Supplementary Material 1 2 SUPPLEMENTARY METHODS 3 4 Single cell GFP analysis. The analysis was performed as shown by Lohse and 5 Johnson (1) with modifications. Fresh opaque single colonies with both allele of WOR1 6 tagged with GFP (AZC90, AZC100, AZC101, and AZC97) were growing in SC+Glu 7 media at 25°C for 4 hrs till OD 600 around 0.6. Cells were harvested and washed once 8 with PBS before images with DeltaVision Imaging System (Applied Precision) under the 9 GFP channel. The GFP intensity was calculated as the average GFP intensity in cell 10 nuclei analyzed with ImageJ64 (NIH) and MATLAB (MathWorks) (scripts upon request). 11 12 SUPPLEMENTAL FIGURES 13 14 FIG S1 Noise in WOR1 expression in selective mutants are no different from WT. Wor1 15 protein levels in single WOR1-GFP tagged opaque cells were analyzed by GFP signal 16 intensity. F-statistics analysis were performed and showed that there were no difference 17 in the variance of WOR1-GFP signals between med16Δ/Δ, med1Δ/Δ, med20Δ/Δ and 18 WT. More than 200 single cells were analyzed for each strain. Boxes represent 25% 19 percentile and 75% percentile with median. Whiskers represent minimum and maximum. 20 21 FIG S2 Morphology of cells with or without Wor1 overexpression in WT, med3Δ/Δ and 22 med3Δ/Δ complementation strain. Cells transformed with Pmal-WOR1-3HA cassette 23 were growing in either SC+Glu (WOR1 OFF) or SC+Mal (WOR1 ON) overnight at 25°C. 24 The pictures are representatives of cells analyzed in Fig. 5D. 25
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Supplementary Material 1
2
SUPPLEMENTARY METHODS 3
4
Single cell GFP analysis. The analysis was performed as shown by Lohse and 5
Johnson (1) with modifications. Fresh opaque single colonies with both allele of WOR1 6
tagged with GFP (AZC90, AZC100, AZC101, and AZC97) were growing in SC+Glu 7
media at 25°C for 4 hrs till OD600 around 0.6. Cells were harvested and washed once 8
with PBS before images with DeltaVision Imaging System (Applied Precision) under the 9
GFP channel. The GFP intensity was calculated as the average GFP intensity in cell 10
nuclei analyzed with ImageJ64 (NIH) and MATLAB (MathWorks) (scripts upon request). 11
12
SUPPLEMENTAL FIGURES 13
14
FIG S1 Noise in WOR1 expression in selective mutants are no different from WT. Wor1 15
protein levels in single WOR1-GFP tagged opaque cells were analyzed by GFP signal 16
intensity. F-statistics analysis were performed and showed that there were no difference 17
in the variance of WOR1-GFP signals between med16Δ/Δ, med1Δ/Δ, med20Δ/Δ and 18
WT. More than 200 single cells were analyzed for each strain. Boxes represent 25% 19
percentile and 75% percentile with median. Whiskers represent minimum and maximum. 20
21
FIG S2 Morphology of cells with or without Wor1 overexpression in WT, med3Δ/Δ and 22
med3Δ/Δ complementation strain. Cells transformed with Pmal-WOR1-3HA cassette 23
were growing in either SC+Glu (WOR1 OFF) or SC+Mal (WOR1 ON) overnight at 25°C. 24
The pictures are representatives of cells analyzed in Fig. 5D. 25
TABLE S3 Plasmid used in this study Name Parent Plasmid desription Target locus Reference pSN69 _ ARG4 marker _ 2
AZE042 pSN69 5'HR-MED16-ARG4-3'HR med16Δ/Δ This study AZE040 pSN69 5'HR-MED9-ARG4-3'HR med9Δ/Δ This study AZE036 pSN69 5'HR-MED20-ARG4-3'HR med2020Δ/Δ This study AZE039 pSN69 5'HR-MED1-ARG4-3'HR med1Δ/Δ This study
AZE030 pSN69 5'HR-MED3-ARG4-3'HR med3Δ/Δ 4
pTTR01 pSN69 5'HR-MED15-ARG4-3'HR med15Δ/Δ This study AZE043 pSN69 5'HR-MED12-ARG4-3'HR med12Δ/Δ This study Ectopic Wor1 _ adh1: Pmal-WOR1-3HA-
SAT1 ADH1 8
pMB126 _ WOR1-GFP-SAT1 tagging cassette WOR1 1
pFA-HA-
SAT1 _ 3XHA-SAT1 tagging
cassette MED3 9
pFA-HA-
ARG4 _ 3XHA-ARG4 tagging
cassette MED3 9
pFA-HF-SAT1 _ 6XHIS-FLAG-SAT1 tagging
cassette MED15 9
pFA-HF-ARG4 _ 6XHIS-FLAG-ARG4 tagging
cassette MED8 9
pFA-HF-HIS3 _ 6XHIS-FLAG-HIS3 tagging
cassette MED8 9
32
33
34
TABLE S4 Doubling time (in minutes) of Mediator mutants
a dual-layer network controls white-opaque switching in Candida albicans. Mol. 60
Microbiol. 74:1-15. 61
62
7. Lu Y, Su C, Liu H. 2012. A GATA transcription factor recruits Hda1 in response to 63
reduced Tor1 signaling to establish a hyphal chromatin state in Candida albicans. 64
PLoS Pathog. 8:e1002663. 65
66
8. Stevenson JS, Liu H. 2011. Regulation of white and opaque cell-type formation in 67
Candida albicans by Rtt109 and Hst3. Mol. Microbiol. 81:1078-91. 68
69
9. Lavoie H, Sellam A, Askew C, Nantel A, Whiteway M. 2008. A toolbox for epitope-70
tagging and genome-wide location analysis in Candida albicans. BMC Genomics 71
9:578. 72
FIG S1 Noise in WOR1 expression in selective mutants are no different from WT. Wor1 protein levels in single WOR1-GFP tagged opaque cells were analyzed by GFP signal intensity. F-statistics analysis were performed and showed that there were no difference in the variance of WOR1-GFP signals between med16Δ/Δ, med1Δ/Δ, med20Δ/Δ and WT. More than 200 single cells were analyzed for each strain. Boxes represent 25% percentile and 75% percentile with median. Whiskers represent minimum and maximum.
FIG S2 Morphology of cells with or without Wor1 overexpression in WT, med3Δ/Δ and med3Δ/Δ complementation strain. Cells transformed with Pmal-WOR1-3HA cassette were growing in either SC+Glu (WOR1 OFF) or SC+Mal (WOR1 ON) overnight at 25°C. The pictures are representatives of cells analyzed in Fig. 5D.