Journal: G3: Genes|Genomes|Genetics

Article Title: RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif

doi: 10.1093/g3journal/jkac292

Figure Lengend Snippet: The def1 attenuator mutant ( def1 atten ) overexpresses mRNA and protein, exacerbating the cell toxicity of processed Def1 ( def1 1–530 ). a) Schematic of def1 mutants ( def1-atten and def1 1–530 ) along with RT-PCR primers (F1, R1, blue) used to detect the DEF1 mRNA readthrough product. Note: the drawing is not to scale. The R1 primer is specific to longer mRNA and not attenuated RNA. DNA positions are numbered relative to the +1 start codon of the DEF1 open reading frame. b) RT-PCR analysis of Def1 mRNA levels. 18S serves as a loading control for total RNA. Reverse Transcriptase (±RT) ensures that signal is dependent on RNA and not genomic DNA template. c) Western blot analysis of Def1 protein levels. Actin serves as a loading control for total protein. d) Spot test assay of def1 mutants on solid plate media. Liquid cultures were grown to saturation at 25°C, serially diluted, spotted onto YPAD, and grown at the temperatures indicated for 3–5 days. e, f) Growth of def1 mutants in liquid culture. Liquid yeast cultures were grown to saturation at 25°C, diluted back, and recovered to exponential phase prior to shifting to (e) 37°C or (f) 39°C for 6 h. Cell density was measured via OD 600 every hour, and doubling times were calculated from exponential lines of best fit for data between 60 and 360 min. Error bars represent SD from 3 biological replicates. Asterisks indicate statistical significance by Welch’s 2 sample t -test (* P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001, ns—not significant).

Article Snippet: After the RNA was isolated, mRNA was converted to cDNA using the OneTaq RT-PCR kit (New England BioLabs).

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Spot Test

Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet: Alternative splicing events produced from the recombined AIv4 allele (A) Scyl1 transcript expression in the cerebella of CMV-Cre+; Scyl1 +/+ and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. Data is expressed as mean ± SEM. RNA extracts from 3 CMV-Cre+; Scyl1 +/+ and 3 CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice were analyzed in triplicates. (B) RT-PCR products generated from cerebellar total RNA extracts of Scyl1 +/+ , Scyl1 +/AIv4 , Scyl1 AIv4/AIv4 , CMV-Cre+; Scyl1 +/+ , CMV-Cre+; Scyl1 +/AIv4Δ , and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. Amplicons of 665, 638, and 488 bp, corresponding to the mature form of the Scyl1 transcript, as well as shorter transcript variants 1 and 2 were obtained. These shorter transcripts were observed only in CMV-Cre+; Scyl1 +/AIv4Δ and CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice. (C) Schematic representation of the five most abundant RNA transcripts observed in CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice (transcripts variants labeled 1–5). RT-PCR products from CMV-Cre+; Scyl1 AIv4Δ/AIv4Δ mice were TOPO cloned. Plasmid DNA was recovered from 24 clones and analyzed by Sanger sequencing. The frequency of each transcript is illustrated to the right of the transcript. 18 out of 24 clones were obtained for transcript variant 1; 3 out of 24 clones were obtained for transcript variant 2; and 1 out of 24 clones were obtained for transcript variants 3, 4, and 5. Transcript variant 1 resulted from the splicing of the AIv4 splice donor site into the splice acceptor site of exon 4. Transcript variant 2 resulted from the splicing of the AIv4 splice donor site into a cryptic splice acceptor site within the 3′ half of exon 3. Transcript variant 3 contained intronic sequences from intron 2 and the recombined AIv4 cassette as well as sequences encoding exon 2–5. Transcript variant 4 contained the expected sequence of the recombined AIv4 cassette. Transcript variant 5 resulted from the splicing of a cryptic splice donor site within the 5′ half of exon 3 into the AIv4 splice acceptor site. (D) Ectopic expression of HA tagged versions of wild type, transcript variant 1, 2 and 5 under the control of the CMV promoter. All three isoforms generated from transcript variants 1, 2 and 5 were expressed at a much lower level than their wild-type, full length, counterpart. The images are representative of 3 independent experiments.

Article Snippet: OneTaq One-Step RT-PCR Kit , New England Biolabs , E5315S.

Techniques: Produced, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Labeling, Clone Assay, Plasmid Preparation, Sequencing, Variant Assay

Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet:

Article Snippet: OneTaq One-Step RT-PCR Kit , New England Biolabs , E5315S.

Techniques: Recombinant, Lysis, Protease Inhibitor, Sequencing, Purification, TA Cloning, Subcloning, Bicinchoninic Acid Protein Assay, Western Blot, Expressing, Mutagenesis, Software

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Effect of SFN and 5-Aza-dC on DNMT3B in human cervical cancer cells (HeLa). (a) 2.5 μ M of SFN and 1.5 μ M 5-Aza-dC treatments significantly inhibit the activity of DNMT in time-dependent manner, respectively. Values are means ± SD of three independent experiments. Symbol ( ∗ ) indicates significant ( P < 0.05) difference of data between control and treated cells. (b) 2.5 μ M SFN treated HeLa cells show significantly time-dependent reduction in the mRNA expression of DNMT3B in comparison to untreated cells. Panel A shows β -actin expression as an internal control, Panel B shows the expression of DNMT3B on treatment with 5-Aza-dC, and Panel C shows the expression of DNMT3B on treatment with SFN. Lane 1 shows the expression of DNMT3B gene in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent alteration in the expression of DNMT3B after treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR.

Article Snippet: Reverse transcription of RNA to synthesize cDNA was performed using the ProtoScriptM-MuLVTaq RT-PCR Kit (New England Bio Labs, USA) from 5 mg of total RNA (at 42°C for 60 min) followed by RT-PCR using gene-specific primers for β -actin, RAR β , CDH1, DAPK1, GSTP1, DNMTB, and HDAC1.

Techniques: Activity Assay, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Effect of SFN and TSA on HDAC1 in human cervical cancer cells (HeLa). (a) 2.5 μ M of SFN and 0.05 μ M TSA treatments significantly inhibit the activity of HDAC in time-dependent manner, respectively. Values are means ± SD of three independent experiments. Symbol ( ∗ ) indicates significant ( P < 0.05) difference of data between control and treated cells. (b) 2.5 μ M SFN treated HeLa cells show significantly time-dependent reduction in the mRNA expression of HDAC1 in comparison to untreated cells. Panel A shows β -actin expression as an internal control, Panel B shows the expression of HDAC1 on treatment with TSA, and Panel C shows the expression of HDAC1 on treatment with SFN. Lane 1 shows the expression of HDAC1 gene in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent decrease in the expression of HDAC1 after treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR.

Article Snippet: Reverse transcription of RNA to synthesize cDNA was performed using the ProtoScriptM-MuLVTaq RT-PCR Kit (New England Bio Labs, USA) from 5 mg of total RNA (at 42°C for 60 min) followed by RT-PCR using gene-specific primers for β -actin, RAR β , CDH1, DAPK1, GSTP1, DNMTB, and HDAC1.

Techniques: Activity Assay, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Alterations of methylation status and mRNA expression levels of RAR β , CDH1, DAPK1, and GSTP1 genes after treatment with SFN. (a) mRNA expression levels before and after the treatment. Lane 1 shows the expression of these genes in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent modulation in the expression of HDAC1 upon treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR. β -actin was used as an internal control. (b) Methylation-specific bands (M) and unmethylation-specific bands (U). Lane 1 shows the methylation status of these genes in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent modulation in the methylation status of RAR β , CDH1, DAPK1, and GSTP1 genes for 24, 48, and 72 h, respectively.

Article Snippet: Reverse transcription of RNA to synthesize cDNA was performed using the ProtoScriptM-MuLVTaq RT-PCR Kit (New England Bio Labs, USA) from 5 mg of total RNA (at 42°C for 60 min) followed by RT-PCR using gene-specific primers for β -actin, RAR β , CDH1, DAPK1, GSTP1, DNMTB, and HDAC1.

Techniques: Methylation, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Effect of SFN and 5-Aza-dC on DNMT3B in human cervical cancer cells (HeLa). (a) 2.5 μ M of SFN and 1.5 μ M 5-Aza-dC treatments significantly inhibit the activity of DNMT in time-dependent manner, respectively. Values are means ± SD of three independent experiments. Symbol ( ∗ ) indicates significant ( P < 0.05) difference of data between control and treated cells. (b) 2.5 μ M SFN treated HeLa cells show significantly time-dependent reduction in the mRNA expression of DNMT3B in comparison to untreated cells. Panel A shows β -actin expression as an internal control, Panel B shows the expression of DNMT3B on treatment with 5-Aza-dC, and Panel C shows the expression of DNMT3B on treatment with SFN. Lane 1 shows the expression of DNMT3B gene in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent alteration in the expression of DNMT3B after treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR.

Article Snippet: RT-PCR (ProtoScriptM-MuLVTaq) kit was obtained from New England Bio Labs (New England Bio Labs, USA).

Techniques: Activity Assay, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Effect of SFN and TSA on HDAC1 in human cervical cancer cells (HeLa). (a) 2.5 μ M of SFN and 0.05 μ M TSA treatments significantly inhibit the activity of HDAC in time-dependent manner, respectively. Values are means ± SD of three independent experiments. Symbol ( ∗ ) indicates significant ( P < 0.05) difference of data between control and treated cells. (b) 2.5 μ M SFN treated HeLa cells show significantly time-dependent reduction in the mRNA expression of HDAC1 in comparison to untreated cells. Panel A shows β -actin expression as an internal control, Panel B shows the expression of HDAC1 on treatment with TSA, and Panel C shows the expression of HDAC1 on treatment with SFN. Lane 1 shows the expression of HDAC1 gene in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent decrease in the expression of HDAC1 after treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR.

Article Snippet: RT-PCR (ProtoScriptM-MuLVTaq) kit was obtained from New England Bio Labs (New England Bio Labs, USA).

Techniques: Activity Assay, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

doi: 10.1155/2015/412149

Figure Lengend Snippet: Alterations of methylation status and mRNA expression levels of RAR β , CDH1, DAPK1, and GSTP1 genes after treatment with SFN. (a) mRNA expression levels before and after the treatment. Lane 1 shows the expression of these genes in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent modulation in the expression of HDAC1 upon treatment for 24, 48, and 72 h, respectively; Lane 5 shows negative control for RT-PCR. β -actin was used as an internal control. (b) Methylation-specific bands (M) and unmethylation-specific bands (U). Lane 1 shows the methylation status of these genes in untreated HeLa cells; Lanes 2, 3, and 4 show the time-dependent modulation in the methylation status of RAR β , CDH1, DAPK1, and GSTP1 genes for 24, 48, and 72 h, respectively.

Article Snippet: RT-PCR (ProtoScriptM-MuLVTaq) kit was obtained from New England Bio Labs (New England Bio Labs, USA).

Techniques: Methylation, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction