Rapid spread of mouse mammary tumor virus in cultured human breast cells-10

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p> was visualized by immunofluorescence staining using a monospecific anti-CA serum. Only a small number of MMTV-positive cells was detected in the third-round infected cells 3 days after infection (A), whereas by week five all the cells expressed MMTV antigen (B). The increase was strictly DEX dependent. Upon cultivation of the cells in DEX-free medium no increase in the number of CA-positive cells could be observed (D). (C) Non-infected Hs578T cells. 24 h prior immunostaining all the cells (A, B, C, D) were grown in medium supplemented with 10M DEX. (Scale bar, 50 μm)

Rapid spread of mouse mammary tumor virus in cultured human breast cells-2

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>) or absence (B) of 10M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10M DEX

Rapid spread of mouse mammary tumor virus in cultured human breast cells-11

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p> or absence (B) of 10M DEX. Genomic DNA was extracted from the infected cells at the indicated time points and semiquantitative PCR was performed. NC: non-transduced HS578T cells. PC: second-round infected Hs578T cells. Equal DNA loading was controlled in a PCR assay with GAPDH-specific primers (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the time-course experiment. (D) Equal loading of the PCR reactions was controlled in a Real-time TaqMan PCR specific for GAPDH gene. (E) The viral RNA was quantified by Real-time RT-PCR in cell culture fluids of the infected Hs578T cells grown either in the presence or absence of 10M DEX

Rapid spread of mouse mammary tumor virus in cultured human breast cells-3

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p> of 10M DEX. One week before immunofluorescence staining the cells were cultivated in the absence of the glucocorticoid analog and 24 h prior immunofluorescence staining with anti-CA antibodies the production of MMTV-specific proteins was either induced (A) or not (B) by addition of 10M DEX in the cell culture media. The nuclei of the cells were counterstained with DAPI. (C) Western blot detecting the expression of gp52 and gp36 Env proteins in the second-round infected HS578T cells. Lane 1, non-infected Hs578T cells, NC; lane 2, infected human cells not stimulated with DEX; lane 3, infected human cells in which the expression of MMTV structural proteins was induced by 10M DEX 24 h before protein harvest

Rapid spread of mouse mammary tumor virus in cultured human breast cells-4

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>ned by PCR. The virus released from the second round infected Hs578T cells was, prior infection, pre-incubated either with anti-MMTV neutralizing antibody (Ab) or PBS. Where indicated AZT was added to the cells infected with the virus. NC: non-infected Hs578T cells. M: 1 kb marker. (B) Spread of the virus was abrogated in medium containing AZT. The third-round infected Hs578T cells were cultured for four weeks in medium containing DEX either supplemented with AZT or not and the presence proviral DNA was monitored by a semiquantitative PCR. GAPDH-specific PCR was used to demonstrate equal loading of all PCR reactions (bottom panels). M: 1 kb marker. (C) Real-time TaqMan PCR quantifying proviral loads in the infected Hs578T cells during the AZT treatment experiment. (D) Equal loading was contolled in a Real-time TaqMan PCR specific for GAPDH gene

Rapid spread of mouse mammary tumor virus in cultured human breast cells-7

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>he cell lysates of GR cells (lane 1) and third-round infected Hs578T cells (lane 2) were submitted to digestion with NdeI. As a digestion control, PCR product obtained by amplification of MTV-8 sequences using pGR16 plasmid as a template, was digested with the same restriction enzyme (lane 3); M, 1 kb marker; black arrow indicates undigested product; open arrows denote fragments resulting from NdeI digestion. (B) Schematic drawing showing NdeI site in the gene of the Mtv-17 and Mtv-8 viruses and the length of the respective restriction fragments

Electron microscopy of viral particles released from the infected Hs578T (A) and GR cells (B)

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p

Rapid spread of mouse mammary tumor virus in cultured human breast cells-0

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>d with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker

Rapid spread of mouse mammary tumor virus in cultured human breast cells-8

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>d for amplification and cloning of MMTV sequences. Sequences of sixteen clones were aligned. Mtv-2 and Mtv-17 (accession number AF263910, sequence is shaded) sequences were included in the alignment. Putative heparin binding domain (HBD) and receptor binding site (RBS) are boxed and amino acid residues representing these regions are shown above the boxes. Non-synonymous mutations in proviral sequences from infected cells resulting in an amino acid exchange are indicated. The coordinates of the Mtv2 nucleotide sequence are according to the MMTV reference strain (accession number M15122)

Rapid spread of mouse mammary tumor virus in cultured human breast cells-9

<p><b>Copyright information:</b></p><p>Taken from "Rapid spread of mouse mammary tumor virus in cultured human breast cells"</p><p>http://www.retrovirology.com/content/4/1/73</p><p>Retrovirology 2007;4():73-73.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2169256.</p><p></p>d with MMTV(GR) were monitored for 20 weeks. Genomic DNA was harvested at week one, six and 20 after infection, respectively, and analyzed by PCR for the presence of MMTV sequences. NC: non-infected cells. M: 1 kb marker. (C) Three infectious cycles were performed in Hs578T cells. The cells infected with MMTV(GR) virus are denoted as first infection cycle. The cell culture supernatant from these Hs578T cells was used in a subsequent infection round. Medium from the second-cycle infected Hs578T cells was used for third infection cycle. M: 1 kb marker. (D) Heat inactivation of the MMTV(GR). Where indicated (heat +) was the virus subjected to the heat treatment (60°C for 10 min). NC: non-infected cells, M: 1 kb marker