Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 1. Rescue of BRCA1 inhibition of ER- transcriptional activity by the HPV E7 and E6 and by other DNA tumor viral oncoproteins. A, domain structure of the DNA tumor virus oncoproteins studied. B, rescue of wtBRCA1 repression of ER- activity by HPV-E7 and HPV-E6 oncogenes. C, rescue of wtBRCA1 repression of ER- activity by adenovirus E1A oncogene. D, rescue of wtBRCA1 repression of ER- activity by the SV40 large T oncogene. E, wtBRCA1 only minimally represses ER- activity in human cervical cancer cell lines. For methodology, subconfluent proliferating cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc)(0.25gDNAforeachplasmid)usingLipofectamineTM,asdescribedunder“MaterialsandMethods.”Thecellswerewashed,incubatedwithoutorwith17-estradiol(E2, 10 nM or 1 M) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed relative to the negative control (0 E2) (B–D) or as a percentage of the E2 positive control (E). The values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. Abbreviations and acronyms are as follows: CDK2, cyclin-dependent kinase 2; CR1/2, conserved region 1/2; CtBP, C-terminal binding protein; C-TERM, C-terminal domain; CXXC, one-half of a zinc coordination motif; cycA, cyclin A; ETQL, PDZ family protein-binding motif; hDLG, homolog of discs large, Drosophila; hScrib, homolog of Scribble, Drosophila; HR, host range domain; Hsc70, heat shock cognate protein 70 kDa; LXCXE, consensus retinoblastoma family protein-binding motif; MAGI-1, membrane-associated guanylate kinase inverted-1; MUPP-1, multiple PDZ domain protein; mut-SV40T, mutant SV40 large T oncogene with defective for RB-binding; NLS, nuclear localization signal; N-TERM, N-terminal domain; PCAF, p300/ CBP-associated factor; PLDLS, CtBP-binding motif; RNA pol II, RNA polymerase II; SV40T, simian virus 40 large T oncogene; TBP, TATA box-binding protein; TEF-1, transcriptional enhancer factor-1; TFIIB, transcription factor IIB; wt, wild-type.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Inhibition, Activity Assay, Virus, Transfection, Expressing, Plasmid Preparation, Luciferase, Negative Control, Positive Control, Binding Assay, Protein Binding, Membrane, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 2. Capture of C-terminally truncated BRCA1 proteins by full-length HPV-E7 and HPV-E6. A, domain structure of BRCA1 protein fragments. B, GST Western blot showing the expression of GST-E7 and GST-E6. The GST-E7 and GST-E6 fusion proteins were visualized by Western blotting using an antibody directed against GST. For reference, 100 l of GST-E7 or GST-E6 was used in the capture assays. C, expression of IVT BRCA1 proteins. The SDS-PAGE autoradiograph shows 10% of the input of each BRCA1 protein used for the GST capture assays. C and E, the arrows indicate the correct bands. D, capture of IVT full-length BRCA1 by GST-E7 and GST-E6. The binding of full-length BRCA1 (amino acids 1–1863) to beadscoatedwithGST-E7orGST-E6wasdeterminedbySDS-PAGEautoradiography.E,captureofsmallerBRCA1proteinfragmentsbyGST-E7andGST-E6.ThebindingofC-truncated BRCA1 proteins to beads coated with GST-E7 or GST-E6 was visualized by SDS-PAGE autoradiography. The abbreviations used are as follows: AD1, activation domain 1; BACH1, BRCA1-associated C-terminal helicase 1; BARD1, BRCA1-associated RING domain 1 protein; BRCT1/2, BRCA1 C-terminal domain 1/2; CtIP, CtBP-interacting protein; HDAC1/2, histone deacetylase 1/2; LMO4, LIM domain only protein 4; NES, nuclear export signal; NLS1, nuclear localization sequence 1; RHA, RNA helicase A; RbAp46/38, retinoblastoma-associated protein 46/48 kDa; TAD, transcriptional activation domain.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Capture-C, Western Blot, Expressing, SDS Page, Autoradiography, Binding Assay, Activation Assay, Histone Deacetylase Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 3. Capture of different IVT BRCA1 protein fragments by full-length GST-E7 and GST-E6. A, domain structure of BRCA1 protein and protein fragments. B, capture of different IVT BRCA1 proteins by full-length GST-E7 protein. The ability of GST-E7 to capture a set of IVT BRCA1 protein fragments spanning the length of the BRCA1 protein was determined. The input lane represents 10% of the IVT protein used in the GST capture assays. C, capture of different IVT BRCA1 proteins by full-length GST-E6 protein. Assays were performed as described in A, except that GST-E6 was used as the bait. D, capture of IVT N-terminal BRCA1 proteins by full-length GST-E7 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E7 protein. E, capture of IVT N-terminal BRCA1 proteins by full-length GST-E6 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E6 protein. F, capture of IVT BRCA1 BRCT1 by full-length GST-E7 and GST-E6 proteins. An IVT BRCA1 protein fragment containing the first BRCT1 was tested for capture by the GST-E7 and GST-E6 proteins. NES, nuclear export signal; AD1, activation domain 1.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 4. Capture of IVT full-length E7 and E6 proteins by various GST-BRCA1 pro- tein. A, capture of full-length IVT E7 by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full-length E7 was tested. B, capture of full-length IVT E6 proteins by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full- length E7 was tested. C, Western blot showing the expression of different GST-BRCA1 proteins.ThedifferentGST-BRCA1proteinsutilizedinAandBwerevisualizedbyWestern blottingusinganantibodydirectedagainstGST.Thecorrectbandisindicatedbyaboxin the figure.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vitro, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 5. In vivo association of BRCA1 and the E6 and E7 proteins. A, IP of endoge- nous E7 co-precipitates E7 and BRCA1 in SiHa cells. Subconfluent proliferating SiHa human cervical cancer cells were subjected to IP using an anti-E7 antibody and Western- blotted to detect E7 and BRCA1. See “Materials and Methods” for details. B, IP of endog- enous BRCA1 co-precipitates BRCA1 and E7 in SiHa cells. Subconfluent proliferating SiHa cells were subject to IP using an anti-BRCA1 antibody and Western-blotted to detect BRCA1 and E7. See “Materials and Methods” for details. C, IP of BRCA1 co-precipitates BRCA1 and exogenous FLAG-tagged E6 in 293T cells. Subconfluent proliferating 293T cells were transfected overnight with FLAG-E6 and wtBRCA1 expression vectors (10 g ofeachvectorper100-mmdish)usingLipofectamineTM,post-incubatedfor24htoallow gene expression, immunoprecipitated using an anti-BRCA1 antibody, and Western-blot- ted to detect the BRCA1 and FLAG-E6 proteins. D, anti-FLAG IP co-precipitates exoge- nous FLAG-E6 and BRCA1 in 293T cells. 293T cells were transfected as described above in C, post-incubated for 24 h to allow gene expression, immunoprecipitated using an anti- FLAG antibody, and Western-blotted to detect the FLAG-E6 and BRCA1 proteins.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vivo, Western Blot, Transfection, Expressing, Gene Expression, Immunoprecipitation, Incubation

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 6. Capture of BRCA1 by truncated and mutant GST-E7 and E6 proteins. A, schematic diagrams of the truncated/mutant E7 proteins used in this study. B, Western blot showing the expression of truncated and mutant GST-E7 proteins. A set of truncated and mutant GST-E7 proteins were visualized by Western blotting, using an anti-GST antibody. C,captureofIVTBRCA1-(1–302)bytruncatedandmutantGST-E7proteins.DifferentGST-E7proteinsshowninAweretestedfortheirabilitytocaptureaninvitrotranslatedN-terminal portion of BRCA1 (amino acids 1–302). D, capture of IVT BRCA1-(1–771) by truncated and mutant GST-E7 proteins. Different GST-E7 proteins from A were tested for their ability to capture an invitrotranslatedN-terminalportionofBRCA1correspondingtoaminoacids1–771.E,schematicdiagramsofthetruncated/mutantE6proteinsusedinthisstudy.F,Western blot showing the expression of truncated and mutant GST-E6 proteins. A set of truncated and mutant GST-E6 proteins were visualized by Western blotting, using an anti-GST antibody. G, capture of IVT BRCA1-(1–302) by truncated and mutant GST-E6 proteins. The various GST-E6 proteins shown in D were tested for their ability to capture IVT BRCA1-(1–302).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Mutagenesis, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 7.RescueofBRCA1repressionofER-activitybywild-typeversusmutantE7andE6.A,rescueofBRCA1repressionofER-bymutantversuswild-typeE7andE6proteins. Subconfluent proliferating MCF-7 cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc) (0.25 g DNA for each plasmid) using LipofectamineTM. The cells were washed, incubated without or with 17-estradiol (E2, 10 nM) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the E2-positive control; and the values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. B and C, dose responses for the rescue of BRCA1 repression of ER- activity by the wild-type E7 (B) and E6 (C) expression vectors. AssayswerecarriedoutinMCF-7andT47DcellsasdescribedaboveinA,exceptusingdifferentquantitiesoftheE7andE6expressionvectors.Thetotalcontentoftransfectedplasmid DNA was kept constant by the addition of the appropriate quantity of control plasmid.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Positive Control, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 8. Effect of HPV oncoproteins E7 and E6 on other functional activities of BRCA1. A, ability of E7 and E6 to rescue the BRCA1 inhibition of E-box-Luc reporter activity. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were trans- fected overnight with the indicated expression vectors and a reporter driven by the c-Myc E-box upstream of a minimal promoter and the luciferase gene (E-box-Luc) (see Ref. 29). After transfection, the cells were washed, post-incubated for 24 h to allow gene expression, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the positive control (E-box-Luc only, no wtBRCA1, E7, or E6); and the values are expressed as means S.E. of four replicate wells. B, ability of E7 and E6 to rescue the BRCA1 repression of hTERT promoter activity. Assays were performed as described in A, except that a reporter containing the human TERT core promoter upstream of the luciferase gene (29) was utilized instead of the E-box-Luc reporter. Lucif- erase activity was expressed as a percentage of the positive control (hTERT-Luc only, no wtBRCA1, E7, or E6), and the values are expressed as means S.E. of four replicate wells.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Functional Assay, Inhibition, Activity Assay, Expressing, Luciferase, Transfection, Incubation, Gene Expression, Positive Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 9. Effects of the E6 and E7 oncoproteins on BRCA1, p53, and RB1 protein levels.SubconfluentproliferatingMCF-7orT47Dcellsweretransientlytransfectedover- night with empty pcDNA3 vector, with FLAG wild-type E6 or zinc finger defective mutant E6 (E6-(C66,136G)) (A), or with FLAG wtE7 or zinc finger defective mutant E7 (E7-(C91G)) (B) expression vectors. The cells were washed, post-incubated for 24 h to allow gene expression,andharvestedforWesternblottingtodetecttheE6andE7oncoproteins,the tumor suppressors BRCA1, p53, and RB1, and -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Incubation, Gene Expression, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 11. Effect of knockdown of BRCA1 on the ability of the E6 and E7 oncopro- teins to stimulate hTERT promoter activity. A, hTERT promoter assays. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were preincubated for 48 h with a previously validated BRCA1 siRNA (29) or the control siRNA (50 nM); transfected over- night with wtE6, wtE7, or pcDNA3 vector; and post-incubated for 24 h to allow gene expression. Luciferase activity was expressed as a percentage of the positive control (hTERT-Luc only, no siRNA, E6, or E7). The values shown are means S.E. of four replicate wells. The data are shown on a linear scale to more easily delineate 2–3-fold changes in reporter gene activity. B, effect of siRNAs on BRCA1 protein levels. Subconfluent prolif- erating MCF-7 cells were treated with no siRNA (vehicle only), BRCA1 siRNA, or control siRNA (50 nM) for 72 h and harvested for Western blotting to detect BRCA1 or -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Knockdown, Activity Assay, Control, Transfection, Plasmid Preparation, Incubation, Gene Expression, Luciferase, Positive Control, Western Blot

Journal: European Journal of Applied Physiology

Article Title: Muscular HSP70 content is higher in elderly compared to young, but is normalized after 12 weeks of strength training

doi: 10.1007/s00421-021-04633-4

Figure Lengend Snippet: Differences in relative content of cytosolic αB-crystallin, HSP70 and HSP27 between young and elderly males based on the mean densitometric intensity. Figures display individual values, as well as mean and standard deviations. Blue indicate male participants. *Different between groups ( p < 0.05)

Article Snippet: HSP27 was measured with an in-house-made double-antibody sandwich ELISA using a monoclonal capture antibody against HSP27 (25 ng/well; ADI-SPA-800, Stressgen) and a polyclonal detection antibody against HSP27 (ADI-SPA-803, Stressgen).

Techniques:

Journal: European Journal of Applied Physiology

Article Title: Muscular HSP70 content is higher in elderly compared to young, but is normalized after 12 weeks of strength training

doi: 10.1007/s00421-021-04633-4

Figure Lengend Snippet: Percentage changes in cytosolic αB-crystallin ( a ), membrane bound αB-crystallin ( b ), cytosolic HSP70 ( c ), membrane bound HSP70 ( d ) and cytosolic HSP27 ( e ) after the training intervention. Representative blots are presented in F. Stippled line indicates baseline (100%). Figures display individual values, as well as mean and standard deviations. STG elderly strength training group. FTG elderly functional strength training group. C elderly control group. YSTG young strength training group. Red indicates female participants. Blue indicates male participants. *Different from pre ( p < 0.05). #Different between groups ( p < 0.05)

Article Snippet: HSP27 was measured with an in-house-made double-antibody sandwich ELISA using a monoclonal capture antibody against HSP27 (25 ng/well; ADI-SPA-800, Stressgen) and a polyclonal detection antibody against HSP27 (ADI-SPA-803, Stressgen).

Techniques: Membrane, Functional Assay, Control

Journal: European Journal of Applied Physiology

Article Title: Muscular HSP70 content is higher in elderly compared to young, but is normalized after 12 weeks of strength training

doi: 10.1007/s00421-021-04633-4

Figure Lengend Snippet: Percentage changes in cytosolic αB-crystallin ( a ), membrane bound αB-crystallin ( b ), cytosolic HSP70 ( c ), membrane bound HSP70 ( d ) and cytosolic HSP27 ( e ) after the training intervention for three different age groups performing strength training (STG and FTG combined). Stippled line indicates baseline (100%). Figures displays individual values, as well as mean and standard deviations. Red indicates female participants. Blue indicates male participants. *Different from pre ( p < 0.05). #Different between groups ( p < 0.05)

Article Snippet: HSP27 was measured with an in-house-made double-antibody sandwich ELISA using a monoclonal capture antibody against HSP27 (25 ng/well; ADI-SPA-800, Stressgen) and a polyclonal detection antibody against HSP27 (ADI-SPA-803, Stressgen).

Techniques: Membrane

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Differentially expressed genes in limbal epithelial stem/progenitor cell clusters compared to basal corneal epithelial cells isolated by laser capture microdissection (n = 5).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Isolation, Laser Capture Microdissection, Standard Deviation, Binding Assay

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Differential expression of SOX family genes in limbal epithelial progenitor cell clusters compared to basal corneal epithelial cells isolated by laser capture microdissection (n = 5).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Quantitative Proteomics, Isolation, Laser Capture Microdissection, Standard Deviation

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Immunohistochemical localization of SoxE family members in corneoscleral tissue sections. ( A ) Immunofluorescence microscopy demonstrates nuclear staining for Sox8 (clone 4E4.1) and Sox9 (clone 3C10) in suprabasal epithelial cells at the limbus (left column) and central cornea (right column), whereas Sox10 (clone BC34) is confined to few cells in the basal limbal epithelium. Higher magnification images of basal limbal regions (middle column), as indicated by boxed areas, show differential cytoplasmic (arrows) and nuclear localization of Sox8 and Sox9 in basal and suprabasal limbal epithelial cells. ( B ) High magnification images of individual channels show cytoplasmic localization of Sox9 in basal stem/progenitor cell clusters and nuclear localization in suprabasal limbal epithelial cells. ( C ) Double labeling experiments show nuclear co-localization of Sox8 (rabbit IgG) and Sox9 (clone 3C10) (left), distinct localization of Sox9 (clone 3C10) and Sox10 (rabbit IgG) (middle), and localization of Sox10 (clone BC34) to Melan A-positive cells (right). Nuclear counterstaining: DAPI. Individual channels of double labeling experiments are shown in Supplementary Fig. .

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Immunohistochemical staining, Immunofluorescence, Microscopy, Staining, Labeling

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Co-localisation of Sox9 with markers related to progenitor cell phenotype, differentiation and proliferation in the limbal epithelium. Double-labelling demonstrates co-localisation (arrows) of cytoplasmic Sox9 (red) with the stem/progenitor cell markers (green) N-cadherin, p75 nerve growth factor receptor (NGF-R), p63α, Oct4 and cytokeratin (CK) 15) in basal epithelial cells at the limbus. Suprabasal epithelial cells revealed co-localisation of nuclear Sox9 (red) with differentiation-related markers (green) CK3 and Pax6 as well as proliferation-related marker Ki-67. Sox9 monoclonal mouse antibody (clone 3C10) was used for double labelling experiments with polyclonal antibodies against Oct4, p63α, Pax6 and Ki-67, Sox9 polyclonal rabbit antibody (1) was used for double labelling experiments with monoclonal antibodies against p75 NGF-R, N-cadherin, CK3 and CK15. Nuclear counterstaining: DAPI. Individual channels of all double labelling experiments are shown in Supplementary Fig. .

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Marker, Bioprocessing

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Expression of Sox9 during limbal epithelial cell expansion and wound healing in vitro . ( A ) Relative expression levels of Sox9 in cultured limbal epithelial cells expanded as clones on a 3T3 feeder layer or as feeder-free monolayer in passage (P) 0 to P2. Expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays and normalized against GAPDH. Data are expressed as means (2 −∆CT × 1,000) ± SD (n = 3) relative to clonal cultures; *p < 0.01, unpaired t -test. ( B ) Limbal epithelial cell (LEPC) clones (dashed lines) on 3T3 feeder cells (3T3) stain positively for Sox9 (clone 3C10, red), preferentially towards the proliferating border of the clones; Sox9-expressing cells partly co-localize with Ki-67 (green). Nuclear staining: DAPI. ( C ) Immunofluorescent staining shows increased levels of nuclear Sox9 (clone 3C10) in basal/suprabasal limbal epithelial cells and central corneal epithelial cells after epithelial debridement and regeneration compared to unwounded control corneas (the background fluorescence seen in the central corneal stroma may be attributed to the epithelial debridement allowing media and serum components to infiltrate the stroma during the wound healing process). Higher magnification images of limbal epithelial progenitor cell clusters show increased nuclear localization of Sox9 in basal epithelial cells in wound healing conditions compared to cytoplasmic retention of Sox9 in control tissues (inserts). Nuclear staining: DAPI. ( D ) Relative expression of SOX9 in limbal epithelial cells of wounded and unwounded corneas as determined by quantitative real-time polymerase chain reaction (qRT-PCR) primer assays. Normalized data are expressed as means (2 −∆CT × 1,000) ± SD (n = 5) relative to unwounded controls; p = 0.08, unpaired t -test.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, In Vitro, Cell Culture, Clone Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Control, Fluorescence

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Changes in mRNA expression following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. ( A ) Results of quantitative real-time polymerase chain reaction (qRT-PCR) showing reduction of SOX9 mRNA transcripts in cultured limbal epithelial cells 24–96 hours after transfection with siRNA to SOX9 relative to control cells transfected with scramble siRNA (Control) (n = 6; Mean ± SD). ( B ) Transcriptional changes following knockdown of SOX9 in cultured limbal epithelial cells as determined by qRT-PCR. Significant or no relevant changes were seen in the expression levels of stemness-related genes ABCG2 (ATP Binding Cassette Subfamily G Member 2), TP63 (ΔNp63α) and CEBPD (CCAAT/enhancer-binding protein delta); progenitor cell marker genes KRT15 (keratin 15), KRT14 and CDH2 (N-cadherin); differentiation marker genes KRT3, KRT12 and IVL (involucrin); and proliferation-related genes PCNA (proliferating cell nuclear antigen), CDKN1A (cyclin-dependent kinase inhibitor 1A, p21) and CDKN1C (p57). Normalized data are expressed as means (2 −∆CT × 1,000) ± SD (n = 6) relative to scramble siRNA-transfected control cells; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t -test.

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Control, Binding Assay, Marker

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Changes in protein expression and proliferation following knockdown of SOX9 in cultured limbal epithelial cells by RNAi. ( A ) Western blot analysis of limbal epithelial cells transfected with either siRNA that targets SOX9 or non-targeting, scrambled siRNA as a control. Protein expression of Sox9, cytokeratin (CK) 15, CK3 and PCNA was detected with monoclonal antibodies, normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD); *p < 0.05, **p < 0.005, ***p < 0.0001, unpaired t -test. Uncropped versions of Western blots are shown in Supplementary Fig. . ( B ) BrdU incorporation (i.e., cell proliferation) was determined by measuring absorbance at 450 nm. Statistically significant differences were observed at 72 (*p = 0.005) and 96 hours (**p = 0.009) between cells transfected with siRNA that targets SOX9 (Si-Sox9) and control cells transfected with scramble siRNA (Scr-Crtl) (n = 3; mean ± SD).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Expressing, Knockdown, Cell Culture, Western Blot, Transfection, Control, Bioprocessing, BrdU Incorporation Assay

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Interactions between Sox9 and cell signaling pathways. ( A ) Changes in mRNA expression of genes centrally involved in Wnt/ß-catenin signaling, i.e., WNT4 (Wnt-4), CTNNB1 (ß-catenin) and GSK3B (glycogen synthase kinase 3 beta), following knockdown of SOX9 in cultured limbal epithelial cells by RNAi relative to mock-transfected control cells. Normalized data are expressed as means (2 −∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t -test. ( B ) Changes in SOX9 mRNA expression following exposure of cultured limbal epithelial cells with signaling activators lithium chloride (LiCl), IM-12, BMP-2, JAG-1 and SAG as well as signaling inhibitors C-59, DMH1, DAPT and Cyclopamine (Cyclo) for 24 hours relative to vehicle-treated control cells (Ctrl). Normalized data are expressed as means (2 −∆CT × 1,000) ± SD (n = 3); *p < 0.05, **p < 0.01, unpaired t -test. ( C ) Changes in Sox9 protein expression following exposure of cultured limbal epithelial cells to Hedgehog signaling activators Sonic hedgehog (SHH), SAG and Purmorphamine (Purmo); BMP-2; Wnt signaling activators Wnt-3a, lithium chloride (LiCl) and IM-12; and Notch signaling ligand JAG-1 for 48 hours relative to vehicle-treated control cells (Ctrl). Sox9 protein expression was detected with the monoclonal antibody (clone 3C10), normalized to the house-keeping gene ß-actin, and expressed as percent of the expression in control cells (Ctrl); (n = 3; mean ± SD). Uncropped versions of Western blots are shown in Supplementary Fig. .

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions, Expressing, Knockdown, Cell Culture, Transfection, Control, Western Blot

Journal: Scientific Reports

Article Title: Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

doi: 10.1038/s41598-018-28596-3

Figure Lengend Snippet: Model illustrating the mutually repressive interaction of Sox9 and Wnt/ß-catenin signaling as well as potentially involved regulatory signaling pathways in the limbal stem cell niche (mod. after Xu, Z. et al ., Elife 4, e10567 (2015) ( https://creativecommons.org/licenses/by/4.0 ).

Article Snippet: Membranes were blocked with SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific) for 1 hour and incubated overnight using monoclonal mouse antibodies against Sox9 (1:5000; clone 3C10; BioRad), Cytokeratin 3/76 (1:5000; clone AE5; Millipore), Cytokeratin 15 (1:1000; clone EPR1614Y; Abcam) and PCNA (1:5000; clone PC10; Abcam).

Techniques: Protein-Protein interactions

Journal: Medical sciences (Basel, Switzerland)

Article Title: Hemin Promotes Higher Effectiveness of Aminolevulinic-Photodynamic Therapy (ALA-PDT) in A549 Lung Cancer Cell Line by Interrupting ABCG2 Expression.

doi: 10.3390/medsci12040066

Figure Lengend Snippet: Figure 4. Changes in relative gene expression of two PpIX transporters, (A) ABCB1 and (B) ABCG2, after ALA and hemin addition. A one-way analysis of variance (ANOVA) and Tukey’s test were performed to detect differences in mean values between the treated and non-treated samples (n = 3, ns, not significant; **, p < 0.01; ***, p < 0.001 and ****, p < 0.0001). The distribution of each experimental replication is presented as the black dots.

Article Snippet: The membrane was incubated in 5% w/v skim milk in TBST [20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% v/v Tween20] at 4 ◦C for 1 h. The target proteins were captured using primary antibodies against human ABCG2 and ABCB1 (Santa Cruz Biotechnology; 1:200 dilution) and human actin (MP Biochemicals, CA, USA; 1:500 dilution).

Techniques: Gene Expression

Journal: Medical sciences (Basel, Switzerland)

Article Title: Hemin Promotes Higher Effectiveness of Aminolevulinic-Photodynamic Therapy (ALA-PDT) in A549 Lung Cancer Cell Line by Interrupting ABCG2 Expression.

doi: 10.3390/medsci12040066

Figure Lengend Snippet: Figure 5. Differences in (A) ABCB1 and (B) ABCG2 protein expression following ALA and hemin addition. The Western blotting images shown in this figure were derived from the complete image provided in Supplementary Materials. A one-way analysis of variance (ANOVA) and Tukey’s test were performed to detect differences in mean values between treated and non-treated samples at two levels of significance (n = 3; ns, not significant; ***, p < 0.001). The distribution of each experimental replication is presented as the black dots.

Article Snippet: The membrane was incubated in 5% w/v skim milk in TBST [20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% v/v Tween20] at 4 ◦C for 1 h. The target proteins were captured using primary antibodies against human ABCG2 and ABCB1 (Santa Cruz Biotechnology; 1:200 dilution) and human actin (MP Biochemicals, CA, USA; 1:500 dilution).

Techniques: Expressing, Western Blot, Derivative Assay