I am trying to see the autophagy levels by inducing with the Rapamycin in a mammalian cells and measure the gene levels by Real time PCR. Has anyone worked with this concept? I need your suggestions?
For autophagy study, you could try mRFP-GFP-LC3 and GFP-LC3 labeling system, which can detect the intensity of autophagy flux in real-time, in which GFP and/or RFP tags are fused at the N-termini of the autophagosome marker LC3. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not. Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.
You could find more on this website: www.genemedi.net/i/autophagy
Genemedi provides the production service of AAV, adenovirus and lentivirus encoding mRFP-GFP-LC3 or GFP-LC3, suitable for monitoring the intensity of autophagy flux in real-time in vivo or in vitro.
Attached file is an image of mRFP-GFP-LC3 labeled A549 cells with nutrition deprivation.
ATG4A, ATG4B, ATG5,ATG7, ATG12, BECN1, LC3, and Vps34 are the essential molecules involved in the autophagy. But I think you should also evaluate the molecules both related to apoptosis and autophagy characterized by BCL2 homology-3 (BH3)-only proteins. TP53, BAD, BAX, BCL2, CASP3, and CASP8 should be checked in order to rule out the possibility that apoptosis is not the significant event in your experiment,
I'm sure there are plenty of microarray datasets published showing the effect of mRNA expression by Rapamycin. Here's an early one in T-cells: http://www.jbc.org/content/277/25/22175/T1.expansion.html
This review has a nice list of genes regulated transcriptionally in autophagy and that are also involved in autophagy: http://www.nature.com/nrm/journal/v15/n1/fig_tab/nrm3716_T1.html
Gene's I've personally seen transcriptionally induced by rapamycin include:
KLH24, also seen by these authors: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2547001/
PDK4, also seen by these authors in response to Torin1: http://nar.oxfordjournals.org/content/40/10/4368.full
Torin1 will give a much more profound effect compared to Rapamycin if you want mTOR inhibition-inhibition induced autophagy.
This dataset shows some nice Torin1-induced genes in MEFs:
http://sabatinilab.wi.mit.edu/pubs/SUPPLEMENTARY/Thoreen%20Table%201.xls
You will want to sort this column largest to smallest: WT Transcript: log2(Torin1/Vehicle)
They don't see Atg genes, but instead (Klhl24, Ccng2, etc.)
I published an article about transcriptional regulation of autophagy genes induced by amino acid starvation, these genes are also regulated by ER stress and a big part of them is regulated by rapamycin (no published results) so check the article published in NAR, 2013...
VMP1 is a new autophagy related gene, which expression triggers autophagy in mammalian cells. We use QPCR of VMP1 to know if VMP1-mediated autophagy is activated.
J Biol Chem. 2007 Dec 21;282(51):37124-33
Hi, my question is similar to this.
I am doing Exps on the relationship between HBV and HCC. And we hypothesis that autophagy and EMT would have relationship involved.
However, my question is that: can I use the genes changes to reflect the subtle autophagy on the cell culture model?
Why I ask this question is that: autophagy mainly occurs in cytosome so it did not include chromosome activity. How to interpret this question?
Anyone can answer me?
For autophagy study, you could try mRFP-GFP-LC3 and GFP-LC3 labeling system, which can detect the intensity of autophagy flux in real-time, in which GFP and/or RFP tags are fused at the N-termini of the autophagosome marker LC3. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not. Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.
You could find more on this website: www.genemedi.net/i/autophagy
Genemedi provides the production service of AAV, adenovirus and lentivirus encoding mRFP-GFP-LC3 or GFP-LC3, suitable for monitoring the intensity of autophagy flux in real-time in vivo or in vitro.
Attached file is an image of mRFP-GFP-LC3 labeled A549 cells with nutrition deprivation.
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