3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities

Influensavaksine, en spørreundersøkelse blant apotekkunder

Hensikt: Å kartlegge apotekkunders erfaring med og vilje til å ta influensavaksine, samt utvalgte faktorers sammenheng med ­vaksineringsvilje, inkludert sannsynlighet for å ta covid-19-vaksine. Materiale og metode: Masterstudenter i apotekpraksis intervjuet apotekkunder ved hjelp av spørreskjema. Spørsmålene omfattet erfaring med influensavaksine, risikogruppestatus, vaksineringsvilje, preferanser ved influensavaksinering, sannsynlighet for å ta covid-19-vaksine, antall faste legemidler og sosiodemografi. Resultater: Blant 871 apotekkunder hadde 562 tatt influensavaksine tidligere. Andelen vaksinerte var høyere med høyere alder. Majoriteten var villig til å ta influensavaksine (54 % garantert, 19 % trolig), og kun 16 % var lite/ikke villig. Vaksineringsviljen var positivt assosiert med å ha tatt influensavaksine tidligere, å være i risikogruppen eller ha forhøyet smitterisiko, høyere betalingsvilje, og sterkere tro på vaksineeffekten. Sannsynlighet for å ta covid-19-vaksine var positivt korrelert med vilje til å ta influensavaksine. Apotek var foretrukket vaksinasjonslokale blant 33 %, mens 42 % foretrakk legekontor. Deltakerne rangerte tilgjengelighet og åpningstider som viktigste faktorer ved influensavaksinering, mens pris var mindre viktig. Konklusjon: Norske apotekkunder er positive til influensavaksine og rapporterer også høy sannsynlighet for å ta covid-19-vaksine. Vaksineringsviljen er sterkere blant tidligere vaksinerte og personer i risikogruppen. Nesten like mange foretrekker vaksinering på apotek som legekontor

Influenza-vaccine – a survey among pharmacy customers

Source at https://www.farmatid.no/vitenskap-og-fag/vitenskapelige-artikler/article-3300.Hensikt: Å kartlegge apotekkunders erfaring med og vilje til å ta influensavaksine, samt utvalgte faktorers sammenheng med ­vaksineringsvilje, inkludert sannsynlighet for å ta covid-19-vaksine. Materiale og metode: Masterstudenter i apotekpraksis intervjuet apotekkunder ved hjelp av spørreskjema. Spørsmålene omfattet erfaring med influensavaksine, risikogruppestatus, vaksineringsvilje, preferanser ved influensavaksinering, sannsynlighet for å ta covid-19-vaksine, antall faste legemidler og sosiodemografi. Resultater: Blant 871 apotekkunder hadde 562 tatt influensavaksine tidligere. Andelen vaksinerte var høyere med høyere alder. Majoriteten var villig til å ta influensavaksine (54 % garantert, 19 % trolig), og kun 16 % var lite/ikke villig. Vaksineringsviljen var positivt assosiert med å ha tatt influensavaksine tidligere, å være i risikogruppen eller ha forhøyet smitterisiko, høyere betalingsvilje, og sterkere tro på vaksineeffekten. Sannsynlighet for å ta covid-19-vaksine var positivt korrelert med vilje til å ta influensavaksine. Apotek var foretrukket vaksinasjonslokale blant 33 %, mens 42 % foretrakk legekontor. Deltakerne rangerte tilgjengelighet og åpningstider som viktigste faktorer ved influensavaksinering, mens pris var mindre viktig. Konklusjon: Norske apotekkunder er positive til influensavaksine og rapporterer også høy sannsynlighet for å ta covid-19-vaksine. Vaksineringsviljen er sterkere blant tidligere vaksinerte og personer i risikogruppen. Nesten like mange foretrekker vaksinering på apotek som legekontor.Aim: To describe pharmacy customers’ experience and inclination toward influenza vaccination and factors associated with willingness to vaccinate, including the probability of taking covid-19-vaccine. Material and methods: Pharmacy students interviewed pharmacy customers using a questionnaire which covered influenza vaccine experience, risk group status, willingness to vaccinate, vaccination preferences, probability of taking covid-19-vaccine, number of medications and socio-demography. Results: Among 871 pharmacy customers, 562 had previous influenza vaccine experience. The vaccinated proportion increased with higher age. The majority, 73%, were willing to take the influenza vaccine while 16% were not/marginally willing. Willingness to vaccinate was positively associated with previous experience, being in a high-risk group or having elevated contagion risk, higher willingness to pay and stronger belief in vaccine effect. The probability of taking covid-19-vaccine was positively correlated with willingness to vaccinate. Pharmacy was the preferred location for influenza vaccination for 33% while 42% preferred the general practitioner’s office. Availability and opening hours were the most important preferences regarding influenza vaccination. Conclusion: Norwegian pharmacy customers are positive toward influenza vaccination, and also report high probability of taking covid-19-vaccine. Willingness to vaccinate is stronger among people with influenza vaccine experience and people at risk. Almost as many prefer to be vaccinated in a pharmacy as in the general practitioner’s office

Pax6 Regulates the Expression of Dkk3 in Murine and Human Cell Lines, and Altered Responses to Wnt Signaling Are Shown in FlpIn-3T3 Cells Stably Expressing Either the Pax6 or the Pax6(5a) Isoform

<div><p>Pax6 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the <i>Dkk3</i> gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of <i>Dkk3</i> expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse <i>Dkk3</i> promoter. The minimal <i>Dkk3</i> promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse <i>Dkk3</i> promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of <i>Dkk3/REIC</i> expression in human cell lines. Six of eight cell lines tested showed changes in <i>Dkk3/REIC</i> expression after <i>PAX6</i> siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active β-catenin and GSK3-β Ser9 phosphorylation were measured after LiCl stimulation.</p></div

Oligonucleotides used in EMSA and <i>in vitro</i> mutagenesis.^#

<p>#The reverse complement of each oligonucleotide was also ordered.</p

FlpIn-3T3 cells stably expressing Pax6 or Pax6(5a) have increased expression of Dkk3.

<p>A, RT-qPCR shows a 6-fold expression of <i>Dkk3</i> in the Pax6 cell line, and a 24-fold expression of <i>Dkk3</i> in the Pax6(5a) cell line, compared to the untransfected FlpIn-3T3 control cell line. B, Western blot confirms higher expression levels of Dkk3 protein in the two Pax6 containing cell lines. The observed molecular weight of Dkk3 is between 60 and 70 kD due to glycosylation. For Pax6 it is 48 kD, Pax6(5a) 50 kD and for Actin 43 kD. C, Western blot of fractionated cell extracts showing expression of Dkk3 and Pax6 (C; cytoplasm, N:E; nuclear extract, N.P; nuclear pellet). The nuclear pellet contains chromatin and remnants of the cell membrane. D, confocal imaging of immunostained FlpIn-3T3Control, -Pax6 and -Pax6(5a) cells demonstrates that the Dkk3 protein is localized in cytoplasm and nucleus. Draq5 (blue) was used to visualize the nuclei. Indicated scalebar = 20 µM.</p

Primers for RT-qPCR.

<p>Abbreviations: <i>h</i>; human, <i>m</i>; mouse, <i>DKK3</i>; Dickkopf 3, <i>Nono</i>; Non-POU-domain-containing octamer binding protein.</p

Qiagen Quantitect primer assays.

<p>Abbreviation: <i>h;</i> human, <i>m;</i> mouse <i>TFRC</i>; Transferrin receptor, <i>GUSB</i>; Glucuronidase beta.</p

PAX6 regulates the expression of Dkk3 in several human cell lines derived from both normal and tumor tissues.

<p>A, RT-qPCR of human cell lines where PAX6 has been knocked down either stably by shRNA (HPAFII), or transiently by siRNA (H460, H661 and GaMG). The result is the mean of 3 independent experiments. B, Western blot of the pancreatic adenocarcinoma cell line HPAFII stably transfected with Pax6 shRNA. C, Western blot of H460 and H661 (lung cancer), PC3 (prostate cancer), GaMG (glioblastoma) and B3 (normal lens epithelium) cell lines with transient knockdown of PAX6 with siRNA. Each western blot in B and C is representative of at least two independent experiments. D, alignment of the human and mouse <i>Dkk3</i> promoter showing the localisation of the bioinformatically predicted and evolutionary conserved BSAP (Pax5)-, Pax6- and Pax6(5a) binding sites. The start point for most transcripts in the mouse <i>Dkk3</i> promoter is marked by an arrow (TSS-2). The human <i>Dkk3/REIC</i> promoter also contains multiple transcripts initiating in this area according to DBTSS. 5aCON½ is a Pax6(5a) consensus halfsite with 2 mismatches.</p