Since Tsix-mutant ES cells devoid of H3K27me3 at Xist hyperactivate Xist transcription on differentiation [ 16], it appears that encroachment of this repressive mark renders Xist activation less efficient, probably by inhibition of transcription elongation.
To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p.
Accordingly, in FLIP experiments the fluorescence decayed homogenously over the entire nuclear envelope, whether they contained circles or not (t50 = 46 ± 9.1 s for gcn5∆ and 41 ± 6.7 s for sgf73∆ mutant cells; Figure 8C), as in wild-type cells devoid of circles (t50 = 35 ± 5.9 s).
These mutants were expressed, at comparable levels, in cells devoid of endogenous EGFR.
Cells devoid of Tsc1 are overgrowing under starvation conditions, similar to PTEN mutant cells.
Stability of these mutant DNMT1 proteins was tested by transient transfections into Dnmt1c/c cells devoid of detectable DNMT1 [18] and immunoblotting with the anti-DNMT1 UPT82 antibody (data not shown).
Cell-biological and biochemical evidence suggests impaired autophgay: mutant cells accumulated triacylglycerides and formed enlarged starch granules; they were devoid of autolysosomes although WT cells showed many; and they showed enlarged mitochondria.
Subsequent studies with the pH-sensitive dye LysoSensor Green indicated that most lysosomes in wild-type and mutant cells had similar signal intensities, reflecting the typical acidic pH, but SPG15 cells also harbored numerous large, round subcellular structures devoid of fluorescence (Fig. 4A).
GH680 did not bind to cells devoid of dsRed expression.
In addition, as it is known that 786-O cells devoid of VHL grow as very compact and cohesive spheroids, while VHL-reintroduced 786-O cells grow as loosely arranged spheroids [28], [29], we compared the 786-O cells with wild type VHL or its mutants.
