Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Hrq1 and Pso2 participate in the same ICL repair pathway for MMC and DEB lesions. A . Mutation of HRQ1 is epistatic to pso2 for MMC sensitivity. Saturated overnight cultures of strains with the genotypes indicated on the left were diluted to OD 660 = 1.0 and then further serially diluted 10-fold to 10 −4 . Equal volumes of each dilution were then spotted onto rich medium containing the solvent control (DMSO) or rich medium supplemented with the indicated concentration of MMC. B . Mutation of HRQ1 is epistatic to pso2 for DEB sensitivity. The assay was performed as described above, and the DMSO control plate is shown again for ease of comparison. Mutation of SGS1 is not epistatic with either hrq1 or pso2 in these assays. These results are representative of ≥ 3 independent experiments.

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Hrq1 stimulates Pso2 nuclease activity. A . Concentration-dependent nuclease activity. Recombinant Pso2 (20-200 nM) was incubated with dsDNA for 30 min, and the nuclease products were separated on a denaturing gel. B . Hrq1-dependent stimulation of Pso2 nuclease activity. Denaturing gel showing the radiolabelled dsDNA substrate incubated alone, with 50 nM Pso2, 150 nM Hrq1, or 50 nM Pso2 and 20-150 nM Hrq1. C . Hrq1 requires catalytic activity to stimulate Pso2 nuclease activity. Quantification of the nuclease activity of Pso2 alone and in the presence of Hrq1 or the inactive Hrq1-K318A mutant. Nuclease-inactive Pso2-H611A was used as a control. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the standard deviation (S.D.).

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay, Concentration Assay, Recombinant, Incubation, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: smFRET analysis of the stimulation of Pso2 nuclease activity by Hrq1. A . Diagram of the smFRET substrate and the effect of Pso2. Short lengths of dsDNA are rigid, keeping the Cy3 and Cy5 FRET pair distal from one another. Pso2 can digest away the unlabelled DNA strand, yielded flexible ssDNA and allowing the FRET pair to come into proximity. B . smFRET signal of the dsDNA substrate and the labelled ssDNA. C . Hrq1 stimulates Pso2 nuclease activity. Pso2 alone slowly generates an increase in the FRET signal by degrading the dsDNA substrate, but the addition of Hrq1 greatly increases the speed at which the high FRET signal appears.

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Hrq1 stimulates the translesion nuclease activity of Pso2. A . Pso2 lacks translesion nuclease activity in the absence of Hrq1. smFRET analysis of Pso2 activity on undamaged DNA and DNA with a site-specific ICL (XL-DNA) in the absence and presence of Hrq1. ATP is required to observe Pso2 stimulation by Hrq1. B . Quantification of the results from A. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the S.D.

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Eukaryotic RecQ4 subfamily helicases specifically stimulate Pso2 nuclease activity. A . Pso2 nuclease activity alone and in the presence of recombinant Hrq1, RECQL4, M. smegmatis SftH, or Sgs1. The radiolabelled dsDNA substrate was incubated with 50 nM Pso2 and/or 100 nM of the indicated helicase for 30 min, and nuclease products were separated on a denaturing gel and visualized by phosphorimaging. B . Quantification of ≥ 3 independent experiments performed as in A. The graphed bars are the averages of the independent experiments (individual data points shown as open circles), and the error bars are the S.D.*, p < 0.05 and **, p < 0.01. Significant differences were determined by multiple t -tests using the Holm-Sidak method, with α = 5% and without assuming a consistent S.D. C . Sgs1 unwinds the dsDNA substrate used in nuclease assays. Equimolar amounts of Hrq1, RECQL4, SftH, or Sgs1 were incubated for 30 min with either 2 nM Fork or dsDNA and quantified for helicase activity. The data are plotted as in B.

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay, Recombinant, Incubation

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Hrq1 and Pso2 physically interact. A . Recombinant Hrq1 and Pso2 can be crosslinked in solution. Molar equivalents of Hrq1 and Pso2 were incubated in the presence or absence of 1 nM poly(dT) 50mer ssDNA and/or the presence or absence of a molar excess (20x or 40x) of the protein-protein crosslinker DSSO. The reactions were then analysed by SDS-PAGE and western blotting with an antibody recognizing the N-terminal 10xHis tag on Hrq1. Hrq1 alone produces a signal at the expected size of ∼135 kDa, but the addition of Pso2 and DSSO yields a band of the approximate size of a Hrq1-Pso2 complex (∼180 kDa). The amount of this slower migrating product increases when Hrq1 is present in a fivefold molar excess of Pso2 (0.2x), consistent with the optimal ratio of Hrq1:Pso2 in nuclease reactions. B . Nuclease activity of Pso2 and Pso2ΔN in the absence and presence of Hrq1 or Hrq1ΔN. The Hrq1ΔN truncation mutant fails to stimulate the nuclease activity of full-length Pso2, and Pso2ΔN alone has nuclease activity equivalent to Pso2+Hrq1. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the S.D. C . The hrq1ΔN and pso2ΔN alleles phenocopy complete deletions of HRQ1 and PSO2 . Cells of the indicated genotypes were grown, diluted, and spotted onto YPD + DEB plates as in .

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

doi: 10.1101/773267

Figure Lengend Snippet: Model of ICL repair in WT, hrq1Δ , and pso2Δ cells. In the first step of ICL repair, the NER machinery cuts one strand of DNA on either side of the ICL. Then, in WT cells, Hrq1 and Pso2 are recruited to the lesion to digest away the incised strand, leaving an adducted base. Translesion synthesis (TLS) polymerases fill the gap, and NER removes the adducted base. In hrq1Δ cells, Pso2 is still recruited to the ICL, but its poor translesion nuclease activity in the absence of Hrq1 yields some amount of incompletely processed substrates, which can lead to mutagenesis or cell death. In cells lacking Pso2, other nucleases ( e . g ., Exo1) may be recruited to ICLs, but there less optimal activity on such substrates can also lead to mutagenesis or cell death.

Article Snippet: Pso2-6xHis was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 mM isopropyl β-D-1- thiogalactopyranoside for 4 h at 30°C.

Techniques: Translesion Synthesis, Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Hrq1 and Pso2 participate in the same ICL repair pathway for MMC and DEB lesions. A, mutation of HRQ1 is epistatic to pso2 for MMC sensitivity. Saturated overnight, cultures of strains with the genotypes indicated on the left were diluted to OD660 = 1.0 and then further serially diluted 10-fold to 10−4. Equal volumes of each dilution were then spotted onto rich medium containing the solvent control (DMSO) or rich medium supplemented with the indicated concentration of MMC. B, mutation of HRQ1 is epistatic to pso2 for DEB sensitivity. The assay was performed as described above, and the DMSO control plate is shown again for ease of comparison. Mutation of SGS1 is not epistatic with either hrq1 or pso2 in these assays. These results are representative of ≥3 independent experiments.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Mutagenesis, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Hrq1 stimulates Pso2 nuclease activity. A, concentration-dependent nuclease activity. Recombinant Pso2 (20–200 nM) was incubated with dsDNA for 30 min, and the nuclease products were separated on a denaturing gel. B, Hrq1-dependent stimulation of Pso2 nuclease activity. Denaturing gel showing the radiolabeled dsDNA substrate incubated alone, with 50 nm Pso2, 150 nm Hrq1, or 50 nM Pso2 and 20–150 nM Hrq1. C, Hrq1 requires catalytic activity to stimulate Pso2 nuclease activity. Quantification of the nuclease activity of Pso2 alone and in the presence of Hrq1 or the inactive Hrq1-K318A mutant. Nuclease-inactive Pso2-H611A was used as a control. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the S.D.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay, Concentration Assay, Recombinant, Incubation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: smFRET analysis of the stimulation of Pso2 nuclease activity by Hrq1. A, diagram of the smFRET substrate and the effect of Pso2. Short lengths of dsDNA are rigid, keeping the Cy3 and Cy5 FRET pair distal from one another. Pso2 can digest away the unlabeled DNA strand, yielded flexible ssDNA and allowing the FRET pair to come into proximity. B, smFRET signal of the dsDNA substrate and the labeled ssDNA. C, Hrq1 stimulates Pso2 nuclease activity. Pso2 alone slowly generates an increase in the FRET signal by degrading the dsDNA substrate, but the addition of Hrq1 greatly increases the speed at which the high FRET signal appears.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Hrq1 stimulates the translesion nuclease activity of Pso2. A, Pso2 lacks translesion nuclease activity in the absence of Hrq1. smFRET analysis of Pso2 activity on undamaged DNA and DNA with a site-specific ICL (XL-DNA) in the absence and presence of Hrq1. ATP is required to observe Pso2 stimulation by Hrq1. B, quantification of the results from A. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the S.D.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Eukaryotic RecQ4 subfamily helicases specifically stimulate Pso2 nuclease activity. A, Pso2 nuclease activity alone and in the presence of recombinant Hrq1, RECQL4, M. smegmatis SftH, or Sgs1. The radiolabeled dsDNA substrate was incubated with 50 nm Pso2 and/or 100 nm of the indicated helicase for 30 min, and nuclease products were separated on a denaturing gel and visualized by phosphorimaging. B, quantification of ≥3 independent experiments performed as in A. The graphed bars are the averages of the independent experiments (individual data points shown as open circles), and the error bars are the S.D. *, p < 0.05 and **, p < 0.01. Significant differences were determined by multiple t tests using the Holm-Sidak method, with α = 5% and without assuming a consistent S.D. C, Sgs1 unwinds the dsDNA substrate used in nuclease assays. Equimolar amounts of Hrq1, RECQL4, SftH, or Sgs1 were incubated for 30 min with either 2 nm Fork or dsDNA and quantified for helicase activity. The data are plotted as in B.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Activity Assay, Recombinant, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Hrq1 and Pso2 physically interact. A, recombinant Hrq1 and Pso2 can be crosslinked in solution. Molar equivalents of Hrq1 and Pso2 were incubated in the presence or absence of 1 nm poly(dT) 50 mer ssDNA and/or the presence or absence of a molar excess (20× or 40×) of the protein-protein crosslinker DSSO. The reactions were then analyzed by SDS-PAGE and Western blotting with an antibody recognizing the N-terminal His10 tag on Hrq1. Hrq1 alone produces a signal at the expected size of ∼135 kDa, but the addition of Pso2 and DSSO yields a band of the approximate size of a Hrq1-Pso2 complex (∼180 kDa). The amount of this slower migrating product increases when Hrq1 is present in a 5-fold molar excess of Pso2 (0.2×), consistent with the optimal ratio of Hrq1:Pso2 in nuclease reactions. B, nuclease activity of Pso2 and Pso2ΔN in the absence and presence of Hrq1 or Hrq1ΔN. The Hrq1ΔN truncation mutant fails to stimulate the nuclease activity of full-length Pso2, and Pso2ΔN alone has nuclease activity equivalent to Pso2+Hrq1. The graphed bars are the averages of three independent experiments (individual data points shown as open circles), and the error bars are the S.D. C, the hrq1ΔN and pso2ΔN alleles phenocopy complete deletions of HRQ1 and PSO2. Cells of the indicated genotypes were grown, diluted, and spotted onto YPD + DEB plates as in Fig. 1.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

doi: 10.1074/jbc.RA120.013626

Figure Lengend Snippet: Model of ICL repair in WT, hrq1Δ, and pso2Δ cells. In the first step of ICL repair, the NER machinery cuts one strand of DNA on either side of the ICL. Then, in WT cells, Hrq1 and Pso2 are recruited to the lesion to digest away the incised strand, leaving an adducted base. Translesion synthesis (TLS) polymerases fill the gap, and NER removes the adducted base. In hrq1Δ cells, Pso2 is still recruited to the ICL, but its poor translesion nuclease activity in the absence of Hrq1 yields some amount of incompletely processed substrates, which can lead to mutagenesis or cell death. In cells lacking Pso2, other nucleases (e.g. Exo1) may be recruited to ICLs, but their less optimal activity on such substrates can also lead to mutagenesis or cell death.

Article Snippet: Pso2-His 6 was expressed in Rosetta 2(DE3) pLysS (Novagen) cells by growing cultures to an OD 600 of 0.6 at 37°C followed by induction with 1 m m isopropyl β- d -1-thiogalactopyranoside for 4 h at 30°C.

Techniques: Translesion Synthesis, Activity Assay, Mutagenesis

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: SOX9 protein and SOX9 mRNA expression in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells. ( A ) Western blot analysis of the expression of E-cadherin (CDH1), cytokeratin-19 (KRT19), vimentin (VIM), SOX9, and GAPDH in the investigated cell lines. Pancreatic cancer cells were seeded in 6-well plates (0.5 × 10 6 cells per well). After 48 h, cell lysates were prepared by adding SDS sample buffer (200 µL per well). ( B ) Densitometric quantitation of Western blots from ( A ). The levels of the SOX9 protein were normalized to the levels of the GAPDH protein. ( C ) Real-time qPCR analysis of the mRNA expression of SOX9 in pancreatic cancer lines. The results are presented as the mean ± SEM of three independent experiments and normalized against the HPRT expression. ( D ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 100 µm.

Article Snippet: pCMV6-SOX9 human tagged ORF clone (RC208994) and control pCMV6-Entry plasmid vector (PS100001) were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Quantitation Assay, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of protein markers of differentiation and developmental transcription factors in pancreatic cancer cells. ( A ) Western blot analysis of the expression of SOX9 in AsPC-1, BxPC-3, Colo357, Capan-2, MiaPaCa-2, and Panc1 cells transfected with control siNeg and siSOX9 ( n = 3). GAPDH and beta-tubulin (TUBB) were used as loading and normalization controls. ( B ) Immunofluorescence imaging of the SOX9 expression in Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for total cytokeratin (green) and SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 µm. ( C ) Western blot analysis of the expression of epithelial cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( D ) Heatmap of differentially expressed epithelial cell proteins from ( C ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( E ) Western blot analysis of the expression of mesenchymal cell protein markers in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( F ) Heatmap of differentially expressed mesenchymal cell proteins from ( E ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( G ) Western blot analysis of the expression of developmental transcription factors in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( H ) Heatmap of differentially expressed developmental transcription factors from ( G ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression.

Article Snippet: pCMV6-SOX9 human tagged ORF clone (RC208994) and control pCMV6-Entry plasmid vector (PS100001) were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Transfection, Immunofluorescence, Imaging, Staining

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: The effect of SOX9 downregulation on the expression levels of cell cycle protein regulators. ( A ) Western blot analysis of the expression of cell cycle protein regulators in the investigated cell lines transfected with control siNeg and siSOX9 ( n = 3). ( B ) Heatmap of differentially expressed cell cycle protein regulators from ( B ). Color code: red, upregulation; blue, downregulation; gray, no change. ND = undetected expression. ( C ) Immunofluorescence imaging of p21 Waf1/Cip1 (CDKN1A) expression in Colo357 and Panc1 cells transfected with control siNeg and siSOX9. Cells were stained for SOX9 (red). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. ( D , E ) Relative Western blot, RT-qPCR, and RNA-Seq data quantification of the levels of SOX9, SNAI1, SNAI2, FOXA2, GATA4, CDKN1A, TP53, and PTEN in control siNeg and siSOX9 transfected Colo357 ( D ) and Panc1 ( E ) cells. RT-qPCR and RNA-Seq data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control.

Article Snippet: pCMV6-SOX9 human tagged ORF clone (RC208994) and control pCMV6-Entry plasmid vector (PS100001) were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Transfection, Immunofluorescence, Imaging, Staining, Quantitative RT-PCR, RNA Sequencing Assay

Journal: Biomedicines

Article Title: SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner

doi: 10.3390/biomedicines10071466

Figure Lengend Snippet: Effect of SOX9 expression on cell proliferation, cellular senescence, apoptosis, and cell migration. ( A ) Effect of SOX9 downregulation on the cell proliferation of BxPC-3, Colo357, MiaPaCa-2, and Panc1. Pancreatic cancer cells seeded in 6-well plates were transfected with siSOX9 and control siNeg. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. Data were expressed as the percentage from control cell growth. Data are means ± SEM from three technical replicates and representative of at least three experiments. * p ≤ 0.05; ** p ≤ 0.01 compared with siNeg control. ( B ) Effect of SOX9 downregulation on the senescence-associated β-galactosidase (SA-β-Gal) activity of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. SA-β-Gal activity data were expressed relative to SA-β-Gal activity levels in siNeg and siSOX9 transfected cells. ( C ) Effect of SOX9 downregulation on caspase 3/7, caspase 8, and caspase 9 activities of BxPC-3, Colo357, MiaPaCa-2, and Panc1 cells. Caspase activity data were expressed relative to caspase activity levels in siNeg and siSOX9 transfected cells, respectively. ( D ) Panc1 cells were transfected with siSOX9 and siNeg for 72 h and then analyzed by annexin V–AF488/DAPI staining with flow cytometry analysis. The lower right area (Q3) shows early apoptotic cells, and the upper right area (Q2) shows late apoptotic cells. ( E ) Summary graphs of the flow cytometry results. ( F ) Western blot analysis of SOX9 and CDKN1A expression in Panc1 cells transfected with pCMV6 empty plasmid and pCMV6-SOX9-FLAG after 72 h post-transfection. GAPDH was used as control. ( G ) Effect of SOX9 upregulation on cell proliferation of BxPC-3 and Colo357. Pancreatic cancer cells seeded in 6-well plates were transfected with empty pCMV6 vector and pCMV6-SOX9-FLAG. After 72 h, the cell number was measured by counting cells using the TC20 automated cell counter. ( H ) Western blot analysis of the expression of SOX9, TP53, CDKN1A, and CDKN1B in Panc1 cells transfected with control siNeg and siSOX9. Cell lysates were prepared at 72, 96, and 120 h after first siRNA transfection. GAPDH was used as loading control. ( I ) The representative images displayed the GFP distributions of transplanted Panc1-EGFP control siNeg transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence. ( J ) The representative image displayed the GFP distributions of transplanted Panc1-EGFP siSOX9 transfected cells in Danio rerio embryo. The microinjection site Danio rerio embryo is indicated with a red circle. The bioimaging of injected embryos was performed 2 days after cell transplantation. PH, phase contrast; FL, fluorescence.

Article Snippet: pCMV6-SOX9 human tagged ORF clone (RC208994) and control pCMV6-Entry plasmid vector (PS100001) were obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Migration, Transfection, Activity Assay, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Injection, Transplantation Assay, Fluorescence