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E. coli strains were incubated at 37 °C in Luria Bertani medium containing doxycycline at concentrations of 0, 20, 100 and 400 ng mL−1, as indicated.
In general, medium containing doxycycline was changed 2 3 times per week.
For experiments, the cells were induced with complete medium containing doxycycline (100 ng/mL) for 26 28 h for the expression of MAS.
Since Necdin expression was inducible, we chose to maintain all populations in this experiment in medium containing doxycycline to avoid variation.
To measure sister chromatid cohesion, we first arrested strains containing a 7.5 kb centromere-containing minichromosome in G1 with α factor at 25°C and depleted functional Smc proteins by transferring the cells to medium containing doxycycline and galactose at 37°C.
Fresh medium containing Doxycycline was replaced every day.
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Medium containing fresh doxycycline was replenished every 2 days.
Cells were subsequently transferred to medium containing galactose, doxycycline, and nocodazole (instead of α factor) at 37°C.
Cells were harvested, washed once in sterile water and resuspended in the SMM medium containing no doxycycline or glucose for 36 h to allow Gis1 overexpression.
This was replaced by medium containing increasing doxycycline (0 100 μM) to generate a standard curve, against which the mouse serum samples were analysed.
Myogenic differentiation was induced in these cells by administration of doxycycline for 24 hours to activate MYOD1 expression, and then switched to DM containing doxycycline and BETi compounds (or control treatments) for a further 72 hours.
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