This expands the role of Skp1 and its modifications selleck bio in developmental regulation, and supports the model that O2 regulates its modification in cells. Cell development Cells were harvested by centrifugation at 4 C, resuspended in PDF buffer, re centrifuged and resuspended in PDF at 108 ml, and deposited on 0. 45 um pore Millipore cellulose ni trate filters for standard development at an air water interface. For submerged development, washed cells were resuspended in PDF at 2 �� 107 ml and 1. 4 ml was deposited into each well of a 6 well bacteriological or tissue culture plate. Plates were incubated for up to 72 h in a sealed plastic box, with in let and outlet ports for gas flow, under room fluorescent lights at 22 C.
The inlet valve was connected via a bub bling water humidifier to a compressed gas tank formu lated with the indicated percentage of O2, with the balance made up of N2. Previously it was shown that in clusion of 1% CO2 did not affect the O2 dependence of culmination. The outlet tube was connected to a Pasteur pipette held under water to monitor gas flow. Cultures were kept unstirred to prevent contact of cells or cell aggregates with the buffer surface, which led to polarization and or floating fruiting bodies. Volume and cell density were optimized for maximal spore differentiation at 100% O2. Alternate buffers, including KP, or Agg buffer, yielded lower spore numbers. Cell aggregates were visualized in a stereomicroscope using transmitted light, or using phase contrast illumin ation on an inverted microscope.
For detection of cellu losic cell walls, samples were analyzed under epifluorescence illumination in the presence of 0. 1% Calcofluor White ST in 10 mM po tassium phosphate, using DAPI filters. Multipho ton confocal microscopy was performed at the OUHSC Imaging Laboratory on a Leica SP2 MP Confocal microscope. For determining spore numbers, samples were supple mented with 0. 2% NP 40, and spores were counted in a hemacytometer. Spores were identified based on their resistance to detergent, shape, refractility, and labeling with Calcofluor White ST or anti spore coat Abs. Spore plating efficiency was determined by spreading an ali quot of detergent treated spores on SM agar in associ ation with Klebsiella aerogenes, and dividing the number of colonies by the counted number of input spores.
Immunofluorescence Spores were released from cysts by probe sonication in 0. 2% NP 40 in KP, centrifuged at 13,000 g �� 10 s, and resuspended in KP buffer. Spores were recovered from fruiting bodies on non nutrient agar by slapping the inverted Petri plate on a counter and washing the spores from the lid, and processed in parallel. An aliquot was treated with Cilengitide 6 M urea, 1% 2 mercaptoethanol in TBS for 3 min at 100 C prior to dilution in cold TBS and recovery by centrifugation.