Notes- Wed March 18

I created this page in hopes of getting someone's notes over the article "Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1" for Figures 1A and 1B… which I missed. Thanks!!!

Charts

1A: Technique: rtPCR— IN VIVO
- Turning on or turning off SIRT1. But are indirectly looking at SIRT1
o Looking at downstream effects of SIRT1 activation
- rtPCR:Looks at mRNa levels for genes
- Brighter band = more mRNA
- Use HeLa cells (cancer cell line)
- Start with mRNA that were treated
o Treatments: Heat Shock, Celastrol, CdCl2, MG132, 18S (ribosome small subunit; positive control)
o GAPDH: enzyme used in glycolysis by all eukaryotes
o Celastrol: antioxidant, plant-derived, anti-inflammatory
 Antioxidant: chemicals that soak up free radicals (thought to cause cell aging)
MG132: inhibits chaperones, stops folding of proteins
CdCl2: general cell toxin

1B: HeLa Cells, rtPCR using siRNA(small interfering RNA)- In VIVO
- Introduced DNA that will transcribe to antisense sirt1 mRNA
- So dsRNA of sirt1, so no translation to SIRT1 protein

1C: Chromatin Immunoprecipitation
- Chromatin: DNA that is wound around protein
- Using antibodies to precipitate out protein/DNA of interest
- Use qPCR to count DNA
- Sirt1 is an in vivo regulator of HSF1 DNA binding activity and hsp70 expression.
- Looking at the interaction of HSF1 DNA binding activity and hsp70 gene.

2A:

AcK= Acetylated lysine

Fusion protein: proteins created through the joining of two or more genes which originally coded for separate proteins
- Flag: a protein that is attached to HSF1 that allows us to see HSF1
- IP: flag  antibody used to drop out HSF1 is against fusion protein
- Primary antibodies used in WB are different in two cases
o Primary antibody is the first used to treat protein
o First Line: level of protein over all; whether lysine are acetylated
o Second Line: lysines are not acetylated
2B: Myc gene:
TSA

2C: SIRT1 H363Y: Histidine  Tyrosine at 363 codon

2D: IP
- Out fusion proteins for looking at HSF protein
- Amount of protein does not change
- AcK
2E:
HSF1 is bound to DNA
- WEstern blot
- cells were treated either with ethanol or resveratrol

2F: Measures how many times HSF1 binds to DNA in a Mock cell vs a cell treated with SIRT1
- heat shock and hours

3A:EMSA: Electrophoretic Mobility Shift Assay
- K80R: Lysine  Arginine at 80 codon
- K80Q: Lysine  Glutamine at 80 codon
- HSF1 is being expressed and is present; however it is not binding to DNA
- Under what circumstances does HSF1 bind to DNA (promoter sequences) to make HS proteins?
- Cell with normal HS factor 1 gene treated with heat
- Acetylation of lycine (needs correct charge) causes expression of HSF1 gene
- Western blot being used

3B: Recombinant HSF1 EMSA and WB — IN VITRO
- Compares EMSA and WB
- 2 transcriptional activators which activate two proteins
- HSF1: binds to DNA because it is a tran. Activator
o Positively charged
o Does not bind to DNA at K80Q
 Therefore it is acetylated; (wont bind while acetylated)
o K80Q mutants is like irreversible acetylation of lysine
- WB: shows that there were more HSF1 protein levels added

3C: treated each version of proteins with Mock, WT, K80R, K80Q
- Wild type is the only DNA that showed relative expression
- In vivo; has to look at expression through living cells

4A: Shows presents of SIRT1 reduces cell death
- Why? SIRT1 deacetylates HSF1
- shocked cells and trypan blue uptake assay (dye that colors dead cells)
o live cells won’t absorb trypan blue and the trypan blue won't pass thru the membranes.
- in dead cells trypan blue can pass thru membranes

4B: Senescent cells: very little Hsp70; doesn’t bind HSF1
- very little Hsp70 produced when compared to early passages
- process: obtain fibroblasts from mice muscles
o plate fibroblasts and fibroblasts begin layering in petri dishes,
o grow my cultures in new dishes from original dishes (early passage  senescenct)
- Unknown: age of rat
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