High Throughput Screening System for Inhibitors of Human Heat Shock Factor 2

High throughput screening system for inhibitors of human Heat Shock Factor 2

Levi M. Smith, Dwipayan Bhattacharya, Daniel J. Williams, Ivan J. Dixon, Nicholas R. Powell,

Tamara Y. Erkina, and Alexandre M. Erkine1*

1 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,

Butler University, Indianapolis, IN 46208, USA

* To whom correspondence should be addressed. Tel: 317-9408569; Fax: 317-9406172; Email: [email protected]

GROWTH RESTORATION ASSAY (OD600) PROTOCOL

WORK MUST BE CONDUCTED USING ASEPTIC TECHNIQUE (sterile media, equipment, gloves). DAY 1

STARTER CULTURES

1. Inoculate starter culture for each strain from fresh, isolated colony. (See NOTE 1.)  LNL4-pYES – empty pYES vector LNL4-pYES-hHSF2 – hHSF2 overexpressing  Choose large colonies of the same size.  Transfer colony to 5mL synthetic –ura dropout Raffinose growth medium.

Note: growth medium is sterilized after reconstitution from the following

chemicals (see manufacturer labels for reconstitution instructions): (i) Yeast

Nitrogen Base without amino acids, without carbohydrate, and with Ammonium

Sulphate (US Biological Cat # Y2025), (ii) Drop-out mix synthetic minus Uracil without nitrogen base (US Biological Cat# D9535), (iii) Raffinose (US Biological

Cat# R1030) or Galactose (Sigma Cat#G0750-500G).

2. Grow o/n with shaking @ 30°C.

 Grow >18h for culture in late log phase (OD600 3.0).  Grow ~12h for culture in early log phase (OD 0.1-0.4).

 Note: An OD600 reading over 0.5 is inaccurate. Be sure the read value is

0.1-0.5. In the case of a higher OD reading, dilute cells appropriately for

OD600 reading to be within 0.1-0.5 range.

3. Pellet cells by centrifugation 5min at 1000 × g. and resuspend pellet in the same amount of sterile water. 4. Wash cells twice with sterile water, repeating centrifugation.

5. Inoculate –ura dropout Galactose growth media with LNL4-pYES and with LNL4-

pYES-hHSF2 (1 ml of culture with OD600 0.2 per 50 ml of medium). Since LNL4- pYES-hHSF2 has slow growth phenotype, inoculate twice as much cells for this

strain in comparison to LNL4-pYES, shooting for the same OD600 after overnight growth. (See NOTE 1A).

DISPENSING AND GROWTH

1. Verify that cultures have similar OD600 (within 0.2 OD difference, if OD difference exceeds 0.2, restart cultures using appropriate dilution of overnight cultures. (See NOTE 2.)

2. Calculate volume of cells required to inoculate 20mL media to OD600 0.01 for

each strain.

3. Transfer 20mL of each required sterile media to sterile 50mL conical tubes. Label. (See NOTE 4.)  USE STERILE PIPETTE.  Be mindful of Glucose contamination. If using Glucose-containing media for controls, use SEPARATE pipettes for control culture media and experimental culture media.

4. Transfer calculated volume of each strain into labeled tube/media from step 7.  VORTEX cells × 20s IMMEDIATELY prior to transfer. 5. Prepare necessary sterile 96-well plate for dispensing. Label layout.  BE SURE to use appropriate plate type (clear, flat bottom for OD; white opaque for LUM)  It may be helpful to print a copy of the planned plate layout, if complex.

 If small-molecule compounds are not to be used in the assay, proceed to step 13.

6. Prepare 1mM stock of compounds to be tested, in DMSO. (See NOTE 5.)

7. Calculate required volume of 1mM stock to reach desired final concentration in 200μL cultures. (See NOTE 6.)  Ensure volume of compound to be transferred is 2μL.  FINAL concentration of DMSO in each well should be 1%

8. Dispense calculated volume of compounds into wells, according to plate layout.

9. Dispense 2μL DMSO into each well that does not have an experimental or control compound.  At this point, visually ensure that ALL wells are properly loaded with either compound or DMSO.

10. Dispense 200μL appropriate growth media into media-only control wells. (See NOTE 7.)  Control well(s) are necessary to use as “blank” values in the plate reader.

11. Dispense 200μL cell culture (OD600 0.01) into each well.  Dispense using Eppendorf repeater with STERILE combitips. (See NOTE 8.)  VORTEX cell culture × 20s IMMEDIATELY PRIOR to dispensing. Cells are precipitating within minutes, and delay from mixing to dispensing will severely affect noisiness of the assay.  If dispensing manually: continuously shake or stir culture in sterile beaker during dispensing.

12. Apply film seal, or plate cover with tape around perimeter to seal. (See NOTE 9.)

13. Place plate in 30°C with orbital shaking × 22h. (See NOTE 10.)  If using plate reader, set shaking to ORBITAL; FAST; 1mm magnitude.

TERMINAL READING AND DATA COLLECTION For OD readings, it is important:  to add Tween 20 to final concentration of 0.5% into each well, as it will eliminate errors caused by uneven menisci between wells.  to shake plates so that cells are evenly suspended and there is no cell collection on the walls or on specific spots on the well floor.

14. Dispense 150μL Tween 20 into each well of one column in an empty microplate.

15. Turn on plate reader, open GEN5 and prepare “new experiment” as follows:  SHAKE: orbital; fast setting; 4mm amplitude × 30s  READ: Absorbance; endpoint; 600nm.

16. Remove plate cover; resuspend cells by pipetting × 20, using 8-channel 10 μL pipette.  Ensure culture in each tip is fully expelled after resuspending.  Cells pellet on well bottoms; pipetting must resuspend cells from ENTIRE surface of well floor. (See NOTE 11.)

17. Quickly dispense 10μL 10% Tween 20 into wells using 8-channel 10μL pipette.  Do not eject

past first stop when dispensing, to prevent bubbles. (See NOTE 12.)

18. Place plate on tray; click green arrow in GEN5 to read plate.  When finished, be sure to save data.  GEN5 will not function with plate reader turned off.  TURN OFF PLATE READER when data is saved and last plate is removed.

1. Selected colonies should be freshly streaked, ideally 2-3 days prior. pYES contains the URA3 gene, allowing selective plating using –ura agar media. For consistency, the carbon source when growing colonies can be raffinose. With raffinose there is no difference in growth rate of LNL4-pYES and LNL4-pYES- hHSF2 strains, while with galactose containing medea the LNL4-pYES-hHSF2 strain growth significantly slower.

1A. Based on our time-course experiments the over-expression of hHSF2 reaches maximum (based on Western blotting) after 16 hours of growth in Galactose containing medium, so the maximum growth rate difference between the LNL4-pYES and LNL4-pYES-hHSF2 strains also reaches maximum after the overnight growth on galactose containing medium.

2. To obtain reliable results, it is imperative that all starter cultures (including control

strains) are grown to the same growth phase (OD600 0.2-0.5). The closer cultures

are to the stationary phase (OD600 3.0) the more difference in growth rate can be observed between LNL4 pYES and LNL4 pYES-hHSF2 strains, but that merely reflects that it takes longer time for LNL4 pYES-hHSF4 than for LNL4 pYES to transition to Log phase.

3. Cell dilutions must be accurate. When determining OD600 of cells (reconstituted in sterile water), take at least three OD readings of the sample, and average the values. Use this averaged value to calculate the volume needed to generate a cell dilution of 0.01 in 20 mL of media (step 5). If enough extra cells are available, it is highly advisable to prepare more than one sample for OD measurement. 4. Experience, to date, suggests that freshly prepared media is favorable. While no definite “expiration time” has been determined for sterilized media, in our hands, contamination was not seen in media stored less than two weeks. 5. Stock solutions of control compounds should be prepared in aliquots for single- use. This minimizes freeze-thaw cycles that may structurally compromise the compounds. 6. Recent studies support a final concentration of 10μM for experimental compounds in high-throughput screens involving HSF, but concentrations of 1μM-10mM have been used. {[Qingyan Au. J Biomol Screen 2009 14: 1165.]; [Barbara Calamini. Nature Chemical Biology 2012 8: 185-196.]; [Daniel W. Neef. PLOS Biology 2010 8(1):e1000291.]}

7. “Blank” wells, containing only growth media, serve two functions: provision of wells to establish a zero value for blanking spectrophotometric readings, as well as demonstration of sterility of media and verification of aseptic technique. Despite a practiced hand, this verification is NECESSARY to control for contamination. 8. Despite the use of a repeater/dispenser, dispensing can generate significant error. Work at a steady (not hurried) pace, and dispense each strain in the same geometric pattern (e.g. column-wise, left to right). Be sure to expel the first two and last aliquots from the Combitip back into the dilution (50mL conical)—these likely will contain air, and therefore dispense less than the desired volume. 9. If assay is incubated in plate reader, a film cover must be used. Do not insert plates with lids into the plate-reader chamber! If performing a growth curve using the plate reader, expect the film cover to distort as the cells produce CO2. This inevitably produces inaccuracy, but does so with both control and experimental wells. This phenomenon can be reduced by changing the film covers during the curve at regular 8 or 12 hour intervals. 10. Be aware that temperature variations exist between incubators, and also within an incubator’s chamber. Consecutive assays should be performed using the same incubator, as well as the same location within the incubator. 11. To ensure consistency in methods, hold pipette vertically. Pipette up and down 10x as you move the tips around the perimeter of the wells, then move to the center of the well for the remaining 10x, to resuspend all cells bound to well floors. 12. Use of a technique called “reverse pipetting” can further reduce bubble formation. When first pulling Tween 20 into tips, push past first stop, to load MORE than desired volume into tips; then, when dispensing, simply depress until first stop. Accuracy is maintained, and NO air bubbles reach the opening of the tips.