) a UV-irradiated embryo injected with xNorrin mRNA (500 pg) as in (C) (arrow: head). (E) Summary of (A–D). Fraction of the population within each group is indicated. (F) Histological analysis of stage 40 embryos. Arrowhead: muscle; arrows: brain and eye. (G–J) Whole-mount in situ hybridizations to Sox3 . (G) A wild-type (WT) embryo (100%, n = 65); (H) a UV-treated embryo (4% Sox3 positive, n = 70); (I) a UV+ Wnt11 (500 pg) rescued embryo (45% Sox3 positive, n = 77); (J) a UV+ xNorrin (500 pg) rescued embryo (83% Sox3 positive, n = 69). All embryos are shown with the anterior pole to the left. Arrowhead: posterior neural structure; arrow: anterior neural structure. (K) Neural marker expression detected by RT-PCR. xNorrin induced expression of anterior neural and pan-neural markers ( En2 , otx2 , Xpax6 , NCAM , and Sox2 ) in UV-irradiated embryos. Wnt11 induced only the hindbrain marker Krox20 in UV-irradiated embryos.

Techniques Used: Irradiation, Injection, In Situ, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction

xNorrin induces neural formation independent of β-catenin signaling. (A) xNorrin dose-dependently induced neural marker ( Xpax6 , otx2 , NCAM , and Sox2 ) expression. The induction is independent of mesoderm formation ( m-actin : muscle actin) and could occur in the presence of a β-catenin-MO. RNA and MO were injected at the one-cell stage, and the caps were dissected around stage 8 and cultured until they reached stage 15. –RT, no reverse transcription; WE, whole embryo; WT, wild-type. (B) xNorrin induced Sox3 expression in animal caps (89% of xNorrin injected caps showed the expression, n = 45). xNorrin mRNA (300 pg) was injected into the animal pole of two-cell embryos. Sox3 expression in stage 15 animal caps was analyzed with in situ hybridization. WT, wild-type animal caps; xNorrin, xNorrin -mRNA-injected caps. (C) xNorrin induced the expression of ectopic neural and anterior markers in whole embryos. xNorrin mRNA (300 pg) was injected into the ventral animal tier cells of 32-cell embryos. Expression of Sox3 (by Δ BMPR : 80%, n = 30; by xNorrin : 73%, n = 30) and XAG1 (by Δ BMPR : 69%, n = 35; by xNorrin : 79%, n = 34) were induced at ectopic sites. β-gal, β-gal -mRNA-injected embryos; ΔBMPR: Δ BMPR -mRNA-injected embryos; WT, wild-type uninjected embryos; xNorrin: xNorrin -mRNA-injected embryos. Arrows: ectopically induced Sox3 or XAG1 . Sox3 expression in wild type is shown in a dorsal view, while XAG1 expression in wild type is shown in an anterior view. All other embryos are shown in a ventral view, except the embryo in the β-gal / Sox3 panel, which is in a lateral view.
Figure Legend Snippet: xNorrin induces neural formation independent of β-catenin signaling. (A) xNorrin dose-dependently induced neural marker ( Xpax6 , otx2 , NCAM , and Sox2 ) expression. The induction is independent of mesoderm formation ( m-actin : muscle actin) and could occur in the presence of a β-catenin-MO. RNA and MO were injected at the one-cell stage, and the caps were dissected around stage 8 and cultured until they reached stage 15. –RT, no reverse transcription; WE, whole embryo; WT, wild-type. (B) xNorrin induced Sox3 expression in animal caps (89% of xNorrin injected caps showed the expression, n = 45). xNorrin mRNA (300 pg) was injected into the animal pole of two-cell embryos. Sox3 expression in stage 15 animal caps was analyzed with in situ hybridization. WT, wild-type animal caps; xNorrin, xNorrin -mRNA-injected caps. (C) xNorrin induced the expression of ectopic neural and anterior markers in whole embryos. xNorrin mRNA (300 pg) was injected into the ventral animal tier cells of 32-cell embryos. Expression of Sox3 (by Δ BMPR : 80%, n = 30; by xNorrin : 73%, n = 30) and XAG1 (by Δ BMPR : 69%, n = 35; by xNorrin : 79%, n = 34) were induced at ectopic sites. β-gal, β-gal -mRNA-injected embryos; ΔBMPR: Δ BMPR -mRNA-injected embryos; WT, wild-type uninjected embryos; xNorrin: xNorrin -mRNA-injected embryos. Arrows: ectopically induced Sox3 or XAG1 . Sox3 expression in wild type is shown in a dorsal view, while XAG1 expression in wild type is shown in an anterior view. All other embryos are shown in a ventral view, except the embryo in the β-gal / Sox3 panel, which is in a lateral view.

Techniques Used: Marker, Expressing, Injection, Cell Culture, In Situ Hybridization

Maternal xNorrin is required for dorsal ectoderm specification. (A) The xNor-MO target sequence (green line) in xNorrin mRNA. (B) xNor-MO dose-dependently suppresses xNorrin-Myc (1.5 ng) mRNA translation in Xenopus embryos. xNorrin-Myc was detected using an anti-c-Myc monoclonal antibody. Uni, no xNorrin-Myc injected. (C–F) xNorrin is required for head formation. (C) A wild-type (WT) stage 35 tadpole. (D) xNor-MO (20 ng) caused anterior truncation (61%, n = 64) when injected into the animal regions of two dorsal cells in four- to eight-cell-stage embryos. (E) xNor-misMO-injected embryos are generally normal (88%, n = 66). (F) The anterior defects caused by NorMO were rescued by xNorrin (25 pg) mRNA (77%, n = 81). (G) Dorsal view of a wild-type tadpole at stage 45. (H) Anterior defects on only one side (arrow) were generated by injecting xNor-MO (10 ng) into one dorsal cell at the four- to eight-cell stage (63% of injected embryos showed defects in the injected side, n = 30). The other side shows normal morphology. The anterior end is to the left. (I) xNor-MO inhibits Wnt signaling in dorsal animal cells. xNor-MO and SuperTopFlash (STF) reporter plasmids were co-injected into the dorsal animal cells of eight-cell embryos. F/R luciferase: ratio of firefly luciferase reading to renilla luciferase reading. (J) Whole-mount in situ hybridization shows that Chordin expression is reduced at stage 9 (53% of injected embryos, n = 80) and stage 10 (61% of injected embryos, n = 79) in xNor-MO-injected embryos, compared to xNor-misMO-injected embryos or uninjected embryos. (K–N) Whole-mount in situ hybridization for Chordin in bisected xNor-MO-injected embryos (stage 9.5) showing that xNor-MO inhibits Chordin expression in neuroectoderm precursors (arrow) (reduction in 66% of injected embryos, n = 104) (L) compared to wild-type embryos (K) and embryos with xNor-misMO injected into dorsal animal cells at the eight- to 16-cell stage (reduction in 13% of injected embryos, n = 78) (M). Note that xNorrin mRNA (100 pg) rescues Chordin expression in the dorsal ectoderm (80% of co-injected embryos showed expression comparable to wild-type embryos, n = 50) (N). Embryos are oriented such that their dorsal side is on the right. Dotted lines indicate the boundaries between the deep mesoderm and the superficial ectoderm.
Figure Legend Snippet: Maternal xNorrin is required for dorsal ectoderm specification. (A) The xNor-MO target sequence (green line) in xNorrin mRNA. (B) xNor-MO dose-dependently suppresses xNorrin-Myc (1.5 ng) mRNA translation in Xenopus embryos. xNorrin-Myc was detected using an anti-c-Myc monoclonal antibody. Uni, no xNorrin-Myc injected. (C–F) xNorrin is required for head formation. (C) A wild-type (WT) stage 35 tadpole. (D) xNor-MO (20 ng) caused anterior truncation (61%, n = 64) when injected into the animal regions of two dorsal cells in four- to eight-cell-stage embryos. (E) xNor-misMO-injected embryos are generally normal (88%, n = 66). (F) The anterior defects caused by NorMO were rescued by xNorrin (25 pg) mRNA (77%, n = 81). (G) Dorsal view of a wild-type tadpole at stage 45. (H) Anterior defects on only one side (arrow) were generated by injecting xNor-MO (10 ng) into one dorsal cell at the four- to eight-cell stage (63% of injected embryos showed defects in the injected side, n = 30). The other side shows normal morphology. The anterior end is to the left. (I) xNor-MO inhibits Wnt signaling in dorsal animal cells. xNor-MO and SuperTopFlash (STF) reporter plasmids were co-injected into the dorsal animal cells of eight-cell embryos. F/R luciferase: ratio of firefly luciferase reading to renilla luciferase reading. (J) Whole-mount in situ hybridization shows that Chordin expression is reduced at stage 9 (53% of injected embryos, n = 80) and stage 10 (61% of injected embryos, n = 79) in xNor-MO-injected embryos, compared to xNor-misMO-injected embryos or uninjected embryos. (K–N) Whole-mount in situ hybridization for Chordin in bisected xNor-MO-injected embryos (stage 9.5) showing that xNor-MO inhibits Chordin expression in neuroectoderm precursors (arrow) (reduction in 66% of injected embryos, n = 104) (L) compared to wild-type embryos (K) and embryos with xNor-misMO injected into dorsal animal cells at the eight- to 16-cell stage (reduction in 13% of injected embryos, n = 78) (M). Note that xNorrin mRNA (100 pg) rescues Chordin expression in the dorsal ectoderm (80% of co-injected embryos showed expression comparable to wild-type embryos, n = 50) (N). Embryos are oriented such that their dorsal side is on the right. Dotted lines indicate the boundaries between the deep mesoderm and the superficial ectoderm.

Techniques Used: Sequencing, Injection, Generated, Luciferase, In Situ Hybridization, Expressing

Maternal xNorrin activates the canonical Wnt signaling pathway. (A) RT-PCR analysis of mRNAs from equatorially bisected oocytes (Egg). x Norrin mRNA is present in the animal half (Ani) of fully grown oocytes, while Xcat-2 mRNA is present in the vegetal half (Veg). –RT: no reverse transcription. (B) Both xNorrin mRNA and Wnt11 mRNA are enriched in the dorsal cells of 16-cell embryos. Embryos are evenly bisected into dorsal and ventral halves. D, dorsal half; V, ventral half; WE, whole embryo. (C) Spatial and temporal expression patterns of xNorrin mRNA from fertilized eggs to the late blastula stage (stage 9) revealed by whole-mount in situ hybridization. (D) xNorrin mRNA (500 pg) injection into the animal region of UV-ventralized embryos at one-cell stage reactivates the expression of Siamois , Chordin , Noggin , and Xnr3 at the late blastula stage. (E) xNorrin injection enhanced Chordin expression (detected by in situ hybridization) at stage 9 (81%, n = 36) and stage 10 (80%, n = 35) compared to wild-type embryos. UV, UV-irradiated embryos; WT, wild-type embryos; xNorrin, wild-type embryos injected with xNorrin (500 pg) into the dorsal-animal region at the four- to eight-cell stage.
Figure Legend Snippet: Maternal xNorrin activates the canonical Wnt signaling pathway. (A) RT-PCR analysis of mRNAs from equatorially bisected oocytes (Egg). x Norrin mRNA is present in the animal half (Ani) of fully grown oocytes, while Xcat-2 mRNA is present in the vegetal half (Veg). –RT: no reverse transcription. (B) Both xNorrin mRNA and Wnt11 mRNA are enriched in the dorsal cells of 16-cell embryos. Embryos are evenly bisected into dorsal and ventral halves. D, dorsal half; V, ventral half; WE, whole embryo. (C) Spatial and temporal expression patterns of xNorrin mRNA from fertilized eggs to the late blastula stage (stage 9) revealed by whole-mount in situ hybridization. (D) xNorrin mRNA (500 pg) injection into the animal region of UV-ventralized embryos at one-cell stage reactivates the expression of Siamois , Chordin , Noggin , and Xnr3 at the late blastula stage. (E) xNorrin injection enhanced Chordin expression (detected by in situ hybridization) at stage 9 (81%, n = 36) and stage 10 (80%, n = 35) compared to wild-type embryos. UV, UV-irradiated embryos; WT, wild-type embryos; xNorrin, wild-type embryos injected with xNorrin (500 pg) into the dorsal-animal region at the four- to eight-cell stage.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ Hybridization, Injection, Irradiation

TGF-β inhibition is implicated in Norrie disease. (A) xNorrin point mutants showed various levels of Wnt activation activity. Wild-type (WT) or mutant xNorrin and Fizzled4 and Lrp5 (FL) were injected into animal poles. The expression of Xnr3 , Saimois , and Chordin in animal caps was analyzed by RT-PCR. xNorrin R40K and xNorrin L60P showed slightly decreased and no Wnt activation, respectively. xNorrin K57N moderately increased Wnt activation. –RT, no reverse transcription; WE, whole embryo. (B–G) xNorrin point mutants showed various levels of mesoderm inhibition activity. Individual xNorrin point mutant mRNAs and β-gal mRNA were co-injected into the vegetal halves of two-cell-stage embryos. The expression of Xbra was analyzed at stage 10.5 by whole-mount in situ hybridization. While wild-type xNorrin inhibited Xbra expression (83% of the injected embryos showed very low or no Xbra expression, n = 35) (D), xNorrin K57N failed to inhibit Xbra expression (13% of the injected embryos showed reduced Xbra expression, n = 39) (F). xNorrin R40K (61% of the injected embryos, n = 33) and L60P (41% of the injected embryos, n = 32) also showed decreased Xbra expression (E and G). Uninjected (B) and β-gal -injected embryos (C). β-gal is stained in red. The embryos are in vegetal views, but slightly tilted toward marginal zones to show Xbra signal.
Figure Legend Snippet: TGF-β inhibition is implicated in Norrie disease. (A) xNorrin point mutants showed various levels of Wnt activation activity. Wild-type (WT) or mutant xNorrin and Fizzled4 and Lrp5 (FL) were injected into animal poles. The expression of Xnr3 , Saimois , and Chordin in animal caps was analyzed by RT-PCR. xNorrin R40K and xNorrin L60P showed slightly decreased and no Wnt activation, respectively. xNorrin K57N moderately increased Wnt activation. –RT, no reverse transcription; WE, whole embryo. (B–G) xNorrin point mutants showed various levels of mesoderm inhibition activity. Individual xNorrin point mutant mRNAs and β-gal mRNA were co-injected into the vegetal halves of two-cell-stage embryos. The expression of Xbra was analyzed at stage 10.5 by whole-mount in situ hybridization. While wild-type xNorrin inhibited Xbra expression (83% of the injected embryos showed very low or no Xbra expression, n = 35) (D), xNorrin K57N failed to inhibit Xbra expression (13% of the injected embryos showed reduced Xbra expression, n = 39) (F). xNorrin R40K (61% of the injected embryos, n = 33) and L60P (41% of the injected embryos, n = 32) also showed decreased Xbra expression (E and G). Uninjected (B) and β-gal -injected embryos (C). β-gal is stained in red. The embryos are in vegetal views, but slightly tilted toward marginal zones to show Xbra signal.

Techniques Used: Inhibition, Activation Assay, Activity Assay, Mutagenesis, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Staining

xNorrin activity is restricted to the ectoderm. (A–F) Injection of NFL into ventral vegetal cells at the eight-cell stage induced partial secondary axes. Wild-type (WT) embryos (stage 28) (A and D). Injection of Wnt8 mRNA (10 pg) into ventral vegetal cells in eight-cell embryos induces complete secondary axis formation (stage 28) (69% of injected embryos showed secondary axes, n = 85) (B and E). NFL mRNA (600 pg total; 200 pg each) co-injection of ventral vegetal cells in eight-cell embryos induces partial secondary axis formation (stage 28) (45% of co-injected embryos showed secondary axes, n = 105) (C and F). Whole-mount in situ hybridization for Sox3 in stage 28 embryos (D–F). Note that NFL induces Sox3 expression in the partial secondary axis (arrowhead in [F]). The black lines in (A–C) indicate the section planes in (G–I), respectively. (G–I) Histological sections of embryos at stage 30. A wild-type embryo (G). Wnt8 mRNA injection induced both secondary neural tube and notochord (H). NFL co-injection induced secondary neural tube but not notochord formation (I). Arrowhead: neural tube; arrow: notochord. (J–L) Immunostaining of notochords with the monoclonal antibody MZ15. Wild-type embryo with single notochord (all examined embryos) (J and J'). An embryo injected ventrally with Wnt8 mRNA (10 pg) showed two notochords (induced secondary notochord and the primary notochord) (all examined embryos with secondary axes) (K and K'). An embryo injected ventrally with NFL (200 pg each) showed only the primary notochord (arrows), with notochord tissue absent in the partial secondary axis (arrowheads) (all examined embryos with partial secondary axes) (L and L'). Embryos are at stage 30. (M–R) NFL and Wnt8 induced Chordin expression in different domains in the ventral cells. Whole-mount in situ hybridization was used to evaluate Chordin expression in stage 10 embryos. Chordin expression in wild-type early gastrula (M and P). Wnt8 mRNA (10 pg) injection into ventral vegetal cells induced Chordin expression both in the superficial layer and in the deep layer of the marginal zone (87.5% of injected embryos showed the expression in both layers, n = 24) (N). Injection of NFL mRNAs (200 pg each) into the same domain induced Chordin expression mainly in the superficial layer (76% of injected embryos showed the expression, n = 30) (O). Injection of Wnt8 into the ventral-animal cells of eight-cell embryos induced Chordin expression both in the ectoderm and in the mesoderm (84% of injected embryos showed the expression in both layers, n = 25) (Q). Injection of NFL mRNAs (200 pg each) into the same domain of eight-cell embryos induced Chordin expression only in the ectoderm (72% of injected embryos showed the expression, n = 32) (R). Arrowheads: superficial layer (O) or ventral ectoderm (R); arrows: deep layers on the ventral side. All embryos in (M–R) are shown with their dorsal sides to the right. Dotted lines delineate the superficial layer and deeper layer on the ventral side.
Figure Legend Snippet: xNorrin activity is restricted to the ectoderm. (A–F) Injection of NFL into ventral vegetal cells at the eight-cell stage induced partial secondary axes. Wild-type (WT) embryos (stage 28) (A and D). Injection of Wnt8 mRNA (10 pg) into ventral vegetal cells in eight-cell embryos induces complete secondary axis formation (stage 28) (69% of injected embryos showed secondary axes, n = 85) (B and E). NFL mRNA (600 pg total; 200 pg each) co-injection of ventral vegetal cells in eight-cell embryos induces partial secondary axis formation (stage 28) (45% of co-injected embryos showed secondary axes, n = 105) (C and F). Whole-mount in situ hybridization for Sox3 in stage 28 embryos (D–F). Note that NFL induces Sox3 expression in the partial secondary axis (arrowhead in [F]). The black lines in (A–C) indicate the section planes in (G–I), respectively. (G–I) Histological sections of embryos at stage 30. A wild-type embryo (G). Wnt8 mRNA injection induced both secondary neural tube and notochord (H). NFL co-injection induced secondary neural tube but not notochord formation (I). Arrowhead: neural tube; arrow: notochord. (J–L) Immunostaining of notochords with the monoclonal antibody MZ15. Wild-type embryo with single notochord (all examined embryos) (J and J'). An embryo injected ventrally with Wnt8 mRNA (10 pg) showed two notochords (induced secondary notochord and the primary notochord) (all examined embryos with secondary axes) (K and K'). An embryo injected ventrally with NFL (200 pg each) showed only the primary notochord (arrows), with notochord tissue absent in the partial secondary axis (arrowheads) (all examined embryos with partial secondary axes) (L and L'). Embryos are at stage 30. (M–R) NFL and Wnt8 induced Chordin expression in different domains in the ventral cells. Whole-mount in situ hybridization was used to evaluate Chordin expression in stage 10 embryos. Chordin expression in wild-type early gastrula (M and P). Wnt8 mRNA (10 pg) injection into ventral vegetal cells induced Chordin expression both in the superficial layer and in the deep layer of the marginal zone (87.5% of injected embryos showed the expression in both layers, n = 24) (N). Injection of NFL mRNAs (200 pg each) into the same domain induced Chordin expression mainly in the superficial layer (76% of injected embryos showed the expression, n = 30) (O). Injection of Wnt8 into the ventral-animal cells of eight-cell embryos induced Chordin expression both in the ectoderm and in the mesoderm (84% of injected embryos showed the expression in both layers, n = 25) (Q). Injection of NFL mRNAs (200 pg each) into the same domain of eight-cell embryos induced Chordin expression only in the ectoderm (72% of injected embryos showed the expression, n = 32) (R). Arrowheads: superficial layer (O) or ventral ectoderm (R); arrows: deep layers on the ventral side. All embryos in (M–R) are shown with their dorsal sides to the right. Dotted lines delineate the superficial layer and deeper layer on the ventral side.

Techniques Used: Activity Assay, Injection, In Situ Hybridization, Expressing, Immunostaining

Reciprocal inhibition between xNorrin and TGF-β. (A–E) xNorrin inhibits Activin-B -mRNA-induced mesoderm formation. A wild-type (WT) embryo at a neurula stage (A). Wild-type animal caps with elongation (5% of the caps showed elongation, n = 60) (B). Elongated animal caps induced by Activin-B mRNA (25 pg) injection (82% of the injected caps showed elongation, n = 55) (C). Animal cap elongation was blocked in animal caps injected with Activin-B (25 pg) and xNorrin (200 pg) mRNAs (10% of the co-injected caps showed elongation, n = 58) (D). The Activin-B -mRNA-induced expression of mesoderm markers ( Wnt8 , Xbra , m-actin , and MyoD ) was inhibited by xNorrin (E). RNAs were injected into the animal pole of one-cell embryos, and animal caps were cut around stage 8 and cultured in 1× MMR until the sibling embryos reached neurula stage. (F and G) xNorrin inhibits BMP4 signaling. xNorrin mRNA (500 pg), like Δ BMPR mRNA (200 pg) and Chordin mRNA (100 pg), inhibited BRE-Luc reporter activity in Xenopus embryos (F). xNorrin mRNA (500 pg) inhibited BMP4-induced Smad1 phosphorylation in animal caps (G). P-Smad1, phosphorylated Smad1; T-Smad1, total Smad1; WE, whole embryo. (H) BMP4 inhibited xNorrin-induced otx2 , Xpax6 , and NCAM RNA expression in animal caps of stage 15 embryos. –RT, no reverse transcription; WE, whole embryo; WT, wild-type animal caps. (I) xNorrin interacts with BMP4. BMP4-Flag and xNorrin-Myc mRNAs or xNorrin-Flag and BMP4-Myc mRNAs were injected into adjacent cells of four-cell embryos. FLAG-tagged proteins were immunoprecipitated (IP) from later gastrula embryos with a FLAG antibody. The proteins were PAGE separated and immunoblotted (IB) with an anti-c-Myc antibody. Arrowheads indicate xNorrin-Myc (top) or BMP4-Myc (bottom). L.C., IgG light chain.
Figure Legend Snippet: Reciprocal inhibition between xNorrin and TGF-β. (A–E) xNorrin inhibits Activin-B -mRNA-induced mesoderm formation. A wild-type (WT) embryo at a neurula stage (A). Wild-type animal caps with elongation (5% of the caps showed elongation, n = 60) (B). Elongated animal caps induced by Activin-B mRNA (25 pg) injection (82% of the injected caps showed elongation, n = 55) (C). Animal cap elongation was blocked in animal caps injected with Activin-B (25 pg) and xNorrin (200 pg) mRNAs (10% of the co-injected caps showed elongation, n = 58) (D). The Activin-B -mRNA-induced expression of mesoderm markers ( Wnt8 , Xbra , m-actin , and MyoD ) was inhibited by xNorrin (E). RNAs were injected into the animal pole of one-cell embryos, and animal caps were cut around stage 8 and cultured in 1× MMR until the sibling embryos reached neurula stage. (F and G) xNorrin inhibits BMP4 signaling. xNorrin mRNA (500 pg), like Δ BMPR mRNA (200 pg) and Chordin mRNA (100 pg), inhibited BRE-Luc reporter activity in Xenopus embryos (F). xNorrin mRNA (500 pg) inhibited BMP4-induced Smad1 phosphorylation in animal caps (G). P-Smad1, phosphorylated Smad1; T-Smad1, total Smad1; WE, whole embryo. (H) BMP4 inhibited xNorrin-induced otx2 , Xpax6 , and NCAM RNA expression in animal caps of stage 15 embryos. –RT, no reverse transcription; WE, whole embryo; WT, wild-type animal caps. (I) xNorrin interacts with BMP4. BMP4-Flag and xNorrin-Myc mRNAs or xNorrin-Flag and BMP4-Myc mRNAs were injected into adjacent cells of four-cell embryos. FLAG-tagged proteins were immunoprecipitated (IP) from later gastrula embryos with a FLAG antibody. The proteins were PAGE separated and immunoblotted (IB) with an anti-c-Myc antibody. Arrowheads indicate xNorrin-Myc (top) or BMP4-Myc (bottom). L.C., IgG light chain.

Techniques Used: Inhibition, Injection, Expressing, Cell Culture, Activity Assay, RNA Expression, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

5) Product Images from "TGFβ1 Promotes Gemcitabine Resistance through Regulating the LncRNA-LET/NF90/miR-145 Signaling Axis in Bladder Cancer"

Article Title: TGFβ1 Promotes Gemcitabine Resistance through Regulating the LncRNA-LET/NF90/miR-145 Signaling Axis in Bladder Cancer

Journal: Theranostics

doi: 10.7150/thno.19542

NF90 regulates miR-145 biogenesis. ( A ) Cluster map of microarray showed the altered miRNAs ≥ 2 folds in NF90 knockdown cells, compared with control group (siNC). ( B ) mRNA level of miR-145 was measured by qRT-PCR in control (siNC) and NF90 knockdown (siNF90) T24 and 5637 cells. ( C ) qRT-PCR showing the level of miR-145 in control (siNC) and lncRNA-LET knockdown (siLET-#1 and siLET-#2) T24 cells. ( D ) The levels of miR-145 and miR-143 in control (shCTL) and lncRNA-LET stable knockdown (shLET) T24 cells. ( E ) RIP combined with qRT-PCR assays of NF90 binding to pri-miR-143 or pri-miR-145 in control (shCTL) or lncRNA-LET knockdown (shLET) T24 cells. Relative enrichment of pri-miRNAs in anti-NF90 group, compared with IgG group. The control GAPDH mRNA level was used for normalization. ( F ) Schematic illustration of wild-type (WT) and mutant (Mut) miR-145 with consensus binding motif of NF90. ( G ) qRT-PCR analysis of exogenous miR-145 levels in T24 cells, transfected with wild-type or mutant NF90 binding site pri-miR-145 vector. Expression of FLAG in Western blotting was used to represent the NF90 transfection efficiency. Data are shown as mean ± SD and represent at least two independent experiments with similar results. * P
Figure Legend Snippet: NF90 regulates miR-145 biogenesis. ( A ) Cluster map of microarray showed the altered miRNAs ≥ 2 folds in NF90 knockdown cells, compared with control group (siNC). ( B ) mRNA level of miR-145 was measured by qRT-PCR in control (siNC) and NF90 knockdown (siNF90) T24 and 5637 cells. ( C ) qRT-PCR showing the level of miR-145 in control (siNC) and lncRNA-LET knockdown (siLET-#1 and siLET-#2) T24 cells. ( D ) The levels of miR-145 and miR-143 in control (shCTL) and lncRNA-LET stable knockdown (shLET) T24 cells. ( E ) RIP combined with qRT-PCR assays of NF90 binding to pri-miR-143 or pri-miR-145 in control (shCTL) or lncRNA-LET knockdown (shLET) T24 cells. Relative enrichment of pri-miRNAs in anti-NF90 group, compared with IgG group. The control GAPDH mRNA level was used for normalization. ( F ) Schematic illustration of wild-type (WT) and mutant (Mut) miR-145 with consensus binding motif of NF90. ( G ) qRT-PCR analysis of exogenous miR-145 levels in T24 cells, transfected with wild-type or mutant NF90 binding site pri-miR-145 vector. Expression of FLAG in Western blotting was used to represent the NF90 transfection efficiency. Data are shown as mean ± SD and represent at least two independent experiments with similar results. * P

Techniques Used: Microarray, Quantitative RT-PCR, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot

6) Product Images from "MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients"

Article Title: MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients

Journal: Asian Journal of Andrology

doi: 10.4103/1008-682X.185001

The expression of CRISP2 in the ejaculated spermatozoa between asthenoteratozoospermic patients and normozoospermic volunteers. ( a ) The expression level of CRISP2 mRNA is detected by qRT-PCR in the ejaculated spermatozoa between ATZ patients and normozoospermic volunteers (Student's t -test, data are shown as mean ± s.e.m.). ( b ) The CRISP2 protein expression is detected by Western blot analysis in 24 (12 Norm vs 12 ATZ) randomly selected ejaculated spermatozoa. CRISP2: cysteine-rich secretory protein 2; Norm: normozoospermic control group; ATZ: asthenoteratozoospermic patients group; s.e.m.: standard error of the mean.
Figure Legend Snippet: The expression of CRISP2 in the ejaculated spermatozoa between asthenoteratozoospermic patients and normozoospermic volunteers. ( a ) The expression level of CRISP2 mRNA is detected by qRT-PCR in the ejaculated spermatozoa between ATZ patients and normozoospermic volunteers (Student's t -test, data are shown as mean ± s.e.m.). ( b ) The CRISP2 protein expression is detected by Western blot analysis in 24 (12 Norm vs 12 ATZ) randomly selected ejaculated spermatozoa. CRISP2: cysteine-rich secretory protein 2; Norm: normozoospermic control group; ATZ: asthenoteratozoospermic patients group; s.e.m.: standard error of the mean.

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

7) Product Images from "Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter *"

Article Title: Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.528257

Expression of SLC44A4 transcript variants in NCM460 cells. Semiquantitative PCR was performed to estimate mRNA expression of SLC44A4 transcript variants 1 and 3 (variant 2 is not expressed in these cells). The image is from a representative set of experiments
Figure Legend Snippet: Expression of SLC44A4 transcript variants in NCM460 cells. Semiquantitative PCR was performed to estimate mRNA expression of SLC44A4 transcript variants 1 and 3 (variant 2 is not expressed in these cells). The image is from a representative set of experiments

Techniques Used: Expressing, Polymerase Chain Reaction, Variant Assay

8) Product Images from "CircPSMC3 suppresses the proliferation and metastasis of gastric cancer by acting as a competitive endogenous RNA through sponging miR-296-5p"

Article Title: CircPSMC3 suppresses the proliferation and metastasis of gastric cancer by acting as a competitive endogenous RNA through sponging miR-296-5p

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0958-6

CircPSMC3 expression is down regulated in clinical GC specimens and cell lines. a Scatter plot analyses of circRNAs microarray data showing differentially expressed circRNA in lymph node metastasis group and normal group . High expression level is indicated by “red” and low levels by “green”. b Scatter plot analyses of circRNA microarray data showing differentially expressed circRNAs in lymph node metastasis group and no lymph node metastasis group. c GO analysis of circRNAs in lymph node metastasis group and normal group. d GO analysis of circRNAs in lymph node metastasis group and no lymph node metastasis group. e The spliced mature sequence length of circPSMC3 derived from the PSMC3 gene is 502 bp. f QRT-PCR for the abundance of circPSMC3 in GC cells treated with RNase R. g QRT-PCR for the abundance of PSMC3 mRNA in GC cells treated with RNase R. h CircPSMC3 expressions were evaluated using qRT-PCR in 106 pairs of gastric cancer plasmas and 21 pairs of normal plasmas. i CircPSMC3 expressions were measured using qRT-PCR in 106 pairs of gastric cancer (Tumor) and matched noncancerous tissue (Normal). j The expressions of circPSMC3 were evaluated in cell lines using qRT-PCR. k The area under the ROC curve (AUC) in distinguishing GC plasmas and normal ones was 0.9326. l Kaplan Meier survival curve showed the relationship between circPSMC3 and survival rates. ** p
Figure Legend Snippet: CircPSMC3 expression is down regulated in clinical GC specimens and cell lines. a Scatter plot analyses of circRNAs microarray data showing differentially expressed circRNA in lymph node metastasis group and normal group . High expression level is indicated by “red” and low levels by “green”. b Scatter plot analyses of circRNA microarray data showing differentially expressed circRNAs in lymph node metastasis group and no lymph node metastasis group. c GO analysis of circRNAs in lymph node metastasis group and normal group. d GO analysis of circRNAs in lymph node metastasis group and no lymph node metastasis group. e The spliced mature sequence length of circPSMC3 derived from the PSMC3 gene is 502 bp. f QRT-PCR for the abundance of circPSMC3 in GC cells treated with RNase R. g QRT-PCR for the abundance of PSMC3 mRNA in GC cells treated with RNase R. h CircPSMC3 expressions were evaluated using qRT-PCR in 106 pairs of gastric cancer plasmas and 21 pairs of normal plasmas. i CircPSMC3 expressions were measured using qRT-PCR in 106 pairs of gastric cancer (Tumor) and matched noncancerous tissue (Normal). j The expressions of circPSMC3 were evaluated in cell lines using qRT-PCR. k The area under the ROC curve (AUC) in distinguishing GC plasmas and normal ones was 0.9326. l Kaplan Meier survival curve showed the relationship between circPSMC3 and survival rates. ** p

Techniques Used: Expressing, Microarray, Sequencing, Derivative Assay, Quantitative RT-PCR

9) Product Images from "Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway"

Article Title: Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

Journal: Scientific Reports

doi: 10.1038/srep21888

FMDV 3A protein inhibits the SeV-induced IFN-β signaling pathway. ( a,b ) Effects of overexpression of FMDV nonstructural proteins on SeV-triggered IFN-β promoter or IRSE activation. HEK293T cells (5 × 10 4 ) were transfected with the IFN-β or ISRE reporter (0.1 μg), 10 ng of pRL-TK (as an internal control) and the indicated expression plasmids (0.1 μg) for 24 h. Cells were infected or uninfected with SeV for 12 h before luciferase assays were performed. ( c ) Effects of overexpression of FMDV proteins on IFNγ-triggered IRF1 activation. The experiments were similarly performed as in a. ( d–f ) FMDV 3A inhibits SeV- or IFNγ-induced activation of IFN-β, ISRE, and IRF1 promoter in a dose-dependent manner. The experiments were similarly performed as in a. ( g ) FMDV 3A inhibits SeV-induced transcription of endogenous Ifnb , Cxcl-10 , Isg56 and Rantes genes. HEK293T cells were transfected with 4 μg FMDV 3A or empty vector (EV) for 24 h followed by SeV infection for 12 h before a relative quantitative RT-PCR experiments were performed. ( h ) 3A inhibited the transcriptional level of the endogenous IFN-β mRNA in PK-15 cells. PK-15 (2 × 10 5 ) cells were transfected with 4 μg pMSCV-3A plasmid using lipofectamine 2000 for 12 h, subsequently treated with puromycin (1 mg/ml) for 24 h. Cells were then infected with the type O FMDV (moi: 0.1) for 6 h. IFN-β mRNA abundance in PK-15 cells was assessed using a relative quantitative RT-PCR. ( i ) Increased FMDV genome copies in 3A-transfected PK-15 cells. The experiments were similarly performed as in i. FMDV genome copies were assessed using a quantitative RT-PCR assay. ( j,k ) FMDV 3A inhibits phosphorylation and dimerization of IRF3 and the expression of RIG-I and MDA5 after SeV stimulation. HEK293T (2 × 10 5 ) cells were transfected with 4 μg the FMDV 3A plasmid or EV for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by western blotting. Values are presented as mean ± SD of three independent experiments. Rel. Luc. Act., relative luciferase activity.
Figure Legend Snippet: FMDV 3A protein inhibits the SeV-induced IFN-β signaling pathway. ( a,b ) Effects of overexpression of FMDV nonstructural proteins on SeV-triggered IFN-β promoter or IRSE activation. HEK293T cells (5 × 10 4 ) were transfected with the IFN-β or ISRE reporter (0.1 μg), 10 ng of pRL-TK (as an internal control) and the indicated expression plasmids (0.1 μg) for 24 h. Cells were infected or uninfected with SeV for 12 h before luciferase assays were performed. ( c ) Effects of overexpression of FMDV proteins on IFNγ-triggered IRF1 activation. The experiments were similarly performed as in a. ( d–f ) FMDV 3A inhibits SeV- or IFNγ-induced activation of IFN-β, ISRE, and IRF1 promoter in a dose-dependent manner. The experiments were similarly performed as in a. ( g ) FMDV 3A inhibits SeV-induced transcription of endogenous Ifnb , Cxcl-10 , Isg56 and Rantes genes. HEK293T cells were transfected with 4 μg FMDV 3A or empty vector (EV) for 24 h followed by SeV infection for 12 h before a relative quantitative RT-PCR experiments were performed. ( h ) 3A inhibited the transcriptional level of the endogenous IFN-β mRNA in PK-15 cells. PK-15 (2 × 10 5 ) cells were transfected with 4 μg pMSCV-3A plasmid using lipofectamine 2000 for 12 h, subsequently treated with puromycin (1 mg/ml) for 24 h. Cells were then infected with the type O FMDV (moi: 0.1) for 6 h. IFN-β mRNA abundance in PK-15 cells was assessed using a relative quantitative RT-PCR. ( i ) Increased FMDV genome copies in 3A-transfected PK-15 cells. The experiments were similarly performed as in i. FMDV genome copies were assessed using a quantitative RT-PCR assay. ( j,k ) FMDV 3A inhibits phosphorylation and dimerization of IRF3 and the expression of RIG-I and MDA5 after SeV stimulation. HEK293T (2 × 10 5 ) cells were transfected with 4 μg the FMDV 3A plasmid or EV for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by western blotting. Values are presented as mean ± SD of three independent experiments. Rel. Luc. Act., relative luciferase activity.

Techniques Used: Over Expression, Activation Assay, Transfection, Expressing, Infection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Activated Clotting Time Assay, Activity Assay

FMDV 3A inhibits the expression of the RIG-I, MDA5, and VISA by disrupting their mRNA level. ( a–c ) FMDV 3A inhibits the expressions of the RIG-I, MDA5, and VISA not at translational level. HEK293T cells were transfected with FMDV 3A or empty vector (1.0 μg), pRK-HA-actin (0.015 μg) and the indicated expression plasmids (0.2 μg). As a control, pRK-HA-actin (0.015 μg) was also transfected with above plasmids. At 18 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO), MG-132 (20 μM), 3-MA (0.5 mg/ml), or NH 4 Cl (20 mM) for 6 h and subjected to western blotting analysis. ( d ) FMDV 3A inhibits the mRNA abundances of RIG-I, MDA5, and VISA. HEK293T cells were transfected with 3A or an empty vector (EV) for 24 h. Total RNA was extracted form cells and RT-PCR was performed to test the expression levels of Rig-i , Mda5 , Visa , Tbk1 , Traf3 , Irf3 , and Gapdh . Results were expressed as the relative fold change in mRNA levels compared with those in cells transfected with EV. The expression levels were normalized to GAPDH. Values are presented as the mean ± SD from three independent experiments.
Figure Legend Snippet: FMDV 3A inhibits the expression of the RIG-I, MDA5, and VISA by disrupting their mRNA level. ( a–c ) FMDV 3A inhibits the expressions of the RIG-I, MDA5, and VISA not at translational level. HEK293T cells were transfected with FMDV 3A or empty vector (1.0 μg), pRK-HA-actin (0.015 μg) and the indicated expression plasmids (0.2 μg). As a control, pRK-HA-actin (0.015 μg) was also transfected with above plasmids. At 18 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO), MG-132 (20 μM), 3-MA (0.5 mg/ml), or NH 4 Cl (20 mM) for 6 h and subjected to western blotting analysis. ( d ) FMDV 3A inhibits the mRNA abundances of RIG-I, MDA5, and VISA. HEK293T cells were transfected with 3A or an empty vector (EV) for 24 h. Total RNA was extracted form cells and RT-PCR was performed to test the expression levels of Rig-i , Mda5 , Visa , Tbk1 , Traf3 , Irf3 , and Gapdh . Results were expressed as the relative fold change in mRNA levels compared with those in cells transfected with EV. The expression levels were normalized to GAPDH. Values are presented as the mean ± SD from three independent experiments.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Pressure mediated hypertrophy and mechanical stretch up-regulate expression of the long form of leptin receptor (ob-Rb) in rat cardiac myocytes"

Article Title: Pressure mediated hypertrophy and mechanical stretch up-regulate expression of the long form of leptin receptor (ob-Rb) in rat cardiac myocytes

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-13-37

Effect of cyclic mechanical stretch in cardiac myocytes. Effect of cyclic mechanical stretch on ob, ob-Ra, and ob-Rb mRNA expression in neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were treated with mechanical stretch for 1 to 24 h. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control wells. n=4 dishes in each group. Values are reported as means ± SD. *p
Figure Legend Snippet: Effect of cyclic mechanical stretch in cardiac myocytes. Effect of cyclic mechanical stretch on ob, ob-Ra, and ob-Rb mRNA expression in neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were treated with mechanical stretch for 1 to 24 h. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control wells. n=4 dishes in each group. Values are reported as means ± SD. *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

mRNA expression of ob and ob-R isoforms in pressure-overloaded hearts. mRNA expression of the ob and ob-R isoforms in the normal rat heart ( A ). mRNA levels of ob ( B ), ob-Ra ( C ), and ob-Rb ( D ) isoforms in pressure-overloaded rat hearts. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control groups at 2 or 4 weeks after surgery. 2w: control n=11, banding n=9, 4w: control n=7, banding n=8. Values are reported as means ± SD. *p
Figure Legend Snippet: mRNA expression of ob and ob-R isoforms in pressure-overloaded hearts. mRNA expression of the ob and ob-R isoforms in the normal rat heart ( A ). mRNA levels of ob ( B ), ob-Ra ( C ), and ob-Rb ( D ) isoforms in pressure-overloaded rat hearts. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control groups at 2 or 4 weeks after surgery. 2w: control n=11, banding n=9, 4w: control n=7, banding n=8. Values are reported as means ± SD. *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Effect of ANG-II and ET-1 in cardiac myocytes. Effect of neurohumoral factors on ob, ob-Ra, and ob-Rb mRNA expression in neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were treated with ANGII (1 to 10 μM), ET-1 (0.01 to 0.1 μM) for 24 h. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control dishes. n=4 dishes in each group. Values are reported as means ± SD. *p
Figure Legend Snippet: Effect of ANG-II and ET-1 in cardiac myocytes. Effect of neurohumoral factors on ob, ob-Ra, and ob-Rb mRNA expression in neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were treated with ANGII (1 to 10 μM), ET-1 (0.01 to 0.1 μM) for 24 h. All quantitative PCR assay data for ob, ob-Ra, and ob-Rb mRNA is normalized by GAPDH mRNA of the same sample, and expressed as fold increase compared with the mean level of control dishes. n=4 dishes in each group. Values are reported as means ± SD. *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

11) Product Images from "Suppression of iASPP-dependent aggressiveness in cervical cancer through reversal of methylation silencing of microRNA-124"

Article Title: Suppression of iASPP-dependent aggressiveness in cervical cancer through reversal of methylation silencing of microRNA-124

Journal: Scientific Reports

doi: 10.1038/srep35480

Oncogene iASPP is a direct target of miR-124 in CC cells. ( a ) Summary of the number of miRNAs that were predicted to bind to the 3′-UTR of iASPP by TargetScan and microRNA.org (upper panel). The 6 predicted miRNAs were common to these two algorithms. Schematic representation of the 3′-UTR of iASPP with the predicted target site for miR-124 (lower panel). ( b ) In silico prediction and molecular pathway enrichment analysis was performed on predicted target genes regulated by miR-124. The top 10 ranking KEGG pathways are listed. ( c ) qPCR analysis of miR-124 expression in CC cell lines and a normal cell line. The results are presented as the fold-change in expression compared with normal cell. ( d ) Expression of the iASPP mRNA and protein in CC cells after the transient overexpression or knockdown of miR-124. Hela ( e ) and Siha ( f ) cells were cotransfected with reporter plasmids containing wild-type iASPP or a mutant iASPP 3′-UTR together with a miR-124 mimic, miR-124 inhibitor, or respective negative controls. The relative luciferase activity was assayed. Data are shown as mean ± SEM; n = 3; ** P
Figure Legend Snippet: Oncogene iASPP is a direct target of miR-124 in CC cells. ( a ) Summary of the number of miRNAs that were predicted to bind to the 3′-UTR of iASPP by TargetScan and microRNA.org (upper panel). The 6 predicted miRNAs were common to these two algorithms. Schematic representation of the 3′-UTR of iASPP with the predicted target site for miR-124 (lower panel). ( b ) In silico prediction and molecular pathway enrichment analysis was performed on predicted target genes regulated by miR-124. The top 10 ranking KEGG pathways are listed. ( c ) qPCR analysis of miR-124 expression in CC cell lines and a normal cell line. The results are presented as the fold-change in expression compared with normal cell. ( d ) Expression of the iASPP mRNA and protein in CC cells after the transient overexpression or knockdown of miR-124. Hela ( e ) and Siha ( f ) cells were cotransfected with reporter plasmids containing wild-type iASPP or a mutant iASPP 3′-UTR together with a miR-124 mimic, miR-124 inhibitor, or respective negative controls. The relative luciferase activity was assayed. Data are shown as mean ± SEM; n = 3; ** P

Techniques Used: In Silico, Real-time Polymerase Chain Reaction, Expressing, Over Expression, Mutagenesis, Luciferase, Activity Assay

12) Product Images from "The pro-metastasis effect of circANKS1B in breast cancer"

Article Title: The pro-metastasis effect of circANKS1B in breast cancer

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0914-x

USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
Figure Legend Snippet: USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p

Techniques Used: Expressing, Luciferase, Cotransfection, Plasmid Preparation, Mutagenesis, Transfection, Multiple Displacement Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control, Quantitative RT-PCR

13) Product Images from "miR-203 Inhibits Alcohol-Induced Hepatic Steatosis by Targeting Lipin1"

Article Title: miR-203 Inhibits Alcohol-Induced Hepatic Steatosis by Targeting Lipin1

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00275

miR-203 inhibited hepatocyte lipids accumulation in vitro . (A) Transfection effect of miR-203 mimics was confirmed by qRT-PCR. (B) The cellular Oil Red staining (x200). (C) The cellular TG and TCH levels. (D,E) qRT-PCR and “Western blot” analysis for mRNA and protein expression of lipid metabolism markers PPAR-α ∗ SREBP-1, inflammation markers IL-6, TNP-α. ∗ p
Figure Legend Snippet: miR-203 inhibited hepatocyte lipids accumulation in vitro . (A) Transfection effect of miR-203 mimics was confirmed by qRT-PCR. (B) The cellular Oil Red staining (x200). (C) The cellular TG and TCH levels. (D,E) qRT-PCR and “Western blot” analysis for mRNA and protein expression of lipid metabolism markers PPAR-α ∗ SREBP-1, inflammation markers IL-6, TNP-α. ∗ p

Techniques Used: In Vitro, Transfection, Quantitative RT-PCR, Staining, Expressing

miR-203 inhibited liver lipids accumulation in vivo . (A) Lenti-mir-203 and Lenti-NC expression in mice liver. (B) Expression of miR-203 was confirmed by qRT-PCR. (C) The liver tissues H E and Oil Red O staining (x100). (D) Liver triglyceride (TG) levels. (E,F) qRT-PCR and Western blot analysis for mRNA and protein expression of lipid metabolism markers PPAR-α ∗ SREBP-1, inflammation markers IL-6, TNF-α. ∗ p
Figure Legend Snippet: miR-203 inhibited liver lipids accumulation in vivo . (A) Lenti-mir-203 and Lenti-NC expression in mice liver. (B) Expression of miR-203 was confirmed by qRT-PCR. (C) The liver tissues H E and Oil Red O staining (x100). (D) Liver triglyceride (TG) levels. (E,F) qRT-PCR and Western blot analysis for mRNA and protein expression of lipid metabolism markers PPAR-α ∗ SREBP-1, inflammation markers IL-6, TNF-α. ∗ p

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

miR-203 directly regulated the expression of Lipin1. (A) Bioinformatics analyses show the seed sequence of miR-203 bind to the 3′-UTR of LPIN1 mRNA. (B) “Wild type” 3′-UTR of LPIN1 gene was cloned into the firefly and Renilla reporter plasmid. The LPIN1-3′ UTR constructs or blank plasmid were transfected into AML-12 cells with control or miR-203 mimics, followed by dual luciferase assays. (C,D) Western blot analysis for protein expression of LPIN1 after transfected with miR-203 mimics or miR-NC, mir-203 inhibitor or inhibitor-NC. ∗ p
Figure Legend Snippet: miR-203 directly regulated the expression of Lipin1. (A) Bioinformatics analyses show the seed sequence of miR-203 bind to the 3′-UTR of LPIN1 mRNA. (B) “Wild type” 3′-UTR of LPIN1 gene was cloned into the firefly and Renilla reporter plasmid. The LPIN1-3′ UTR constructs or blank plasmid were transfected into AML-12 cells with control or miR-203 mimics, followed by dual luciferase assays. (C,D) Western blot analysis for protein expression of LPIN1 after transfected with miR-203 mimics or miR-NC, mir-203 inhibitor or inhibitor-NC. ∗ p

Techniques Used: Expressing, Sequencing, Clone Assay, Plasmid Preparation, Construct, Transfection, Luciferase, Western Blot

14) Product Images from "Altered Expression of TXNIP in the peripheral leukocytes of patients with coronary atherosclerotic heart disease"

Article Title: Altered Expression of TXNIP in the peripheral leukocytes of patients with coronary atherosclerotic heart disease

Journal: Medicine

doi: 10.1097/MD.0000000000009108

The mRNA expression levels of TXNIP gene of peripheral leucocytes in patients and healthy controls. The mRNA levels of TXNIP gene were normalized to β-actin , which was used as an internal control. Data were expressed as means ± SD. ∗ P
Figure Legend Snippet: The mRNA expression levels of TXNIP gene of peripheral leucocytes in patients and healthy controls. The mRNA levels of TXNIP gene were normalized to β-actin , which was used as an internal control. Data were expressed as means ± SD. ∗ P

Techniques Used: Expressing

The sensitivity and specificity of the RT-qPCR and western blot were compared using the receiver operating characteristic (ROC) curves. Area under the curve (AUC) for the level of thioredoxin interacting protein (TXNIP) mRNA was significantly higher than protein levels (AUCmRNA = 0.791, AUCProtein = 0.656, P
Figure Legend Snippet: The sensitivity and specificity of the RT-qPCR and western blot were compared using the receiver operating characteristic (ROC) curves. Area under the curve (AUC) for the level of thioredoxin interacting protein (TXNIP) mRNA was significantly higher than protein levels (AUCmRNA = 0.791, AUCProtein = 0.656, P

Techniques Used: Quantitative RT-PCR, Western Blot

15) Product Images from "The Circadian Binding of CLOCK Protein to the Promoter of C/ebpα Gene in Mouse Cells"

Article Title: The Circadian Binding of CLOCK Protein to the Promoter of C/ebpα Gene in Mouse Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0058221

Clock mutation damps daily variation in C/ebpα mRNA expression. Total RNA was extracted from the livers of three wild-type (WT; open symbols) and three Clock mutant ( Clk ; closed symbols) mice maintained under a 12 h light/12 h dark cycle (lights on at 0∶00 and lights off at 12∶00) and then C/ebpα expression was determined by RT-PCR. Maximal value for wild-type mice is expressed as 100%. Each time point comes from average of three mice. Data are presented as mean ± SEM (n = 3, *: p
Figure Legend Snippet: Clock mutation damps daily variation in C/ebpα mRNA expression. Total RNA was extracted from the livers of three wild-type (WT; open symbols) and three Clock mutant ( Clk ; closed symbols) mice maintained under a 12 h light/12 h dark cycle (lights on at 0∶00 and lights off at 12∶00) and then C/ebpα expression was determined by RT-PCR. Maximal value for wild-type mice is expressed as 100%. Each time point comes from average of three mice. Data are presented as mean ± SEM (n = 3, *: p

Techniques Used: Mutagenesis, Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

16) Product Images from "Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9, et al. Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9"

Article Title: Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9, et al. Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13552

Nucleolin binded to VEGF ‐A and MMP ‐9 mRNA and up‐regulated the expressions of VEGF ‐A and MMP ‐9 mRNA through enhancing their stability. (A) and (B) IP ‐ RT ‐ PCR analysis demonstrated that nucleolin binded to VEGF ‐A and MMP ‐9 mRNA . Nucleolin in the HUVEC s was precipitated by nucleolin antibody and magnetic A/G magnetic beads, Western blotting was performed to identify the precipitation efficiency of nucleolin, and the mRNA expressions of MMP ‐9 and VEGF ‐A were detected in the extracted from the magnetic beads, the representative images (A) and the analysis of results (B) (n = 3 per group; * P
Figure Legend Snippet: Nucleolin binded to VEGF ‐A and MMP ‐9 mRNA and up‐regulated the expressions of VEGF ‐A and MMP ‐9 mRNA through enhancing their stability. (A) and (B) IP ‐ RT ‐ PCR analysis demonstrated that nucleolin binded to VEGF ‐A and MMP ‐9 mRNA . Nucleolin in the HUVEC s was precipitated by nucleolin antibody and magnetic A/G magnetic beads, Western blotting was performed to identify the precipitation efficiency of nucleolin, and the mRNA expressions of MMP ‐9 and VEGF ‐A were detected in the extracted from the magnetic beads, the representative images (A) and the analysis of results (B) (n = 3 per group; * P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Magnetic Beads, Western Blot

Hydroxysafflor Yellow A elevated the expressions of nucleolin, VEGF ‐A and MMP ‐9 in HUVEC s. (A) Real‐time PCR analysis demonstrated the effect of different concentrations (0, 0.025, 0.1, 0.2 mmol/L) of HSYA on the mRNA expressions of nucleolin, VEGF ‐A and MMP ‐9 in HUVEC s (n = 3 per group; ** P
Figure Legend Snippet: Hydroxysafflor Yellow A elevated the expressions of nucleolin, VEGF ‐A and MMP ‐9 in HUVEC s. (A) Real‐time PCR analysis demonstrated the effect of different concentrations (0, 0.025, 0.1, 0.2 mmol/L) of HSYA on the mRNA expressions of nucleolin, VEGF ‐A and MMP ‐9 in HUVEC s (n = 3 per group; ** P

Techniques Used: Real-time Polymerase Chain Reaction

17) Product Images from "Dipeptide-modified nanoparticles to facilitate oral docetaxel delivery: new insights into PepT1-mediated targeting strategy"

Article Title: Dipeptide-modified nanoparticles to facilitate oral docetaxel delivery: new insights into PepT1-mediated targeting strategy

Journal: Drug Delivery

doi: 10.1080/10717544.2018.1480675

(A) Competitive study in hPepT1-Hela cells in the presence of typical substrate GlySar (GS); (B) Influence of proton in the culture medium on the cellular uptake of dipeptide modified NPs in hPepT1-Hela cells; The variation of relative PepT1 mRNA expression versus β-actin (C) and the variations of membrane and cytosol PepT1 protein expression (D,E) after treatments with NSPV1000 NPs for different time over 24 h. Data are shown as mean ± SD. * p
Figure Legend Snippet: (A) Competitive study in hPepT1-Hela cells in the presence of typical substrate GlySar (GS); (B) Influence of proton in the culture medium on the cellular uptake of dipeptide modified NPs in hPepT1-Hela cells; The variation of relative PepT1 mRNA expression versus β-actin (C) and the variations of membrane and cytosol PepT1 protein expression (D,E) after treatments with NSPV1000 NPs for different time over 24 h. Data are shown as mean ± SD. * p

Techniques Used: Modification, Expressing

18) Product Images from "Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer"

Article Title: Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer

Journal: Cancer Biology & Therapy

doi: 10.4161/cbt.29691

Figure 1. CCNB1 is overexpressed in colorectal cancer. ( A ) qRT-PCR analysis of CCNB1 mRNA expression in 30 paired human colorectal cancer tissues and the matched adjacent normal tissues. GAPDH was used as internal control and for normalization
Figure Legend Snippet: Figure 1. CCNB1 is overexpressed in colorectal cancer. ( A ) qRT-PCR analysis of CCNB1 mRNA expression in 30 paired human colorectal cancer tissues and the matched adjacent normal tissues. GAPDH was used as internal control and for normalization

Techniques Used: Quantitative RT-PCR, Expressing

19) Product Images from "miR-148a and miR-17–5p synergistically regulate milk TAG synthesis via PPARGC1A and PPARA in goat mammary epithelial cells"

Article Title: miR-148a and miR-17–5p synergistically regulate milk TAG synthesis via PPARGC1A and PPARA in goat mammary epithelial cells

Journal: RNA Biology

doi: 10.1080/15476286.2016.1276149

Functional evaluation of miR-17–5p (A) TAG levels in cells transfect with miR-17–5p mimic (60nM) or inhibitor (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars:miR-17–5p mimic or inhibitor. (B) Cholesterol levels in cells transfect with miR-17–5p mimic (60nM) or inhibitor (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars:miR-17–5p mimic or inhibitor. (C) GMECs are transfected with miR-17–5p mimic or inhibitor for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: miR-17–5p mimic or inhibitor. (D) Western blot analysis of β -Casein expression in the miR-17–5p mimic or inhibitor treatment experiments The effect of miR-17–5p mimic or inhibitor for 48h on β -Casein protein expression is evaluated by western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) Changes in the lipid contents of GMECs transfect with miR-17–5p mimic or inhibitor for 48h. Cells were stained by oil red. After examined microscopically, the oil red o is extracted with 400 µl of isopropanol and its absorbance is determined at 510 nm. The relative fat droplet contents are normalized to control transfected cells. a: NC mimic treatment, b: miR-17–5p mimic treatment, c: NC inhibitor treatment, d: miR-17–5p inhibitor treatment (F) Expression of fat metabolism related genes GMECs are transfected with miR-17–5p mimic or inhibitor for 48h, and the mRNA expression of ACOX1, CPT1, ATGL, DGAT1 and LPL are quantified by RT-qPCR (n = 6). White bars:miR-17–5p mimic; black bars:miR-17–5p inhibitor. All experiments are duplicated and repeated for 3 times. Values are presented as means ± standard errors, *, P
Figure Legend Snippet: Functional evaluation of miR-17–5p (A) TAG levels in cells transfect with miR-17–5p mimic (60nM) or inhibitor (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars:miR-17–5p mimic or inhibitor. (B) Cholesterol levels in cells transfect with miR-17–5p mimic (60nM) or inhibitor (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars:miR-17–5p mimic or inhibitor. (C) GMECs are transfected with miR-17–5p mimic or inhibitor for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: miR-17–5p mimic or inhibitor. (D) Western blot analysis of β -Casein expression in the miR-17–5p mimic or inhibitor treatment experiments The effect of miR-17–5p mimic or inhibitor for 48h on β -Casein protein expression is evaluated by western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) Changes in the lipid contents of GMECs transfect with miR-17–5p mimic or inhibitor for 48h. Cells were stained by oil red. After examined microscopically, the oil red o is extracted with 400 µl of isopropanol and its absorbance is determined at 510 nm. The relative fat droplet contents are normalized to control transfected cells. a: NC mimic treatment, b: miR-17–5p mimic treatment, c: NC inhibitor treatment, d: miR-17–5p inhibitor treatment (F) Expression of fat metabolism related genes GMECs are transfected with miR-17–5p mimic or inhibitor for 48h, and the mRNA expression of ACOX1, CPT1, ATGL, DGAT1 and LPL are quantified by RT-qPCR (n = 6). White bars:miR-17–5p mimic; black bars:miR-17–5p inhibitor. All experiments are duplicated and repeated for 3 times. Values are presented as means ± standard errors, *, P

Techniques Used: Functional Assay, Negative Control, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Staining

Functional evaluation of PPARGC1A (A) TAG levels in cells transfect with Si-NC (60nM) or SiRNA- PPARGC1A (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (B) Cholesterol levels in cells transfect with Si-NC (60nM) or SiRNA- PPARGC1A (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (C) GMECs are transfected with Si-NC (60nM) or SiRNA- PPARGC1A (60nM) for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (D) Western blot analysis of β -Casein expression in the Si-NC (60nM) or SiRNA- PPARGC1A (60nM) treatment experiments The effect of Si-NC (60nM) or SiRNA- PPARGC1A (60nM) for 48h on β -Casein protein expression is evaluated by Western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) TAG levels in cells transfect with Control inhibitor (50nM) + Control siRNA (50nM), Inhibitor-miR-148a (50nM) + Control siRNA (50nM) and Inhibitor-miR-148a (50nM) + siRNA- PPARGC1A (50nM); TAG levels are compared with that of control (n = 6).
Figure Legend Snippet: Functional evaluation of PPARGC1A (A) TAG levels in cells transfect with Si-NC (60nM) or SiRNA- PPARGC1A (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (B) Cholesterol levels in cells transfect with Si-NC (60nM) or SiRNA- PPARGC1A (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (C) GMECs are transfected with Si-NC (60nM) or SiRNA- PPARGC1A (60nM) for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: SiRNA- PPARGC1A . (D) Western blot analysis of β -Casein expression in the Si-NC (60nM) or SiRNA- PPARGC1A (60nM) treatment experiments The effect of Si-NC (60nM) or SiRNA- PPARGC1A (60nM) for 48h on β -Casein protein expression is evaluated by Western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) TAG levels in cells transfect with Control inhibitor (50nM) + Control siRNA (50nM), Inhibitor-miR-148a (50nM) + Control siRNA (50nM) and Inhibitor-miR-148a (50nM) + siRNA- PPARGC1A (50nM); TAG levels are compared with that of control (n = 6).

Techniques Used: Functional Assay, Negative Control, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Functional evaluation of miR-148a (A) TAG concentrations in cells transfect with miR-148a mimic (60 nM) or inhibitor (60 nM); TAG concentrations were compared with that of control (n = 6). White bars: negative control; black bars:miR-148a mimic or inhibitor. (B) Cholesterol concentrations in cells transfected with miR-148a mimic (60 nM) or inhibitor (60 nM); cholesterol concentrations were compared with that of control (n = 6). White bars: negative control; black bars: miR-148a mimic or inhibitor. (C) mRNA expression of β -Casein quantified by RT-qPCR (n = 6) in GMECs transfected with miR-148a mimic or inhibitor for 48 h. White bars: negative control; black bars: miR-148a mimic or inhibitor. (D) Western blot analysis of β -Casein expression in the miR-148a mimic or inhibitor treatment experiments. The effect of miR-148a mimic or inhibitor for 48 h on β -Casein protein expression in GMECs was evaluated by Western blot analysis. Total protein was harvested for 48 after post-treatment. (E) Changes in the lipid content of GMECs transfected with miR-148a mimic or inhibitor for 48 h. Cells were stained with oil red O. After examination microscopically, the oil red was extracted with 400 µl of isopropanol and its absorbance determined at 510 nm. The relative fat droplet content was normalized to control transfected cells. a: NC mimic treatment, b: miR-148a mimic treatment, c: NC inhibitor treatment, d: miR-148a inhibitor treatment (F) Expression of fat metabolism related genes GMECs are transfected with miR-148a mimic or inhibitor for 48h, and the mRNA expression of ACSL1, CPT1, HSL, SCD and CD36 are quantified by RT-qPCR (n = 6). White bars: miR-148a mimic; black bars: miR-148a inhibitor. All experiments were duplicated and repeated 3 times. Values are presented as means ± standard errors, *, P
Figure Legend Snippet: Functional evaluation of miR-148a (A) TAG concentrations in cells transfect with miR-148a mimic (60 nM) or inhibitor (60 nM); TAG concentrations were compared with that of control (n = 6). White bars: negative control; black bars:miR-148a mimic or inhibitor. (B) Cholesterol concentrations in cells transfected with miR-148a mimic (60 nM) or inhibitor (60 nM); cholesterol concentrations were compared with that of control (n = 6). White bars: negative control; black bars: miR-148a mimic or inhibitor. (C) mRNA expression of β -Casein quantified by RT-qPCR (n = 6) in GMECs transfected with miR-148a mimic or inhibitor for 48 h. White bars: negative control; black bars: miR-148a mimic or inhibitor. (D) Western blot analysis of β -Casein expression in the miR-148a mimic or inhibitor treatment experiments. The effect of miR-148a mimic or inhibitor for 48 h on β -Casein protein expression in GMECs was evaluated by Western blot analysis. Total protein was harvested for 48 after post-treatment. (E) Changes in the lipid content of GMECs transfected with miR-148a mimic or inhibitor for 48 h. Cells were stained with oil red O. After examination microscopically, the oil red was extracted with 400 µl of isopropanol and its absorbance determined at 510 nm. The relative fat droplet content was normalized to control transfected cells. a: NC mimic treatment, b: miR-148a mimic treatment, c: NC inhibitor treatment, d: miR-148a inhibitor treatment (F) Expression of fat metabolism related genes GMECs are transfected with miR-148a mimic or inhibitor for 48h, and the mRNA expression of ACSL1, CPT1, HSL, SCD and CD36 are quantified by RT-qPCR (n = 6). White bars: miR-148a mimic; black bars: miR-148a inhibitor. All experiments were duplicated and repeated 3 times. Values are presented as means ± standard errors, *, P

Techniques Used: Functional Assay, Negative Control, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Staining

Functional evaluation of PPARA (A) TAG levels in cells transfect with Si-NC (60nM) or SiRNA- PPARA (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (B) Cholesterol levels in cells transfect with Si-NC (60nM) or SiRNA- PPARA (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (C) GMECs are transfected with Si-NC (60nM) or SiRNA- PPARA (60nM) for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (D) Western blot analysis of β -Casein expression in the Si-NC (60nM) or SiRNA- PPARA (60nM) treatment experiments The effect of Si-NC (60nM) or SiRNA- PPARA (60nM) for 48h on β -Casein protein expression is evaluated by Western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) TAG levels in cells transfect with Control inhibitor(50nM) + Control siRNA (50nM), Inhibitor-miR-17–5p (50nM) + Control siRNA (50nM) and Inhibitor-miR-17–5p (50nM)+ SiRNA- PPARA (50nM); TAG levels are compared with that of control (n = 6).
Figure Legend Snippet: Functional evaluation of PPARA (A) TAG levels in cells transfect with Si-NC (60nM) or SiRNA- PPARA (60nM); TAG levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (B) Cholesterol levels in cells transfect with Si-NC (60nM) or SiRNA- PPARA (60nM); cholesterol levels are compared with that of control (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (C) GMECs are transfected with Si-NC (60nM) or SiRNA- PPARA (60nM) for 48h, and the mRNA expression of β -Casein is quantified by RT-qPCR (n = 6). White bars: negative control; black bars: SiRNA- PPARA . (D) Western blot analysis of β -Casein expression in the Si-NC (60nM) or SiRNA- PPARA (60nM) treatment experiments The effect of Si-NC (60nM) or SiRNA- PPARA (60nM) for 48h on β -Casein protein expression is evaluated by Western blot analysis in GMECs. Total proteins are harvested for 48h post-treatment, respectively. (E) TAG levels in cells transfect with Control inhibitor(50nM) + Control siRNA (50nM), Inhibitor-miR-17–5p (50nM) + Control siRNA (50nM) and Inhibitor-miR-17–5p (50nM)+ SiRNA- PPARA (50nM); TAG levels are compared with that of control (n = 6).

Techniques Used: Functional Assay, Negative Control, Transfection, Expressing, Quantitative RT-PCR, Western Blot

20) Product Images from "A Transcriptome-Level Study Identifies Changing Expression Profiles for Ossification of the Ligamentum Flavum of the Spine"

Article Title: A Transcriptome-Level Study Identifies Changing Expression Profiles for Ossification of the Ligamentum Flavum of the Spine

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.07.018

Differentially Expressed miRNAs in OLF and the Function of miR-19b-3p (A) Sequencing analysis for miRNA was performed with RNA extracted from ossification (n = 4) and normal (n = 4) LF. Hierarchical cluster analysis of significantly differentially expressed lncRNAs: bright green, under-expression; gray, no change; bright red, overexpression. (B) Differential expression of ten representative miRNAs was validated in human ossified and normal LF tissues by qPCR (n = 10 per group). (C) The differentially expressed miRNAs were distributed on different chromosomes. (D and E) qPCR analysis of miR-19b-3p was performed with total RNA isolated from ossified and normal LF (D) and hMSCs treated with osteogenic medium for 0 and 7 days (E). (F) hMSCs were transfected with miR-19b-3p mimic or the negative control and further cultured in osteogenic medium for 7 days. The RNA levels of miR-19b-3p, ALP, Col1α1, BGLAP, and RUNX2 were detected by qPCR. U6 was used as an internal control for miR-19b-3p. GAPDH was used as an internal control for mRNA. (G) Representative images of ALP staining of hMSCs after transfection with miR-19b-3p mimic or the negative controls and further culture in osteogenic medium for 7 days. Scale bars, 400 μm. Data are presented as mean ± SEM for at least triplicate experiments. The p values were analyzed by Student’s t test; *p
Figure Legend Snippet: Differentially Expressed miRNAs in OLF and the Function of miR-19b-3p (A) Sequencing analysis for miRNA was performed with RNA extracted from ossification (n = 4) and normal (n = 4) LF. Hierarchical cluster analysis of significantly differentially expressed lncRNAs: bright green, under-expression; gray, no change; bright red, overexpression. (B) Differential expression of ten representative miRNAs was validated in human ossified and normal LF tissues by qPCR (n = 10 per group). (C) The differentially expressed miRNAs were distributed on different chromosomes. (D and E) qPCR analysis of miR-19b-3p was performed with total RNA isolated from ossified and normal LF (D) and hMSCs treated with osteogenic medium for 0 and 7 days (E). (F) hMSCs were transfected with miR-19b-3p mimic or the negative control and further cultured in osteogenic medium for 7 days. The RNA levels of miR-19b-3p, ALP, Col1α1, BGLAP, and RUNX2 were detected by qPCR. U6 was used as an internal control for miR-19b-3p. GAPDH was used as an internal control for mRNA. (G) Representative images of ALP staining of hMSCs after transfection with miR-19b-3p mimic or the negative controls and further culture in osteogenic medium for 7 days. Scale bars, 400 μm. Data are presented as mean ± SEM for at least triplicate experiments. The p values were analyzed by Student’s t test; *p

Techniques Used: Sequencing, Expressing, Over Expression, Real-time Polymerase Chain Reaction, Isolation, Transfection, Negative Control, Cell Culture, ALP Assay, Staining

Integrated circRNA-microRNA-mRNA ceRNA Network Analysis (A) The competing endogenous RNA network has been based on circRNA-miRNA and miRNA-mRNA interactions. In this network, the circRNAs connect expression-correlated mRNAs via miRNAs. Square nodes represent miRNAs, diamonds represent circRNAs, and blue circles represent mRNAs. Red and green colors represent up- and downregulation, respectively. The shade of darkness represents fold change. The size of circles represents p values, with larger size owing smaller p value. Solid lines represent directed relationships, and dashed lines represent undirected relationships. (B) A simplified scheme was constructed, including the upregulated ENST00000608133, ENST00000599584, circ_0050139 and RUNX2 and the downregulated miR-19b-3p. (C) hMSCs were transfected with miR-19b-3p mimic or the negative control, and the RNA levels of ENST00000608133, ENST00000599584, and circ_0050139 were detected by qPCR. GAPDH was used as an internal control. Data are presented as mean ± SEM for at least triplicate experiments. The p values were analyzed by Student’s t test; *p
Figure Legend Snippet: Integrated circRNA-microRNA-mRNA ceRNA Network Analysis (A) The competing endogenous RNA network has been based on circRNA-miRNA and miRNA-mRNA interactions. In this network, the circRNAs connect expression-correlated mRNAs via miRNAs. Square nodes represent miRNAs, diamonds represent circRNAs, and blue circles represent mRNAs. Red and green colors represent up- and downregulation, respectively. The shade of darkness represents fold change. The size of circles represents p values, with larger size owing smaller p value. Solid lines represent directed relationships, and dashed lines represent undirected relationships. (B) A simplified scheme was constructed, including the upregulated ENST00000608133, ENST00000599584, circ_0050139 and RUNX2 and the downregulated miR-19b-3p. (C) hMSCs were transfected with miR-19b-3p mimic or the negative control, and the RNA levels of ENST00000608133, ENST00000599584, and circ_0050139 were detected by qPCR. GAPDH was used as an internal control. Data are presented as mean ± SEM for at least triplicate experiments. The p values were analyzed by Student’s t test; *p

Techniques Used: Expressing, Construct, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Expression Profile of mRNAs in OLF and Normal LF (A) Microarray analysis for mRNA was performed with RNA extracted from ossified (n = 4) and normal (n = 4) ligamentum flavum . Hierarchical cluster analysis of significantly differentially expressed mRNAs: bright green, under-expression; gray, no change; bright red, overexpression. (B) Ten differentially expressed representative mRNAs were validated in OLF and normal LF tissues by qPCR (n = 10 per group). GAPDH was used as an internal control. (C) GO annotation of the linear counterparts of upregulated mRNAs with the top ten enrichment scores covering domains of biological processes, CCs, and MFs was performed. Data are presented as mean ± SEM; p values were analyzed by Student’s t test; *p
Figure Legend Snippet: Expression Profile of mRNAs in OLF and Normal LF (A) Microarray analysis for mRNA was performed with RNA extracted from ossified (n = 4) and normal (n = 4) ligamentum flavum . Hierarchical cluster analysis of significantly differentially expressed mRNAs: bright green, under-expression; gray, no change; bright red, overexpression. (B) Ten differentially expressed representative mRNAs were validated in OLF and normal LF tissues by qPCR (n = 10 per group). GAPDH was used as an internal control. (C) GO annotation of the linear counterparts of upregulated mRNAs with the top ten enrichment scores covering domains of biological processes, CCs, and MFs was performed. Data are presented as mean ± SEM; p values were analyzed by Student’s t test; *p

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

21) Product Images from "A Novel Circular RNA Generated by FGFR2 Gene Promotes Myoblast Proliferation and Differentiation by Sponging miR-133a-5p and miR-29b-1-5p"

Article Title: A Novel Circular RNA Generated by FGFR2 Gene Promotes Myoblast Proliferation and Differentiation by Sponging miR-133a-5p and miR-29b-1-5p

Journal: Cells

doi: 10.3390/cells7110199

CircFGFR2 promotes myoblast differentiation. ( A ) Overexpression of circFGFR2 promotes mRNA expression of MYOD and MYOG. ( B ) Knockdown of circFGFR2 inhibits the mRNA expression of MYOD and MYOG. ( C , D ) Overexpression of circFGFR2 facilitates the formation of myotubes. ( E , F ) Down-regulation of circFGFR2 suppresses the formation of myotubes. In all panels, data are presented as mean ± S.E.M. of three biological replicates. Statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p
Figure Legend Snippet: CircFGFR2 promotes myoblast differentiation. ( A ) Overexpression of circFGFR2 promotes mRNA expression of MYOD and MYOG. ( B ) Knockdown of circFGFR2 inhibits the mRNA expression of MYOD and MYOG. ( C , D ) Overexpression of circFGFR2 facilitates the formation of myotubes. ( E , F ) Down-regulation of circFGFR2 suppresses the formation of myotubes. In all panels, data are presented as mean ± S.E.M. of three biological replicates. Statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p

Techniques Used: Over Expression, Expressing

CircFGFR2 eliminates the inhibition effect of miR-133a-5p and miR-29b-1-5p on myoblast differentiation. ( A ) The mRNA expression of MYOD and MYOG of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). ( B ) The mRNA expression of MYOD and MYOG of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). ( C , D ) The myotube area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). ( E , F ) The myotubes area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector, and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). In all panels, results are expressed as the mean ± S.E.M. of three independent experiments, and statistical significance of differences between means were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p
Figure Legend Snippet: CircFGFR2 eliminates the inhibition effect of miR-133a-5p and miR-29b-1-5p on myoblast differentiation. ( A ) The mRNA expression of MYOD and MYOG of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). ( B ) The mRNA expression of MYOD and MYOG of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). ( C , D ) The myotube area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). ( E , F ) The myotubes area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector, and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). In all panels, results are expressed as the mean ± S.E.M. of three independent experiments, and statistical significance of differences between means were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p

Techniques Used: Inhibition, Expressing, Cotransfection, Over Expression, Plasmid Preparation, Software

22) Product Images from "NCAM(CD56) and RUNX1(AML1) Are Up-Regulated in Human Ischemic Cardiomyopathy and a Rat Model of Chronic Cardiac Ischemia"

Article Title: NCAM(CD56) and RUNX1(AML1) Are Up-Regulated in Human Ischemic Cardiomyopathy and a Rat Model of Chronic Cardiac Ischemia

Journal: The American Journal of Pathology

doi:

RNase Protection Assay ( a ) and Western blot ( b ) of normal human control hearts ( lanes 1 to 4 in a and b ) and ICM hearts ( a , lanes 5 to 10 ; b , lanes 5 to 8 ). Strong overexpression of NCAM(CD56) message and protein in ICM. Slight but significant NCAM(CD56) mRNA and protein overexpression in CCM ( a , lanes 11 to 14 ; b , lanes 9 to 10 ). Equal loading of mRNA or protein, respectively, per lane checked by GAPDH ( a ) and muscle-specific actin Western blot ( b ). up, unprotected radioactive probe; p, protected radioactive probe.
Figure Legend Snippet: RNase Protection Assay ( a ) and Western blot ( b ) of normal human control hearts ( lanes 1 to 4 in a and b ) and ICM hearts ( a , lanes 5 to 10 ; b , lanes 5 to 8 ). Strong overexpression of NCAM(CD56) message and protein in ICM. Slight but significant NCAM(CD56) mRNA and protein overexpression in CCM ( a , lanes 11 to 14 ; b , lanes 9 to 10 ). Equal loading of mRNA or protein, respectively, per lane checked by GAPDH ( a ) and muscle-specific actin Western blot ( b ). up, unprotected radioactive probe; p, protected radioactive probe.

Techniques Used: Rnase Protection Assay, Western Blot, Over Expression

Detection of NCAM(CD56)-related transcription factors NF-kappa B, HOXD9, PAX ( a ), and RUNX1(AML1) ( b and c ) in normal and ICM hearts. a: Similar amounts of NF-kappa B p65 and p50, HOXD9, and PAX 2/5/8 in normal ( lanes 1 to 4 ) and ICM hearts ( lanes 5 to 8 ) (Western blots). b: Overexpression of RUNX1(AML1) mRNA in ICM ( lanes 3 to 6 ) compared to normal human hearts ( lanes 7 to 10 ) and slight overexpression in CCM ( lanes 11 to 12 ). Jurkat-derived ( lane 1 ) and TE671 rhabdomyosarcoma-derived ( lane 2 ) control mRNAs (RNase protection assays). c: in ICM ( lanes 6 to 9 ) compared to normal human hearts ( lanes 2 to 5 ) and again either very slight or no overexpression in CCM ( lanes 10 to 11 ) (Western blots). Jurkat cell extract ( lane 1 , positive control). Strong expression of an additional 38-kd band in all samples, with down-regulation in ICM ( lanes 6 to 9 ) and CCM ( lanes 10 to 11 ). up, unprotected radioactive probe; p, protected radioactive probe
Figure Legend Snippet: Detection of NCAM(CD56)-related transcription factors NF-kappa B, HOXD9, PAX ( a ), and RUNX1(AML1) ( b and c ) in normal and ICM hearts. a: Similar amounts of NF-kappa B p65 and p50, HOXD9, and PAX 2/5/8 in normal ( lanes 1 to 4 ) and ICM hearts ( lanes 5 to 8 ) (Western blots). b: Overexpression of RUNX1(AML1) mRNA in ICM ( lanes 3 to 6 ) compared to normal human hearts ( lanes 7 to 10 ) and slight overexpression in CCM ( lanes 11 to 12 ). Jurkat-derived ( lane 1 ) and TE671 rhabdomyosarcoma-derived ( lane 2 ) control mRNAs (RNase protection assays). c: in ICM ( lanes 6 to 9 ) compared to normal human hearts ( lanes 2 to 5 ) and again either very slight or no overexpression in CCM ( lanes 10 to 11 ) (Western blots). Jurkat cell extract ( lane 1 , positive control). Strong expression of an additional 38-kd band in all samples, with down-regulation in ICM ( lanes 6 to 9 ) and CCM ( lanes 10 to 11 ). up, unprotected radioactive probe; p, protected radioactive probe

Techniques Used: Western Blot, Over Expression, Derivative Assay, Positive Control, Expressing

23) Product Images from "MicroRNA-18a-5p functions as an oncogene by directly targeting IRF2 in lung cancer"

Article Title: MicroRNA-18a-5p functions as an oncogene by directly targeting IRF2 in lung cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.145

IRF2 is a direct target of miR-18a-5p. ( a ) IRF2 WT 3'-UTR contains predicted miR-18a-5p binding sites. The figure shows alignment of miR-18a-5p with the IRF2 WT 3'-UTR, with arrows indicating the mutagenesis nucleotides. ( b ) Dual luciferase reporter assay. HEK293T cells were co-transfected with luciferase reporter constructs containing the pGL3-IRF2 WT 3'-UTR (IRF2 WT) and the pGL3-IRF2 mut 3'-UTR (IRF2 mut) with miR-18a-5p mimic or NC mimic. Relative firefly luciferase expression is displayed, normalized to Renilla luciferase expression, n =3 independent experiments. ( c ) Immunoblotting analysis of IRF2 levels in H23, H1299 and A549 cells transfected with NC mimic/miR-18a-5p mimic or NC inhibitor/miR-18a-5p inhibitor for 48 h. ( d ) The relative expression of IRF2 mRNA from qRT-PCR of corresponding non-tumour tissues (NT) and tumour tissues (T). 18 S was used for normalization. ( e-g ) Expression of IRF2 related to tumour size, sex and pathological stage. ( h ) The relative expression of IRF2 mRNA in lung cancer cell lines or a pulmonary epithelial cell line, n =3 independent experiments. ( i ) The effect of IRF2 expression levels on the overall survival in 1926 lung cancer patients (Kaplan–Meier Plotter online database) was analysed and Kaplan–Meier plots were generated using a Kaplan–Meier plotter ( http://www.kmplot.com ). ( j ) MiR-18a-5p had a negative correlation with IRF2 according to Pearson correlation coefficient. Error bars represent the mean±S.E.M. * P
Figure Legend Snippet: IRF2 is a direct target of miR-18a-5p. ( a ) IRF2 WT 3'-UTR contains predicted miR-18a-5p binding sites. The figure shows alignment of miR-18a-5p with the IRF2 WT 3'-UTR, with arrows indicating the mutagenesis nucleotides. ( b ) Dual luciferase reporter assay. HEK293T cells were co-transfected with luciferase reporter constructs containing the pGL3-IRF2 WT 3'-UTR (IRF2 WT) and the pGL3-IRF2 mut 3'-UTR (IRF2 mut) with miR-18a-5p mimic or NC mimic. Relative firefly luciferase expression is displayed, normalized to Renilla luciferase expression, n =3 independent experiments. ( c ) Immunoblotting analysis of IRF2 levels in H23, H1299 and A549 cells transfected with NC mimic/miR-18a-5p mimic or NC inhibitor/miR-18a-5p inhibitor for 48 h. ( d ) The relative expression of IRF2 mRNA from qRT-PCR of corresponding non-tumour tissues (NT) and tumour tissues (T). 18 S was used for normalization. ( e-g ) Expression of IRF2 related to tumour size, sex and pathological stage. ( h ) The relative expression of IRF2 mRNA in lung cancer cell lines or a pulmonary epithelial cell line, n =3 independent experiments. ( i ) The effect of IRF2 expression levels on the overall survival in 1926 lung cancer patients (Kaplan–Meier Plotter online database) was analysed and Kaplan–Meier plots were generated using a Kaplan–Meier plotter ( http://www.kmplot.com ). ( j ) MiR-18a-5p had a negative correlation with IRF2 according to Pearson correlation coefficient. Error bars represent the mean±S.E.M. * P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Transfection, Construct, Expressing, Quantitative RT-PCR, Generated

24) Product Images from "Mir20a/106a-WTX axis regulates RhoGDIa/CDC42 signaling and colon cancer progression"

Article Title: Mir20a/106a-WTX axis regulates RhoGDIa/CDC42 signaling and colon cancer progression

Journal: Nature Communications

doi: 10.1038/s41467-018-07998-x

WTX loss promotes CRC cells, migration and liver metastasis. a Percentage of WTX IHC in CRC and matched adjacent normal colorectal mucosa tissues. b Kaplan–Meier survival analysis of CRC patients with WTX high or WTX low expression. c Imunoblotting (IB) analyzes the WTX expression in human CRC tumor (T) and matched adjacent normal mucosa (N) tissues. d qRT-PCR analyzes WTX mRNA level in human CRC tumor and matched adjacent normal mucosa tissues. ** p = 0.0023, mean ± SEM. Two-tailed Student’s t -test. e IB analyzes WTX expression in CRC cell lines. f , g IB analyzes WTX expression in indicated cell lines. h , i Transwell analyzes the migration of the indicated cell lines. Scale bars, 200 μm. j – m Colony formation analysis of the indicated cell lines. *** p
Figure Legend Snippet: WTX loss promotes CRC cells, migration and liver metastasis. a Percentage of WTX IHC in CRC and matched adjacent normal colorectal mucosa tissues. b Kaplan–Meier survival analysis of CRC patients with WTX high or WTX low expression. c Imunoblotting (IB) analyzes the WTX expression in human CRC tumor (T) and matched adjacent normal mucosa (N) tissues. d qRT-PCR analyzes WTX mRNA level in human CRC tumor and matched adjacent normal mucosa tissues. ** p = 0.0023, mean ± SEM. Two-tailed Student’s t -test. e IB analyzes WTX expression in CRC cell lines. f , g IB analyzes WTX expression in indicated cell lines. h , i Transwell analyzes the migration of the indicated cell lines. Scale bars, 200 μm. j – m Colony formation analysis of the indicated cell lines. *** p

Techniques Used: Migration, Immunohistochemistry, Expressing, Quantitative RT-PCR, Two Tailed Test

25) Product Images from "FGF-dependent left-right asymmetry patterning in zebrafish is mediated by Ier2 and Fibp1"

Article Title: FGF-dependent left-right asymmetry patterning in zebrafish is mediated by Ier2 and Fibp1

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

doi: 10.1073/pnas.0812880106

Functional interaction between Ier2 and Fibp1. ( A–D ) Lateral views at 24 hpf. The ier2 MO-injected embryo has a short trunk ( B ) compared with the control ( A ). The ier2 MO phenotype was rescued by coinjection with zebrafish ier2 mRNA ( C ) and human
Figure Legend Snippet: Functional interaction between Ier2 and Fibp1. ( A–D ) Lateral views at 24 hpf. The ier2 MO-injected embryo has a short trunk ( B ) compared with the control ( A ). The ier2 MO phenotype was rescued by coinjection with zebrafish ier2 mRNA ( C ) and human

Techniques Used: Functional Assay, Injection

26) Product Images from "Circ HIPK3 sponges miR‐558 to suppress heparanase expression in bladder cancer cells"

Article Title: Circ HIPK3 sponges miR‐558 to suppress heparanase expression in bladder cancer cells

Journal: EMBO Reports

doi: 10.15252/embr.201643581

Over‐expression of circHIPK3 inhibits migration and invasion of bladder cancer cell lines in vitro A The expression levels of circHIPK3 and HIPK3 mRNA in T24T and UMUC3 cells after stable transfection of circHIPK3 or vector plasmids were detected by real‐time PCR in mock, vector, and circHIPK3 over‐expression groups treated with or without RNase R digestion. Data are mean ± SEM, n = 3. ** P
Figure Legend Snippet: Over‐expression of circHIPK3 inhibits migration and invasion of bladder cancer cell lines in vitro A The expression levels of circHIPK3 and HIPK3 mRNA in T24T and UMUC3 cells after stable transfection of circHIPK3 or vector plasmids were detected by real‐time PCR in mock, vector, and circHIPK3 over‐expression groups treated with or without RNase R digestion. Data are mean ± SEM, n = 3. ** P

Techniques Used: Over Expression, Migration, In Vitro, Expressing, Stable Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

27) Product Images from "Myocardin Expression Is Regulated by Nkx2.5, and Its Function Is Required for Cardiomyogenesis"

Article Title: Myocardin Expression Is Regulated by Nkx2.5, and Its Function Is Required for Cardiomyogenesis

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.24.9222-9232.2003

Inhibition of cardiomyogenesis by overexpression of a dominant-negative mutant of myocardin. (A) Blots were made with total RNA (10 μg) isolated from P19CL6 cells on the indicated days. Myocardin (arrow) and Nkx2.5 mRNA expressions were first detectable at day 6. 28S RNA was used as a control for assessing RNA loading. (B) Schematic representation of myocardin A, myocardin, and MC129-509. Abbreviations: B, basic region; S, SAP domain; C, coiled-coil domain. P19CL6 cells were transiently cotransfected with the ANF truncated promoter with a functional SRE2 fused to the end (−132 SRE Luc), with or without myocardin A or myocardin expression vector and with or without MC129-509 expression vector. MC129-509 inhibited both actions of myocardin A and myocardin on this promoter. (C) P19CL6[pcDNA3] cells and P19CL6[MC129-509] cells were treated with 1% DMSO. At 16 days after treatment, cells were stained by immunofluorescence with MF20 (upper panels) or TO-PRO-3 (lower panels). About 80% of the P19CL6[pcDNA3] cells were differentiated into cardiac myocytes. In contrast,
Figure Legend Snippet: Inhibition of cardiomyogenesis by overexpression of a dominant-negative mutant of myocardin. (A) Blots were made with total RNA (10 μg) isolated from P19CL6 cells on the indicated days. Myocardin (arrow) and Nkx2.5 mRNA expressions were first detectable at day 6. 28S RNA was used as a control for assessing RNA loading. (B) Schematic representation of myocardin A, myocardin, and MC129-509. Abbreviations: B, basic region; S, SAP domain; C, coiled-coil domain. P19CL6 cells were transiently cotransfected with the ANF truncated promoter with a functional SRE2 fused to the end (−132 SRE Luc), with or without myocardin A or myocardin expression vector and with or without MC129-509 expression vector. MC129-509 inhibited both actions of myocardin A and myocardin on this promoter. (C) P19CL6[pcDNA3] cells and P19CL6[MC129-509] cells were treated with 1% DMSO. At 16 days after treatment, cells were stained by immunofluorescence with MF20 (upper panels) or TO-PRO-3 (lower panels). About 80% of the P19CL6[pcDNA3] cells were differentiated into cardiac myocytes. In contrast,

Techniques Used: Inhibition, Over Expression, Dominant Negative Mutation, Isolation, Functional Assay, Expressing, Plasmid Preparation, Staining, Immunofluorescence

Transactivation of the ANF promoter by myocardin A and myocardin. (A) COS cells were transiently cotransfected with −638 ANF Luc or −638 ANF Luc with mutations in SRE1 (no SRE1 Luc), SRE2 (no SRE2 Luc), or both SRE1 and SRE2 (no SRE1/SRE2 Luc) and with or without myocardin A or myocardin expression vector. Myocardin A and myocardin activated −638 ANF Luc to the same degree. Mutation of the SRE2 reduced responsiveness to myocardin A. Mutation of the SRE1 almost completely reduced responsiveness to myocardin A, and mutation of both SREs completely abolished activation by myocardin A. (B) Blots were made with total RNA (5 μg) isolated from cardiac myocytes infected with AdLacZ or AdMCA. Overexpression of myocardin A induced endogenous ANF mRNA expression. A GAPDH probe was used as a control for assessing RNA loading.
Figure Legend Snippet: Transactivation of the ANF promoter by myocardin A and myocardin. (A) COS cells were transiently cotransfected with −638 ANF Luc or −638 ANF Luc with mutations in SRE1 (no SRE1 Luc), SRE2 (no SRE2 Luc), or both SRE1 and SRE2 (no SRE1/SRE2 Luc) and with or without myocardin A or myocardin expression vector. Myocardin A and myocardin activated −638 ANF Luc to the same degree. Mutation of the SRE2 reduced responsiveness to myocardin A. Mutation of the SRE1 almost completely reduced responsiveness to myocardin A, and mutation of both SREs completely abolished activation by myocardin A. (B) Blots were made with total RNA (5 μg) isolated from cardiac myocytes infected with AdLacZ or AdMCA. Overexpression of myocardin A induced endogenous ANF mRNA expression. A GAPDH probe was used as a control for assessing RNA loading.

Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, Activation Assay, Isolation, Infection, Over Expression

Myocardin mRNA expression in Nkx2.5-null hearts. Myocardin mRNA expression was detected by in situ hybridization (A) and Northern blot analysis (B). (A) 35 S-labeled common probe for myocardin A and myocardin was hybridized to a wild-type embryo (+/+) and a Nkx2.5-null embryo (−/−) at ED 9.5. Bar, 100 μm. (B) Blots were made with total RNA (1 μg) isolated from hearts of wild-type embryos (+/+), Nkx2.5-null embryos (−/−), and Nkx2.5 heterozygote embryos (+/−) at ED 9.5 and 10.5. Myocardin expression was downregulated in Nkx2.5-null hearts. 28S RNA was used as a control for assessing RNA loading.
Figure Legend Snippet: Myocardin mRNA expression in Nkx2.5-null hearts. Myocardin mRNA expression was detected by in situ hybridization (A) and Northern blot analysis (B). (A) 35 S-labeled common probe for myocardin A and myocardin was hybridized to a wild-type embryo (+/+) and a Nkx2.5-null embryo (−/−) at ED 9.5. Bar, 100 μm. (B) Blots were made with total RNA (1 μg) isolated from hearts of wild-type embryos (+/+), Nkx2.5-null embryos (−/−), and Nkx2.5 heterozygote embryos (+/−) at ED 9.5 and 10.5. Myocardin expression was downregulated in Nkx2.5-null hearts. 28S RNA was used as a control for assessing RNA loading.

Techniques Used: Expressing, In Situ Hybridization, Northern Blot, Labeling, Isolation

Myocardin A expression in murine tissues. (A) Blots were made with mRNA (1 μg) isolated from hearts of embryos at ED 10.5 and tissues of 8-week-old mice. An 8-kb transcript (arrow) hybridized to the myocardin A specific probe in both embryo and adult heart. Weaker signals were observed with mRNA from lung, kidney, and testis in adult. A cyclophilin (Cyph) probe was used as a control for assessing RNA loading. (B) Blots were made with total RNA (10 μg) isolated from mouse cultured neonatal cardiac myocytes (CM) and nonmyocytes (NM). An 8-kb myocardin A transcript was observed in cardiac myocytes (arrow), but not in nonmyocytes. A cyclophilin (Cyph) probe was used as a control for assessing RNA loading. (C) RT-PCR was performed with cDNAs from embryonic (ED 10.5), neonatal, and adult hearts as templates with common primers for myocardin A and myocardin that sandwich the myocardin A-specific exon. RT was included or omitted from samples as indicated. Expected sizes of PCR products from myocardin A and myocardin are 569 and 425 bp, respectively. The myocardin A transcript but not the myocardin transcript was detected in this experimental condition.
Figure Legend Snippet: Myocardin A expression in murine tissues. (A) Blots were made with mRNA (1 μg) isolated from hearts of embryos at ED 10.5 and tissues of 8-week-old mice. An 8-kb transcript (arrow) hybridized to the myocardin A specific probe in both embryo and adult heart. Weaker signals were observed with mRNA from lung, kidney, and testis in adult. A cyclophilin (Cyph) probe was used as a control for assessing RNA loading. (B) Blots were made with total RNA (10 μg) isolated from mouse cultured neonatal cardiac myocytes (CM) and nonmyocytes (NM). An 8-kb myocardin A transcript was observed in cardiac myocytes (arrow), but not in nonmyocytes. A cyclophilin (Cyph) probe was used as a control for assessing RNA loading. (C) RT-PCR was performed with cDNAs from embryonic (ED 10.5), neonatal, and adult hearts as templates with common primers for myocardin A and myocardin that sandwich the myocardin A-specific exon. RT was included or omitted from samples as indicated. Expected sizes of PCR products from myocardin A and myocardin are 569 and 425 bp, respectively. The myocardin A transcript but not the myocardin transcript was detected in this experimental condition.

Techniques Used: Expressing, Isolation, Mouse Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

28) Product Images from "Down regulating PHGDH affects the lactate production of sertoli cells in varicocele"

Article Title: Down regulating PHGDH affects the lactate production of sertoli cells in varicocele

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/s12958-020-00625-9

Testicular protein PHGDH was down-regulated in varicocele patients and rat varicocele model. a Hematoxylin and eosin stained testicular tissues in 8-weeks experimental varicocele rats, the varicocele rats showed more degeneration of the seminiferous tubules. b The percentages of degenerating seminiferous tubules in the varicocele group were significantly increased compared to the sham group. c The PHGDH mRNA expression was determined by quantitative Real-Time PCR, rat GAPDH was used as an endogenous reference. d The PHGDH protein expression was determined by western blot, rat GAPDH was used as an endogenous reference. N = 5; * P
Figure Legend Snippet: Testicular protein PHGDH was down-regulated in varicocele patients and rat varicocele model. a Hematoxylin and eosin stained testicular tissues in 8-weeks experimental varicocele rats, the varicocele rats showed more degeneration of the seminiferous tubules. b The percentages of degenerating seminiferous tubules in the varicocele group were significantly increased compared to the sham group. c The PHGDH mRNA expression was determined by quantitative Real-Time PCR, rat GAPDH was used as an endogenous reference. d The PHGDH protein expression was determined by western blot, rat GAPDH was used as an endogenous reference. N = 5; * P

Techniques Used: Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot

29) Product Images from "Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells"

Article Title: Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074968

Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p
Figure Legend Snippet: Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p

Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Over-expression of miR-490-3p reduced Nanog expression. (A)The expression of its host gene Chrm2 mRNA levels was determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (B) Cells were transfected with 50 nM miR-490 mimics or 100 nM miR-490 inhibitor for 24 h. qRT-PCR was performed to evaluate the mRNA level of Nanog. (C) The relative expression levels of miR-490-3p after transfection. (D) Cells were co-transfected with miR-490-3p mimics and negative control, renilla luciferase vector pRL-SV40 and Nanog 3′UTR luciferase reporters for 24 h. Both firefly and Renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. (E) Sequence alignment of miR-490-3p with 3′ UTR of Nanog. Bottom: mutations in the 3′UTR of Nanog in order to create the mutant luciferase reporter constructs. *indicates significant difference compared with that of control cells (P
Figure Legend Snippet: Over-expression of miR-490-3p reduced Nanog expression. (A)The expression of its host gene Chrm2 mRNA levels was determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (B) Cells were transfected with 50 nM miR-490 mimics or 100 nM miR-490 inhibitor for 24 h. qRT-PCR was performed to evaluate the mRNA level of Nanog. (C) The relative expression levels of miR-490-3p after transfection. (D) Cells were co-transfected with miR-490-3p mimics and negative control, renilla luciferase vector pRL-SV40 and Nanog 3′UTR luciferase reporters for 24 h. Both firefly and Renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. (E) Sequence alignment of miR-490-3p with 3′ UTR of Nanog. Bottom: mutations in the 3′UTR of Nanog in order to create the mutant luciferase reporter constructs. *indicates significant difference compared with that of control cells (P

Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Quantitative RT-PCR, Negative Control, Luciferase, Plasmid Preparation, Sequencing, Mutagenesis, Construct

30) Product Images from "Polyphenol-Rich Fraction of Brown Alga Ecklonia cava Collected from Gijang, Korea, Reduces Obesity and Glucose Levels in High-Fat Diet-Induced Obese Mice"

Article Title: Polyphenol-Rich Fraction of Brown Alga Ecklonia cava Collected from Gijang, Korea, Reduces Obesity and Glucose Levels in High-Fat Diet-Induced Obese Mice

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/418912

Effect of CA or G-CA extracts on adipogenic gene expression and AMPK phosphorylation. Three weeks after beginning a high-fat diet, C57BL6 mice were orally administered CA or G-CA extract (200 mg/kg body weight) or PBS daily for 8 weeks. (a) PPAR γ 2, C/EBP α , SREBP-1c, and Fas mRNA levels were measured in epididymal fat pads by quantitative real-time PCR. Values are expressed as fold change compared with the NC group. (b) Phospho (P)-AMPK and AMPK protein expression in epididymal fat pads was analyzed by western blot (upper panel) and quantified (lower panel). Values are mean ± SE. NC: untreated, normal chow diet; PBS: PBS-treated, high-fat diet (HFD); CA: CA-treated, HFD; G-CA: G-CA-treated, HFD. * P
Figure Legend Snippet: Effect of CA or G-CA extracts on adipogenic gene expression and AMPK phosphorylation. Three weeks after beginning a high-fat diet, C57BL6 mice were orally administered CA or G-CA extract (200 mg/kg body weight) or PBS daily for 8 weeks. (a) PPAR γ 2, C/EBP α , SREBP-1c, and Fas mRNA levels were measured in epididymal fat pads by quantitative real-time PCR. Values are expressed as fold change compared with the NC group. (b) Phospho (P)-AMPK and AMPK protein expression in epididymal fat pads was analyzed by western blot (upper panel) and quantified (lower panel). Values are mean ± SE. NC: untreated, normal chow diet; PBS: PBS-treated, high-fat diet (HFD); CA: CA-treated, HFD; G-CA: G-CA-treated, HFD. * P

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot

31) Product Images from "Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer"

Article Title: Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer

Journal: Molecular Cancer

doi: 10.1186/1476-4598-13-126

Expression of ACVR1B and SMAD4 in PC cell lines. (A) mRNA expression levels of the ACVR1B and SMAD4 genes in normal pancreatic tissue (RNA from Clontech) and PC cell lines. The expressions were analyzed using real-time RT-PCR. ACVR1B mRNA was scarcely expressed in the Sui65 and Sui68 cell lines, and SMAD4 mRNA was also scarcely expressed in the Sui65, Sui70, and Sui71 cell lines. Rel mRNA, normalized mRNA expression levels ( ACVR1B or SMAD4 / GAPD × 10 6 ); Columns, mean of independent triplicate experiments; Bars, SD. (B) Western blot analysis of ACVR1B and SMAD4 in PC cell lines. ACVR1B was scarcely expressed in the Sui65 and Sui68 cell lines. SMAD4 was scarcely expressed in the Sui65, Sui70, and Sui71 cell lines. The findings confirmed the array-comparative genomic hybridization results. β-actin was used as an internal control.
Figure Legend Snippet: Expression of ACVR1B and SMAD4 in PC cell lines. (A) mRNA expression levels of the ACVR1B and SMAD4 genes in normal pancreatic tissue (RNA from Clontech) and PC cell lines. The expressions were analyzed using real-time RT-PCR. ACVR1B mRNA was scarcely expressed in the Sui65 and Sui68 cell lines, and SMAD4 mRNA was also scarcely expressed in the Sui65, Sui70, and Sui71 cell lines. Rel mRNA, normalized mRNA expression levels ( ACVR1B or SMAD4 / GAPD × 10 6 ); Columns, mean of independent triplicate experiments; Bars, SD. (B) Western blot analysis of ACVR1B and SMAD4 in PC cell lines. ACVR1B was scarcely expressed in the Sui65 and Sui68 cell lines. SMAD4 was scarcely expressed in the Sui65, Sui70, and Sui71 cell lines. The findings confirmed the array-comparative genomic hybridization results. β-actin was used as an internal control.

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

32) Product Images from "Anti-tumor effects of fibroblast growth factor-binding protein (FGF-BP) knockdown in colon carcinoma"

Article Title: Anti-tumor effects of fibroblast growth factor-binding protein (FGF-BP) knockdown in colon carcinoma

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-144

RNAi-mediated knockdown of FGF-BP in LS174T colon carcinoma cells . LS174T cells stably mass-transfected with shRNA expression plasmids and clonally selected show reduced FGF-BP expression as determined on mRNA (qRT-PCR, A) and protein level (Western blot, B).
Figure Legend Snippet: RNAi-mediated knockdown of FGF-BP in LS174T colon carcinoma cells . LS174T cells stably mass-transfected with shRNA expression plasmids and clonally selected show reduced FGF-BP expression as determined on mRNA (qRT-PCR, A) and protein level (Western blot, B).

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

RNAi-mediated knockdown of FGF-BP in LS174T colon carcinoma cells . LS174T cells stably mass-transfected with shRNA expression plasmids and clonally selected show reduced FGF-BP expression as determined on mRNA (qRT-PCR, A) and protein level (Western blot, B).
Figure Legend Snippet: RNAi-mediated knockdown of FGF-BP in LS174T colon carcinoma cells . LS174T cells stably mass-transfected with shRNA expression plasmids and clonally selected show reduced FGF-BP expression as determined on mRNA (qRT-PCR, A) and protein level (Western blot, B).

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

33) Product Images from "Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression"

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0969-3

circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
Figure Legend Snippet: circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Staining

34) Product Images from "The miR-590/Acvr2a/Terf1 Axis Regulates Telomere Elongation and Pluripotency of Mouse iPSCs"

Article Title: The miR-590/Acvr2a/Terf1 Axis Regulates Telomere Elongation and Pluripotency of Mouse iPSCs

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2018.05.008

Inhibition of Acvr2a Improves Telomere Re-elongation to Promote Pluripotency in Pre-iPSCs (A) MiR-590-3p or miR-590-5p downregulated the mRNA level of Acvr2a detected by qRT-PCR. Data shown are the mean ± SD (n = 3). (B) Detecting the efficiency of Acvr2a knockdown in pre-iPSCs. Data shown are the mean ± SD (n = 3). (C) Downregulation of Acvr2a promoted telomere elongation. Data shown are the mean ± SD (n = 3). (D) FISH staining of telomeres and histogram statistics showed the promotion of telomere elongation by downregulating Acvr2a . Scale bar indicates 10 μm. (E) Stemness markers were upregulated in Acvr2a knockdown pre-iPSCs. Data shown are the mean ± SD (n = 3). (F) Acvr2a knockdown increased the pluripotency of pre-iPSCs. Data shown are the mean ± SD (n = 3). For all data, ∗ p
Figure Legend Snippet: Inhibition of Acvr2a Improves Telomere Re-elongation to Promote Pluripotency in Pre-iPSCs (A) MiR-590-3p or miR-590-5p downregulated the mRNA level of Acvr2a detected by qRT-PCR. Data shown are the mean ± SD (n = 3). (B) Detecting the efficiency of Acvr2a knockdown in pre-iPSCs. Data shown are the mean ± SD (n = 3). (C) Downregulation of Acvr2a promoted telomere elongation. Data shown are the mean ± SD (n = 3). (D) FISH staining of telomeres and histogram statistics showed the promotion of telomere elongation by downregulating Acvr2a . Scale bar indicates 10 μm. (E) Stemness markers were upregulated in Acvr2a knockdown pre-iPSCs. Data shown are the mean ± SD (n = 3). (F) Acvr2a knockdown increased the pluripotency of pre-iPSCs. Data shown are the mean ± SD (n = 3). For all data, ∗ p

Techniques Used: Inhibition, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Staining

35) Product Images from "Involvement of RBP4 in Diabetic Atherosclerosis and the Role of Vitamin D Intervention"

Article Title: Involvement of RBP4 in Diabetic Atherosclerosis and the Role of Vitamin D Intervention

Journal: Journal of Diabetes Research

doi: 10.1155/2018/7329861

Comparison of adipose tissue RBP4 mRNA.
Figure Legend Snippet: Comparison of adipose tissue RBP4 mRNA.

Techniques Used:

36) Product Images from "Circular RNA circTNPO3 Regulates Paclitaxel Resistance of Ovarian Cancer Cells by miR-1299/NEK2 Signaling Pathway"

Article Title: Circular RNA circTNPO3 Regulates Paclitaxel Resistance of Ovarian Cancer Cells by miR-1299/NEK2 Signaling Pathway

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2020.06.002

Confirmation of Subcellular Localization of circTNPO3 (A) qRT-PCR for the abundance of circTNPO3 and TNPO3 in SKOV3/PTX cells treated with Actinomycin D at the indicated time point. (B) qRT-PCR for the abundance of circTNPO3 and TNPO3 in HeyA-8/PTX cells treated with Actinomycin D at the indicated time point. (C) qRT-PCR for the expression of circTNPO3 and TNPO3 mRNA in SKOV3/PTX cells treated with or without RNase R. (D) qRT-PCR for the expression of circTNPO3 and TNPO3 mRNA in HeyA-8/PTX cells treated with or without RNase R. (E) Levels of circTNPO3 in the nuclear and cytoplasmic fractions of SKOV3/PTX cells. (F) Levels of circTNPO3 in the nuclear and cytoplasmic fractions of HeyA-8/PTX. All tests were performed at least three times. Data were expressed as mean ± SD. ∗∗p
Figure Legend Snippet: Confirmation of Subcellular Localization of circTNPO3 (A) qRT-PCR for the abundance of circTNPO3 and TNPO3 in SKOV3/PTX cells treated with Actinomycin D at the indicated time point. (B) qRT-PCR for the abundance of circTNPO3 and TNPO3 in HeyA-8/PTX cells treated with Actinomycin D at the indicated time point. (C) qRT-PCR for the expression of circTNPO3 and TNPO3 mRNA in SKOV3/PTX cells treated with or without RNase R. (D) qRT-PCR for the expression of circTNPO3 and TNPO3 mRNA in HeyA-8/PTX cells treated with or without RNase R. (E) Levels of circTNPO3 in the nuclear and cytoplasmic fractions of SKOV3/PTX cells. (F) Levels of circTNPO3 in the nuclear and cytoplasmic fractions of HeyA-8/PTX. All tests were performed at least three times. Data were expressed as mean ± SD. ∗∗p

Techniques Used: Quantitative RT-PCR, Expressing

circTNPO3 Positively Regulated NEK2 by Interacting with miR-1299 in PTX-Resistant Ovarian Cancer Cells (A) Venn diagram showing five genes that are putative miR-1299 targets computationally predicted by four algorithms (miRanda, RNAhybrid, miRWalk, and TargetScan). (B) mRNA levels of five candidate target genes were detected after silencing circTNPO3. (C) miR-1299 could significantly enrich the 3′ UTR of NEK2 mRNA. (D) The binding sequence between miR-1299 and NEK2. (E) Luciferase reporter assay demonstrated miR-1299 mimics significantly decreased the luciferase activity of NEK2-WT in OC cells. (F) The real-time PCR analysis demonstrated that the NEK2 was higher in PTX-resistant OC tissues. (G) The expression of NEK2 was obviously increased in PTX-resistant OC cell lines than that in their parental OC cell lines. (H) Inhibition of circTNPO3 mediated decrease of NEK2 mRNA expression was significantly recuperated following miR-1299 inhibitors. (I) Inhibition of circTNPO3-mediated decrease of NEK2 protein expression was significantly recuperated following miR-1299 inhibitors. All tests were performed at least three times. Data were expressed as mean ± SD. ∗∗p
Figure Legend Snippet: circTNPO3 Positively Regulated NEK2 by Interacting with miR-1299 in PTX-Resistant Ovarian Cancer Cells (A) Venn diagram showing five genes that are putative miR-1299 targets computationally predicted by four algorithms (miRanda, RNAhybrid, miRWalk, and TargetScan). (B) mRNA levels of five candidate target genes were detected after silencing circTNPO3. (C) miR-1299 could significantly enrich the 3′ UTR of NEK2 mRNA. (D) The binding sequence between miR-1299 and NEK2. (E) Luciferase reporter assay demonstrated miR-1299 mimics significantly decreased the luciferase activity of NEK2-WT in OC cells. (F) The real-time PCR analysis demonstrated that the NEK2 was higher in PTX-resistant OC tissues. (G) The expression of NEK2 was obviously increased in PTX-resistant OC cell lines than that in their parental OC cell lines. (H) Inhibition of circTNPO3 mediated decrease of NEK2 mRNA expression was significantly recuperated following miR-1299 inhibitors. (I) Inhibition of circTNPO3-mediated decrease of NEK2 protein expression was significantly recuperated following miR-1299 inhibitors. All tests were performed at least three times. Data were expressed as mean ± SD. ∗∗p

Techniques Used: Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Inhibition

37) Product Images from "Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion"

Article Title: Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.20-38

The effect of CLPr administration on the expression level of clock genes in the liver after pretreatment with GLP-1 receptor antagonist. Mice were orally administered CLPr at 150 mg/kg body weight or water (5.0 ml/kg body weight) at ZT3. Exendin (9-39), a GLP-1 receptor antagonist, was pre-injected to the mice at 200 nmol/kg body weight 5 min before the CLPr administration. The liver was collected 3 h after the CLPr administration and the expression of Per1 (A), Dbp (B) and Bmal1 (C) was measured by RT-PCR. After the expression level of mRNA was normalized by that of gapdh , the relative expression level was shown. Data are represented as the means ± SE ( n = 5). Different letters indicate significant differences ( p
Figure Legend Snippet: The effect of CLPr administration on the expression level of clock genes in the liver after pretreatment with GLP-1 receptor antagonist. Mice were orally administered CLPr at 150 mg/kg body weight or water (5.0 ml/kg body weight) at ZT3. Exendin (9-39), a GLP-1 receptor antagonist, was pre-injected to the mice at 200 nmol/kg body weight 5 min before the CLPr administration. The liver was collected 3 h after the CLPr administration and the expression of Per1 (A), Dbp (B) and Bmal1 (C) was measured by RT-PCR. After the expression level of mRNA was normalized by that of gapdh , the relative expression level was shown. Data are represented as the means ± SE ( n = 5). Different letters indicate significant differences ( p

Techniques Used: Expressing, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction

38) Product Images from "Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion"

Article Title: Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.20-38

The effect of CLPr administration on the expression level of clock genes in the liver after pretreatment with GLP-1 receptor antagonist. Mice were orally administered CLPr at 150 mg/kg body weight or water (5.0 ml/kg body weight) at ZT3. Exendin (9-39), a GLP-1 receptor antagonist, was pre-injected to the mice at 200 nmol/kg body weight 5 min before the CLPr administration. The liver was collected 3 h after the CLPr administration and the expression of Per1 (A), Dbp (B) and Bmal1 (C) was measured by RT-PCR. After the expression level of mRNA was normalized by that of gapdh , the relative expression level was shown. Data are represented as the means ± SE ( n = 5). Different letters indicate significant differences ( p
Figure Legend Snippet: The effect of CLPr administration on the expression level of clock genes in the liver after pretreatment with GLP-1 receptor antagonist. Mice were orally administered CLPr at 150 mg/kg body weight or water (5.0 ml/kg body weight) at ZT3. Exendin (9-39), a GLP-1 receptor antagonist, was pre-injected to the mice at 200 nmol/kg body weight 5 min before the CLPr administration. The liver was collected 3 h after the CLPr administration and the expression of Per1 (A), Dbp (B) and Bmal1 (C) was measured by RT-PCR. After the expression level of mRNA was normalized by that of gapdh , the relative expression level was shown. Data are represented as the means ± SE ( n = 5). Different letters indicate significant differences ( p

Techniques Used: Expressing, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction

39) Product Images from "Protective Effect of Hainosankyuto, a Traditional Japanese Medicine, on Streptococcus pyogenes Infection in Murine Model"

Article Title: Protective Effect of Hainosankyuto, a Traditional Japanese Medicine, on Streptococcus pyogenes Infection in Murine Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022188

Levels of cytokine mRNA in mouse peritoneal macrophage with Hainosankyuto treatment. Three-week-old ICR mice were force-fed Hainosankyuto for 1 day. Mouse peritoneal macrophage was prepared as a single-cell suspension. Then total RNA was extracted and IL-12, IFN-γ, and TNF-α expression was detected using northern blotting analysis ( Figure 8A ) and quantitative real time RT-PCR ( Figure 8B ), with HPRT as the internal control.
Figure Legend Snippet: Levels of cytokine mRNA in mouse peritoneal macrophage with Hainosankyuto treatment. Three-week-old ICR mice were force-fed Hainosankyuto for 1 day. Mouse peritoneal macrophage was prepared as a single-cell suspension. Then total RNA was extracted and IL-12, IFN-γ, and TNF-α expression was detected using northern blotting analysis ( Figure 8A ) and quantitative real time RT-PCR ( Figure 8B ), with HPRT as the internal control.

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

40) Product Images from "ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action"

Article Title: ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action

Journal: Mediators of Inflammation

doi: 10.1155/2015/967053

The effects of luteolin on iNOS, COX-2, and TNF- α gene expression and transcriptional regulation in LPS-treated RAW264.7 cells. ((a) and (b)) RAW264.7 cells (5 × 10 6 cells/mL) were incubated with LPS (1 μ g/mL) in the presence or absence of luteolin for 6 h. iNOS, COX-2, and TNF- α mRNA levels were determined using RT-PCR (a) and real-time PCR (b). (c) RAW264.7 cells (5 × 10 6 cells/mL) were incubated with LPS (1 μ g/mL) in the presence or absence of luteolin for the indicated times. After preparing nuclear fractions, the levels of total translocated transcription factors (p65, p50, c-Fos, and c-Jun) were determined by immunoblotting analysis. (d) HEK293 cells cotransfected with NF- κ B-Luc (1 μ g/mL) and β -gal (as a transfection control) plasmid constructs were treated with luteolin in the presence or absence of adaptor molecule (MyD88) for 12 h. Luciferase activity was determined via luminometry. All data are expressed as the mean ± SD of experiments. ∗ p
Figure Legend Snippet: The effects of luteolin on iNOS, COX-2, and TNF- α gene expression and transcriptional regulation in LPS-treated RAW264.7 cells. ((a) and (b)) RAW264.7 cells (5 × 10 6 cells/mL) were incubated with LPS (1 μ g/mL) in the presence or absence of luteolin for 6 h. iNOS, COX-2, and TNF- α mRNA levels were determined using RT-PCR (a) and real-time PCR (b). (c) RAW264.7 cells (5 × 10 6 cells/mL) were incubated with LPS (1 μ g/mL) in the presence or absence of luteolin for the indicated times. After preparing nuclear fractions, the levels of total translocated transcription factors (p65, p50, c-Fos, and c-Jun) were determined by immunoblotting analysis. (d) HEK293 cells cotransfected with NF- κ B-Luc (1 μ g/mL) and β -gal (as a transfection control) plasmid constructs were treated with luteolin in the presence or absence of adaptor molecule (MyD88) for 12 h. Luciferase activity was determined via luminometry. All data are expressed as the mean ± SD of experiments. ∗ p

Techniques Used: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay

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Amplification:

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Ligation:

Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
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Synthesized:

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Article Snippet: .. For circRNA and mRNA, cDNA was synthesized by using reverse transcription kit (Takara, Otsu, Japan) and for miRNA, total RNAs were reversed using RiboBio reverse transcription kit (Guangzhou, China). .. Quantification of mRNA and circular RNA was performed by using a SYBR Green PCR Kit (Takara, Otsu, Japan), and miRNA PCR was performed by using a SYBR Green PCR Kit (RiboBio,Guangzhou, China).

Real-time Polymerase Chain Reaction:

Article Title: A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice
Article Snippet: .. The relative abundance of each mRNA in the sample was determined using RQ-PCR with the corresponding primers ( ) and the SYBR® Premix Ex Taq™II (TaKaRa, Dalian, China) on an iQTM Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA). .. Cycle threshold values were obtained from the Bio-Rad iQ5 2.0 Standard Edition Optical System Software (Bio-Rad, Hercules, CA).

Sequencing:

Article Title: Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus
Article Snippet: .. Plasmid and mRNA The initial xNorrin cDNA clone was first amplified from a X. laevis cDNA library using Pyrobest DNA polymerase (TaKaRa) and PCR primers partially based on a predicted X. tropicalis Norrin gene sequence. ( http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Xentr4 & id=158316 ): xNorrin Up: 5′-AGACGAATTCACCTGAGAGGAAGACTGGG-3′ , xNorrin Down: 5′-AGACCTCGAGAGCAACGCAAGCGAATGG-3′ . .. The cDNA for the coding region was amplified using xNorrin Up: 5′-AATCGGATCCATGGGAAATCGTGTCCTTC-3′ and xNorrin Down: 5′-ATATCTCGAGCTATGAATTGCACTCTTC-3′ .

Polymerase Chain Reaction:

Article Title: Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus
Article Snippet: .. Plasmid and mRNA The initial xNorrin cDNA clone was first amplified from a X. laevis cDNA library using Pyrobest DNA polymerase (TaKaRa) and PCR primers partially based on a predicted X. tropicalis Norrin gene sequence. ( http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Xentr4 & id=158316 ): xNorrin Up: 5′-AGACGAATTCACCTGAGAGGAAGACTGGG-3′ , xNorrin Down: 5′-AGACCTCGAGAGCAACGCAAGCGAATGG-3′ . .. The cDNA for the coding region was amplified using xNorrin Up: 5′-AATCGGATCCATGGGAAATCGTGTCCTTC-3′ and xNorrin Down: 5′-ATATCTCGAGCTATGAATTGCACTCTTC-3′ .

cDNA Library Assay:

Article Title: Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus
Article Snippet: .. Plasmid and mRNA The initial xNorrin cDNA clone was first amplified from a X. laevis cDNA library using Pyrobest DNA polymerase (TaKaRa) and PCR primers partially based on a predicted X. tropicalis Norrin gene sequence. ( http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Xentr4 & id=158316 ): xNorrin Up: 5′-AGACGAATTCACCTGAGAGGAAGACTGGG-3′ , xNorrin Down: 5′-AGACCTCGAGAGCAACGCAAGCGAATGG-3′ . .. The cDNA for the coding region was amplified using xNorrin Up: 5′-AATCGGATCCATGGGAAATCGTGTCCTTC-3′ and xNorrin Down: 5′-ATATCTCGAGCTATGAATTGCACTCTTC-3′ .

Reverse Transcription Polymerase Chain Reaction:

Article Title: MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients
Article Snippet: .. RNA was reverse-transcribed using a PrimerScript™ RT Kit (TaKaRa, Dalian, China) for detection of mRNA and using an SYBR® PrimeScript™ miRNA RT-PCR Kit (TaKaRa, Dalian, China) for detection of miRNA. .. Quantitative real-time PCR for mRNA detection was performed on a Mx3005P thermal cycler (Stratagene, Santa Clara, CA, USA) using an SYBR® Premix Ex Taq™ Real-Time PCR Kit (TaKaRa, Dalian, China).

Article Title: miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6
Article Snippet: .. Quantitative reverse transcription-polymerase chain reaction analyses for the miRNA and mRNA were performed using the One Step SYBRPrimeScript RT–PCR Kit II according to the manufacturer's instructions (Takara, Ohtsu, Japan). ..

Plasmid Preparation:

Article Title: Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-? Antagonist, Controls Early Neuroectoderm Specification in Xenopus
Article Snippet: .. Plasmid and mRNA The initial xNorrin cDNA clone was first amplified from a X. laevis cDNA library using Pyrobest DNA polymerase (TaKaRa) and PCR primers partially based on a predicted X. tropicalis Norrin gene sequence. ( http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Xentr4 & id=158316 ): xNorrin Up: 5′-AGACGAATTCACCTGAGAGGAAGACTGGG-3′ , xNorrin Down: 5′-AGACCTCGAGAGCAACGCAAGCGAATGG-3′ . .. The cDNA for the coding region was amplified using xNorrin Up: 5′-AATCGGATCCATGGGAAATCGTGTCCTTC-3′ and xNorrin Down: 5′-ATATCTCGAGCTATGAATTGCACTCTTC-3′ .

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  • 91
    TaKaRa luciferase mrna
    (A) Stable overexpression of <t>CSF-1R</t> <t>mRNA</t> terminal 578 nt 3′UTR wild type sequence (Wt) in BT20 breast cancer cells increases CSF-1R protein level by 4.7-fold compared to that of stable overexpression of CSF-1R mRNA 3′UTR lacking the pyrimidine-rich sequence (3′UTRΔ69). (B-D) Excess CSF-1R mRNA 3′UTR wild-type, but not in the absence of the 69 nt pyrimidine-rich sequence (3′UTRΔ69), regulates in vitro adhesion, motility, and invasiveness of BT20 breast cancer cells . In vitro (B) adhesion, (C) fibronectin-directed motility, and (D) invasion of BT20 breast cancer clones through a human extracellular matrix are shown. (E) Invasion of BT20 cells with excess 3′UTR CSF-1R RNA is ~50% blocked by an anti-human CSF-1R antibody.
    Luciferase Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa mitochondrial copy numbers mrna
    NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of <t>mRNA</t> and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using <t>RT-PCR</t> ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P
    Mitochondrial Copy Numbers Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa cse mrna
    Effects of the miR-30 family member mimics and miR-30 family inhibitor (FI) on <t>CSE</t> expression. (A) CSE protein and (B) <t>mRNA</t> expression levels in primary neonatal rat myocardial cells after transfecting with the miR-30 family member mimics for 48 h ( n =5). (C) The design of locked nucleic acid (LNA)-miR-30 FI. The miR-30 family FI had a 10-mer LNA-anti-miR that was complementary to the seed region of each miR-30 family member. (D) CSE protein and (E) mRNA levels after transfecting with the LNA-miR-30 FI for 48 h ( n =6). (F) The change in the hydrogen sulfide (H 2 S) concentration in the supernatant after transfecting miR-30 family mimics (50 n M ) or the LNA-miR-30 FI (12.5 n M ) for 48 h ( n =5). (G) Representative immunofluorescent images show the CSE expression levels in primary cardiomyocytes. Green: CSE protein that was stained with fluorescein isothiocyanate, Blue: Cell nucleus stained with DAPI. (H) Quantitative analysis of the mean fluorescence intensity in each group ( n =6). The data are expressed as mean±SEM. * p
    Cse Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa mrna sequence
    Effect of recombinant vector for <t>Nanog</t> <t>mRNA</t> in SGC-7901 cells as shown by semi-quantitative RT-PCR. From left to right: 1,8, DNA Marker (DL 2000); 2, transfected with pshRNA-NanogA; 3, transfected with pshRNA-NanogB; 4, transfected with pshRNA-NanogC; 5, transfected with pshRNA-negative control; 6, transfected with pGenesil-1; 7, SGC-7901.
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    (A) Stable overexpression of CSF-1R mRNA terminal 578 nt 3′UTR wild type sequence (Wt) in BT20 breast cancer cells increases CSF-1R protein level by 4.7-fold compared to that of stable overexpression of CSF-1R mRNA 3′UTR lacking the pyrimidine-rich sequence (3′UTRΔ69). (B-D) Excess CSF-1R mRNA 3′UTR wild-type, but not in the absence of the 69 nt pyrimidine-rich sequence (3′UTRΔ69), regulates in vitro adhesion, motility, and invasiveness of BT20 breast cancer cells . In vitro (B) adhesion, (C) fibronectin-directed motility, and (D) invasion of BT20 breast cancer clones through a human extracellular matrix are shown. (E) Invasion of BT20 cells with excess 3′UTR CSF-1R RNA is ~50% blocked by an anti-human CSF-1R antibody.

    Journal: Translational Oncology

    Article Title: Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3′UTR element in CSF-1R mRNA

    doi: 10.1016/j.tranon.2018.09.012

    Figure Lengend Snippet: (A) Stable overexpression of CSF-1R mRNA terminal 578 nt 3′UTR wild type sequence (Wt) in BT20 breast cancer cells increases CSF-1R protein level by 4.7-fold compared to that of stable overexpression of CSF-1R mRNA 3′UTR lacking the pyrimidine-rich sequence (3′UTRΔ69). (B-D) Excess CSF-1R mRNA 3′UTR wild-type, but not in the absence of the 69 nt pyrimidine-rich sequence (3′UTRΔ69), regulates in vitro adhesion, motility, and invasiveness of BT20 breast cancer cells . In vitro (B) adhesion, (C) fibronectin-directed motility, and (D) invasion of BT20 breast cancer clones through a human extracellular matrix are shown. (E) Invasion of BT20 cells with excess 3′UTR CSF-1R RNA is ~50% blocked by an anti-human CSF-1R antibody.

    Article Snippet: For the cloning of the tetracycline-inducible system of the luciferase-CSF-1R mRNA UTRs, CSF-1R mRNA 5′ and 3′UTRs were ligated to both ends of the luciferase mRNA and cloned into the Tet-ON 3G inducible expression plasmid (Clontech).

    Techniques: Over Expression, Sequencing, In Vitro, Clone Assay

    (A) UV crosslinking of the pyrimidine-rich 69 nt from CSF-1R mRNA 3′UTR using recombinant vigilin. 26-Py is 26 nt pyrimidine-rich sequence only. In Py-Mut, pyrimidines are replaced by purines. Same membrane is reprobed with vigilin antibody (lower panel). (B) Competition assay between pyrimidine-rich 69 nt (Biotin-UTP labeled) and 26 nt (unlabeled) from CSF-1R mRNA 3′UTR for vigilin binding (recombinant vigilin protein) determined by UV crosslinking assay. (C) Expression of 69 nt-wild type sequence from CSF-1R mRNA 3′UTR (Py-Wt) results in increase of CSF-1R protein by 5-fold. (D) Expression of 69 nt-wild type sequence CSF-1R mRNA 3′UTR (Py-Wt) results in increase of CSF-1R mRNA by 22% compared to that in Py-Mut in BT20 breast cancer cells.

    Journal: Translational Oncology

    Article Title: Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3′UTR element in CSF-1R mRNA

    doi: 10.1016/j.tranon.2018.09.012

    Figure Lengend Snippet: (A) UV crosslinking of the pyrimidine-rich 69 nt from CSF-1R mRNA 3′UTR using recombinant vigilin. 26-Py is 26 nt pyrimidine-rich sequence only. In Py-Mut, pyrimidines are replaced by purines. Same membrane is reprobed with vigilin antibody (lower panel). (B) Competition assay between pyrimidine-rich 69 nt (Biotin-UTP labeled) and 26 nt (unlabeled) from CSF-1R mRNA 3′UTR for vigilin binding (recombinant vigilin protein) determined by UV crosslinking assay. (C) Expression of 69 nt-wild type sequence from CSF-1R mRNA 3′UTR (Py-Wt) results in increase of CSF-1R protein by 5-fold. (D) Expression of 69 nt-wild type sequence CSF-1R mRNA 3′UTR (Py-Wt) results in increase of CSF-1R mRNA by 22% compared to that in Py-Mut in BT20 breast cancer cells.

    Article Snippet: For the cloning of the tetracycline-inducible system of the luciferase-CSF-1R mRNA UTRs, CSF-1R mRNA 5′ and 3′UTRs were ligated to both ends of the luciferase mRNA and cloned into the Tet-ON 3G inducible expression plasmid (Clontech).

    Techniques: Recombinant, Sequencing, Competitive Binding Assay, Labeling, Binding Assay, Expressing

    (A) Luciferase gene fused with CSF-1R mRNA 5′ and 3′UTRs under the control of tetracycline-inducible pTre3G promoter. Wt = wild type 3′UTR construct. Py-Mut = pyrimidine rich sequence mutated 3′UTR construct. (B) Linear increase of luciferase activity after doxycycline-induction in SKOV3 cells with chromosomally integrated luciferase constructs. (C) Translation rate ( Kv = L/t ) is plotted in both Wt and Py-Mut constructs. (D) Relative distribution of luciferase mRNA in wild type (Wt) and mutant (Py-Mut) constructs in ribosome profile. Mutation of pyrimidine-rich sequence in CSF-1R mRNA 3′UTR affects the distribution of luciferase mRNA in ribosome profile, which was prepared by the sucrose density gradient ultracentrifugation. Fractions: 1–2, unbound RNPs (Unb.); 3–4, monosomes (Mono.); 5–6, low MW polysomes; 7–10, high MW polysomes. (E) Relative distribution of GAPDH mRNA in ribosome profile. (F,H) Overexpression of vigilin (straight line) decreases luciferase activity and translation rate of the wild type luciferase construct compared to the empty vector (dashed line). (G,I) Overexpression of vigilin does not significantly influence the luciferase activity and translation rate of the Py-Mut construct.

    Journal: Translational Oncology

    Article Title: Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3′UTR element in CSF-1R mRNA

    doi: 10.1016/j.tranon.2018.09.012

    Figure Lengend Snippet: (A) Luciferase gene fused with CSF-1R mRNA 5′ and 3′UTRs under the control of tetracycline-inducible pTre3G promoter. Wt = wild type 3′UTR construct. Py-Mut = pyrimidine rich sequence mutated 3′UTR construct. (B) Linear increase of luciferase activity after doxycycline-induction in SKOV3 cells with chromosomally integrated luciferase constructs. (C) Translation rate ( Kv = L/t ) is plotted in both Wt and Py-Mut constructs. (D) Relative distribution of luciferase mRNA in wild type (Wt) and mutant (Py-Mut) constructs in ribosome profile. Mutation of pyrimidine-rich sequence in CSF-1R mRNA 3′UTR affects the distribution of luciferase mRNA in ribosome profile, which was prepared by the sucrose density gradient ultracentrifugation. Fractions: 1–2, unbound RNPs (Unb.); 3–4, monosomes (Mono.); 5–6, low MW polysomes; 7–10, high MW polysomes. (E) Relative distribution of GAPDH mRNA in ribosome profile. (F,H) Overexpression of vigilin (straight line) decreases luciferase activity and translation rate of the wild type luciferase construct compared to the empty vector (dashed line). (G,I) Overexpression of vigilin does not significantly influence the luciferase activity and translation rate of the Py-Mut construct.

    Article Snippet: For the cloning of the tetracycline-inducible system of the luciferase-CSF-1R mRNA UTRs, CSF-1R mRNA 5′ and 3′UTRs were ligated to both ends of the luciferase mRNA and cloned into the Tet-ON 3G inducible expression plasmid (Clontech).

    Techniques: Luciferase, Construct, Sequencing, Activity Assay, Mutagenesis, Over Expression, Plasmid Preparation

    NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P

    Journal: Arthritis Research & Therapy

    Article Title: Increased neutrophil extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still’s disease

    doi: 10.1186/s13075-018-1800-z

    Figure Lengend Snippet: NET DNA activated macrophages are pivotal to the proinflammatory response in AOSD. a THP-1-derived macrophages were cultured with purified neutrophil extracellular trap (NET) DNA. After 4 h the cells were stained for cell surface markers using specific monoclonal antibodies or the corresponding isotype controls (right panels). Cells were analyzed using flow cytometry. The percentages of proinflammatory macrophages (CD68 + CD86 + ) are indicated (left). The levels of mRNA and protein of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in these THP-1-derived macrophages were measured using RT-PCR ( b ) and ELISA analysis ( c ). The histograms show the means ± SD. * P

    Article Snippet: Real-time PCR for mitochondrial copy numbers mRNA was isolated and cDNA was prepared (PrimeScript RT Master Mix transcription kit, TaKaRa, Tokyo, Japan).

    Techniques: Derivative Assay, Cell Culture, Purification, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effects of the miR-30 family member mimics and miR-30 family inhibitor (FI) on CSE expression. (A) CSE protein and (B) mRNA expression levels in primary neonatal rat myocardial cells after transfecting with the miR-30 family member mimics for 48 h ( n =5). (C) The design of locked nucleic acid (LNA)-miR-30 FI. The miR-30 family FI had a 10-mer LNA-anti-miR that was complementary to the seed region of each miR-30 family member. (D) CSE protein and (E) mRNA levels after transfecting with the LNA-miR-30 FI for 48 h ( n =6). (F) The change in the hydrogen sulfide (H 2 S) concentration in the supernatant after transfecting miR-30 family mimics (50 n M ) or the LNA-miR-30 FI (12.5 n M ) for 48 h ( n =5). (G) Representative immunofluorescent images show the CSE expression levels in primary cardiomyocytes. Green: CSE protein that was stained with fluorescein isothiocyanate, Blue: Cell nucleus stained with DAPI. (H) Quantitative analysis of the mean fluorescence intensity in each group ( n =6). The data are expressed as mean±SEM. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: miRNA-30 Family Inhibition Protects Against Cardiac Ischemic Injury by Regulating Cystathionine-γ-Lyase Expression

    doi: 10.1089/ars.2014.5909

    Figure Lengend Snippet: Effects of the miR-30 family member mimics and miR-30 family inhibitor (FI) on CSE expression. (A) CSE protein and (B) mRNA expression levels in primary neonatal rat myocardial cells after transfecting with the miR-30 family member mimics for 48 h ( n =5). (C) The design of locked nucleic acid (LNA)-miR-30 FI. The miR-30 family FI had a 10-mer LNA-anti-miR that was complementary to the seed region of each miR-30 family member. (D) CSE protein and (E) mRNA levels after transfecting with the LNA-miR-30 FI for 48 h ( n =6). (F) The change in the hydrogen sulfide (H 2 S) concentration in the supernatant after transfecting miR-30 family mimics (50 n M ) or the LNA-miR-30 FI (12.5 n M ) for 48 h ( n =5). (G) Representative immunofluorescent images show the CSE expression levels in primary cardiomyocytes. Green: CSE protein that was stained with fluorescein isothiocyanate, Blue: Cell nucleus stained with DAPI. (H) Quantitative analysis of the mean fluorescence intensity in each group ( n =6). The data are expressed as mean±SEM. * p

    Article Snippet: For the quantitative detection of CSE mRNA, the SYBR Green I (Takara, Dalian, China) incorporation method was used with GAPDH as an internal control.

    Techniques: Expressing, Concentration Assay, Staining, Fluorescence

    miR-30b overexpression aggravates MI injury in C57BL/6 mice. (A) miR-30b levels, (B) CSE mRNA and (C) protein levels in the left ventricle of C57BL/6 mice 8 days after injecting LV-miR-30b ( n =3). (D) Representative heart images that were stained by TTC and Evans blue. (E) Infarct size that is expressed as a ratio of the infarct area and area at risk at 2 days after MI surgery ( n =6). (F–H) LDH, CK, and cTn-I release in plasma of different MI groups treated with LV-miR-30b individually or plus SPRC and PAG ( n =6). The data are expressed as mean±SEM. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: miRNA-30 Family Inhibition Protects Against Cardiac Ischemic Injury by Regulating Cystathionine-γ-Lyase Expression

    doi: 10.1089/ars.2014.5909

    Figure Lengend Snippet: miR-30b overexpression aggravates MI injury in C57BL/6 mice. (A) miR-30b levels, (B) CSE mRNA and (C) protein levels in the left ventricle of C57BL/6 mice 8 days after injecting LV-miR-30b ( n =3). (D) Representative heart images that were stained by TTC and Evans blue. (E) Infarct size that is expressed as a ratio of the infarct area and area at risk at 2 days after MI surgery ( n =6). (F–H) LDH, CK, and cTn-I release in plasma of different MI groups treated with LV-miR-30b individually or plus SPRC and PAG ( n =6). The data are expressed as mean±SEM. * p

    Article Snippet: For the quantitative detection of CSE mRNA, the SYBR Green I (Takara, Dalian, China) incorporation method was used with GAPDH as an internal control.

    Techniques: Over Expression, Mouse Assay, Staining

    CSE is a direct target of miR-30 family. (A) Sequence alignment between the miR-30 family members and the 3′-UTRs of CSE of human, rat, and mouse. The sequences in CSE genes complementary to the miR-30 family seed regions are shown in red . (B) CSE protein and (C) mRNA expression levels in the border and noninfarct zones of all MI rats compared with sham-operated rats at 48 h after MI induction ( n =6). (D, E) Real-time polymerase chain reaction shows the aberrant expression of the miR-30 family in different regions of the rat MI model (D) and C57BL/6 mouse MI model (E) compared with sham-operated heart ( n =6). (F) Dual-luciferase reporter activities show the interaction between miR-30 family members and CSE 3′-UTRs ( n =6). (G) CSE protein, (H) CSE mRNA, and (I) miR-30 family expression levels at 8 h after hypoxia in hypoxic cardiomyocyte model compared with normoxia cell ( n =5–6). The data are expressed as mean±standard error of the mean (SEM). * p

    Journal: Antioxidants & Redox Signaling

    Article Title: miRNA-30 Family Inhibition Protects Against Cardiac Ischemic Injury by Regulating Cystathionine-γ-Lyase Expression

    doi: 10.1089/ars.2014.5909

    Figure Lengend Snippet: CSE is a direct target of miR-30 family. (A) Sequence alignment between the miR-30 family members and the 3′-UTRs of CSE of human, rat, and mouse. The sequences in CSE genes complementary to the miR-30 family seed regions are shown in red . (B) CSE protein and (C) mRNA expression levels in the border and noninfarct zones of all MI rats compared with sham-operated rats at 48 h after MI induction ( n =6). (D, E) Real-time polymerase chain reaction shows the aberrant expression of the miR-30 family in different regions of the rat MI model (D) and C57BL/6 mouse MI model (E) compared with sham-operated heart ( n =6). (F) Dual-luciferase reporter activities show the interaction between miR-30 family members and CSE 3′-UTRs ( n =6). (G) CSE protein, (H) CSE mRNA, and (I) miR-30 family expression levels at 8 h after hypoxia in hypoxic cardiomyocyte model compared with normoxia cell ( n =5–6). The data are expressed as mean±standard error of the mean (SEM). * p

    Article Snippet: For the quantitative detection of CSE mRNA, the SYBR Green I (Takara, Dalian, China) incorporation method was used with GAPDH as an internal control.

    Techniques: Sequencing, Expressing, Real-time Polymerase Chain Reaction, Luciferase

    LNA-miR-30 FI protects the heart against MI injury in C57BL/6 mice. (A) The expression levels of miR-30 family members in C57BL/6 mouse heart after injecting 5 mg/kg of LNA-miR-30 FI for 3 consecutive days ( n =3). (B) CSE mRNA and (C) protein expression levels in the C57BL/6 left ventricle after injecting different concentrations of LNA-miR-30 FI for 3 consecutive days ( n =7). (D) Representative heart images that were stained by TTC and Evans blue, C57BL/6 mice were treated with LNA-miR-30 FI (2.5 mg/kg) for 3 consecutive days before MI surgery. The red region is the border zone that was stained by TTC, the white region is the infarct zone that cannot be stained by either TTC or Evans blue, and the black region is the normal tissue that is stained by both TTC and Evans blue. (E) The infarct size is expressed as the ratio of the infarct area and area at risk ( n =6). (F–H) The effects of LNA-miR-30 FI on LDH, creatine kinase (CK), and cardiac troponin-I (cTn-I) release in plasma of mice MI model ( n =6). The data are expressed as the mean±SEM. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: miRNA-30 Family Inhibition Protects Against Cardiac Ischemic Injury by Regulating Cystathionine-γ-Lyase Expression

    doi: 10.1089/ars.2014.5909

    Figure Lengend Snippet: LNA-miR-30 FI protects the heart against MI injury in C57BL/6 mice. (A) The expression levels of miR-30 family members in C57BL/6 mouse heart after injecting 5 mg/kg of LNA-miR-30 FI for 3 consecutive days ( n =3). (B) CSE mRNA and (C) protein expression levels in the C57BL/6 left ventricle after injecting different concentrations of LNA-miR-30 FI for 3 consecutive days ( n =7). (D) Representative heart images that were stained by TTC and Evans blue, C57BL/6 mice were treated with LNA-miR-30 FI (2.5 mg/kg) for 3 consecutive days before MI surgery. The red region is the border zone that was stained by TTC, the white region is the infarct zone that cannot be stained by either TTC or Evans blue, and the black region is the normal tissue that is stained by both TTC and Evans blue. (E) The infarct size is expressed as the ratio of the infarct area and area at risk ( n =6). (F–H) The effects of LNA-miR-30 FI on LDH, creatine kinase (CK), and cardiac troponin-I (cTn-I) release in plasma of mice MI model ( n =6). The data are expressed as the mean±SEM. * p

    Article Snippet: For the quantitative detection of CSE mRNA, the SYBR Green I (Takara, Dalian, China) incorporation method was used with GAPDH as an internal control.

    Techniques: Mouse Assay, Expressing, Staining

    Effect of recombinant vector for Nanog mRNA in SGC-7901 cells as shown by semi-quantitative RT-PCR. From left to right: 1,8, DNA Marker (DL 2000); 2, transfected with pshRNA-NanogA; 3, transfected with pshRNA-NanogB; 4, transfected with pshRNA-NanogC; 5, transfected with pshRNA-negative control; 6, transfected with pGenesil-1; 7, SGC-7901.

    Journal: Oncology Letters

    Article Title: Effect of shRNA-mediated inhibition of Nanog gene expression on the behavior of human gastric cancer cells

    doi: 10.3892/ol.2013.1394

    Figure Lengend Snippet: Effect of recombinant vector for Nanog mRNA in SGC-7901 cells as shown by semi-quantitative RT-PCR. From left to right: 1,8, DNA Marker (DL 2000); 2, transfected with pshRNA-NanogA; 3, transfected with pshRNA-NanogB; 4, transfected with pshRNA-NanogC; 5, transfected with pshRNA-negative control; 6, transfected with pGenesil-1; 7, SGC-7901.

    Article Snippet: According to the mRNA sequence of human Nanog in the NCBI GenBank ( http://www.ncbi.nlm.nih.gov/nuccore/NM_024865.2 ), a pair of short fragment PCR primers were designed with the primer design software Primer Premier 5 and Oligo 6 and then synthesized by Takara ( ).

    Techniques: Recombinant, Plasmid Preparation, Quantitative RT-PCR, Marker, Transfection, Negative Control