Statistical analysis

Flow cytometry

Reagents

Central:

Mesomorphic ectomorph:

Endomorphic ectomorph:

Ectomorphic mesomo~ph:

Endomorphic mesomorph:

Ectomorphic endomorph:

Mesomorphic endomor~:

Endomorph-ectom9££E:

Mesomorph-endomorph:

Mesomorph-endomorph:

Balanced ectomorph:

Balanced mesomorph:

Balanced endomorph:

SOHATOCHART

The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.

luctuation Maps

The lacZ U118 is an amber nonsense mutation(Am) that confers Lac─phenotype and also polarity of the downstream lacYA genes in the operon due to premature Rho-dependent transcription termination within the untranslated region of lacZ. Melibiose is a sugar which can only be utilized in a lacZ (Am) strain at high temperature (39 ̊C, when the native melibiose permease is inactive) if the downstream gene lacY encoded permease is transcribed and translated. Therefore, in lacZ (Am) strains, growth on minimal melibiose plates (0.2%) at 39°C reflects transcriptional polarity relief at the lac locus, and the same was scored after streaking the relevant strains on such medium

lacZ (Am) assay

dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium

The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-

trpE(fs) assay

The galEp3 (galE490∗)mutation represents a 1.3 kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to rho dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3mutant or its derivatives was monitored by one of two means. In the first, MacConkey galactose indicator plates (with 1% galactose) were used, where Gal+ colonies are red, and Gal− colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression. In the second method, growth of strains on minimal-galactose (0.2%) was used as a test for Gal+ phenotype

galEp3 assay

In vivo transcription termination assays

A calibration curve of fluorescence intensity values versuspH was prepared for BCECF-AM-loaded wt cells by incubatingcellsin YPD medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucoseand5 μM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values in the range of 4.0-8.0. Fluorescence intensity values were measured by excitation at 440and 490 nm with emission at 535 nm and a graph was plotted between the ratio of intensity at 490 to 440 nm versuspH. Similar to pHi calibration curve, a polynomial distribution of fluorescent intensity signal and pH was observedfor BCECF-AMprobe

In vivovacuolar pH calibration curve

CgRTT107(3.3 kb),CgRTT109(1.3 kb),CgVPS15(3.4kb) and CgVPS34(2.4kb) ORFs were PCR amplified from genomic DNA of the wild-type strain using high fidelity Platinum Pfx DNA polymerase with primers carrying restriction sites for SmaI-SalI,BamHI-SalI,XmaI-XhoIandSalI-XmaI,respectively.Amplified fragments werecloneddownstream of the PGK1promoterin the pGRB2.2 plasmid. Clones were verified by bacterial colony PCR, sequencing and complementation analysis

Cloningof C. glabrataORFs

Table 2.4: List of double-stranded oligonucleotides used in the present study

The DNA-protein complexes were then separated from free oligonucleotides on 6.6% native PAGE gel. The samples were loaded into a native PAGE gel, which was pre-run at constant current (40-50 mA) for 15-30 minutes. Electrophoresis was performed at constant current (80-100 mA), till the bromophenol blue dye front reached 1-2 cm from bottom of the gel. The glass plate was carefully removed without disturbing the gel and the Whatmann filter paper no. 3, cut to the size of the gel, and wasplaced over it. The paper was pressed gently and the gel, which was now firmly stuck on the paper, was covered with saran wrap and kept for vacuum drying on the gel-dryer at 80oC for 1 h. After drying, gel was exposed on a Phospho-imager screen for 12-24 h and scanned on Phospho-imager to detect the band of interest.To determine the specificity of the transcription factor binding or sub-unit interacting to the desired oligonucleotide, super-shift assay was performed. For this, 8-10 μg of nuclear extracts were first incubated with desired antibodies (concentration varies for different) or their isotype control for 1h at 25°C, followed by incubation with binding reaction mixture. The various oligonucleotide sequences used in the present study are listed in Table2.4below

For detection of protein-nucleic acid interaction, an electrophoretic mobility shift assay (EMSA) was conducted as described by Hellman and Fried (2007). Breifly, 8-10 μg of nuclear extract protein was incubated with binding reaction-mixture containing either 32P end-labeled double-stranded oligonucleotides (NF-κB, AP-1, p53 or SP-1) or unlabeled oligonucleotide as shown in table 2.3. The reaction mixture was incubated at 37°C for 45-60 min. After completion of reaction, 6μl of 6X DNA-loading dye was added and mixed well by gentle tapping.Table 2.3: Binding conditions for DNA-protein complexes in EMSA

Gel shift assay

The desired S.cerevisiae strain was grown overnight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500x gin 15 mL polypropylene tubes. Yeast cells were washed with PBS, suspended in 500 μL lysis buffer (Section 2.1.6.2) and were transferred to a 1.5 mL microcentrifuge tube. Yeast cells were incubated for 15 min on a thermomixer set at 65 ̊C and 750 rpm.After incubation, a volume equivalent to 500 μLof glass beads (0.5 mm) were added and cells were lysedon a beating apparatus by mixing three times for 45 seconds each with intermittent cooling on ice to prevent overheating.Cell lysates were centrifuged at 12,000 x gfor 5 min and upper aqueous phase was transferred carefully to a new 1.5 mLmicrocentrifuge tube, to which 275 μL of 3M sodium acetate was added. To this solution, 500 μL of chloroform was added, mixed well, and centrifuged at 12,000 x gfor 5 min at 4°C (this step was repeated twice). Supernatant was transferred to a new 1.5 mLmicrocentrifuge tubeand500 μL of isopropanolwas added and mixed well by inverting the tube 3-4 times. To precipitate genomic DNA, the suspension was centrifuged at 12000 x gfor 15minat 4°C. Precipitated genomic DNA was washed with 70% ethanol and dried at room temperature. The genomic DNA pellet was dissolved either in 100μLof Sigma molecular biology grade water or TE buffer supplied with Qiagen plasmid mini prep kit, andadd 1 μL of RNase solution (30 mg/mL) was added to this and incubated at 37oC for 1 h. The extracted genomic DNA was checked for integrityon a 0.8%agarose gel by electrophoresis and stored at -20oC

Genomic DNA isolation by glass bead lysis method

44KDa to 670 KDa; Ovalbumin (44KDa), Conalbumin (75KDa), Aldolase (158KDa), Ferritin (440KDa), Thyroglobulin (669KDa)] purchased fromGE Healthcare Life Science. Blue dextran was used for the void volume of the column. The molecular weight of the protein complex fractions was calculated from the plot. The different fractions that were collected as eluates from the column were concentrated,and the presence of WWP2 or WWP1 as a monomer or multimer was identified by western blotting using specific antibodies for WWP2 and WWP1

The WWP2-WWP1 heterodimeric complex formation under normal conditions and upon cisplatin-induced stress conditions was studied by gel exclusion chromatography. HEK293T cells untreated or treated with cisplatin were harvested,and cell lysate was prepared by using the standard protocol. Sephacryl S-200 (GE Healthcare) columns were equilibrated with 1X NETN (without Triton X-100) at a flow rate of 1ml/min. 0.8ml of cell lysate (1mg/ml) was passed through the Sephacryl S-200 column,and different fractions (fraction size; 500μl) were collected using Bio-Rad 2110 fraction collectorat the same flowrate. To determine the molecular weight of the fractions, column was calibrated with high molecular weight markers [range

Gel filtration

flash (Thermoscientific). β-Glucuronidase activity for GUS was expressed as nanomoles of MU produced/minute/108 cells

In planta siderophore gene expression was studied by measuring β-glucuronidase activity. GUS marked BXOR1 strain and wild-typeBXOR1 (control) were inoculated in the leaves of 14 day old susceptible Taichung Native 1 (TN-1) variety of rice. After 10 days of inoculation, leaves were crushed and dissolved in 1 ml of MUG extraction buffer without adding MUG substrate (4-methylumbelliferyl β-D-glucuronide). Subsequently, 250 μl extraction buffer containing MUG was added, and incubated at 37°C for appropriate time (Jefferson et al., 1987). Next, 75-μl aliquots were taken from each reaction mixture, and the reaction was terminated by the addition of 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone (MU; Sigma) as standard at excitation/emission wavelength of 365/455 nm, respectively in

In plantaGUS expression assay for siderophore cluster

shRNA were processed for invasion studies. A suspension of 1×105cells in 200μLof 1%FBS containing DMEM was prepared and plated as described in Section 2.2.16. Cells were incubated at 37ºC with 5% CO2for 24 h to allow invasion. Downstream processing of the samples i.e. removing non-invading cells, staining and imaging were followed as described in Section 2.2.16. The number of cells invadedto the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fields. The total number of cells invaded was normalized to non-targeting control cells and expressed as percentage invasion

Invasion assays were conducted as described previously (Raoet al., 2015).Invasion chambers with pre-coated matrigel (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning) were used to conduct the invasion assays. Packages containing invasion chambers were kept outside and allowed to come to room temperature. Inserts were equilibrated by adding with 500 μLwarm (37ºC) 1% FBS containing DMEM to the upper chamber of the insert and bottom well of the companion plate, and rehydrated for 2 h in a humidified tissue culture incubator at 37ºCwith 5% CO2. The rehydration media was removed carefully without disturbing the pre-coated matrigel matrix on the membrane. Meanwhile, HCT116 cells expressing either non-targeting or IP6K1 targeting

Invasion assay