Validation & Assay Performance Summary CellSensor HSE-bla HeLa Cell Line Cat. no. K Pathway Description Activation of the heat shock response/unfolded protein response (HSR/UPR) occurs in response to a diversity of chemical, environmental, and physiological stress conditions. Transcriptional regulation of the human HSR is mediated by a family of three heat shock transcription factors (HSFs), HSF-, -, and -. Stress-induced activation of quiescent HSF monomers results in their trimerization and accumulation in the nucleus, wherein they bind to and upregulate transcription of target genes (e.g. molecular chaperones, certain proteases, and other stress response genes) harboring a heat shock element (HSE). Downstream expression of heat shock protein family members (e.g. Hsp and Hsp) that function as molecular chaperones to guide conformational states of client proteins is essential to maintaining the health of cells and protecting them from various acute and chronic stress conditions. As a result, HSR activation may provide therapeutic benefit to certain types of tissue trauma (e.g. brain and heart ischemia) and neurodegenerative disorders (e.g. Huntington disease, Alzheimer disease, and Parkinson disease). Conversely, since aberrant expression of chaperones has been associated with tumorigenesis, compounds that down-regulate the HSR and chaperone levels could provide useful tools for combating cancer. Cell Line Description To generate an effective readout for interrogating the HSR pathway, we engineered HeLa cervical cancer cells with an HSE driving beta-lactamase reporter gene expression (HSEbla). A stably integrated pool of heat shock responsive cells was isolated by FACS and further evaluated using an inhibitor of Hsp, -AAG. Hsp inhibition is known to upregulate HSF- activity leading to potent induction of the HSR, which can be readout using HSE-bla HeLa cells. Cell stress (heat shock, oxidative stress, heavy metals, infection) Small molecule inducers (e.g. Hsp inhibitor -AAG) HSF HSFHSF HSF HSE BLA Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
Validation Summary Testing and validation of this assay was evaluated in -well format using LiveBLAzer -FRET B/G Substrate.. Primary agonist dose response under optimized conditions (n=) Average -AAG EC = nm Average Z -Factor (EC ) =. Average =. Recommended cell no. =, cells/well Recommended [DMSO] = up to. % Stimulation Time = hours Max. [Stimulation] = nm -AAG. Activator panel hour stimulation EC -AAG EC Bortezomib/Velcade EC Celastrol Overnight stimulation EC -AAG EC Bortezomib/Velcade EC Celastrol. Inhibitor Dose Response = nm = μm. μm = nm =. μm =. μm Primary Agonist Dose Response Figure -AAG dose response under optimized conditions Log [nm] -AAG day day day HSE-bla HeLa cells were assayed on three separate days in - well assay format in Assay Medium at, cells/well. Following overnight incubation, serial dilutions of the Hsp inhibitor - AAG were applied to the wells (. % final DMSO) for h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). IC Quercetin =. μm. C Heat Shock Response. Stealth RNAi Testing. Assay-ready Cryopreserved Cells Testing. Cell culture and maintenance See Cell Culture and Maintenance Section and Table Assay Testing Summary. Assay performance with variable cell number. Assay performance with variable DMSO concentration. Assay performance with variable substrate loading time. Assay performance with variable stimulation time Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
Activator Panel Figure A Activator Panel Dose Response ( h stim) Inhibitor Dose Response Figure Inhibitor Dose Response Log [nm] compound -AAG Puromycin Celastrol Arachidonic acid Bortezomib/Velcade Prostaglandin PGA - - - Log [um] Quercetin Quercetin HSE-bla HeLa cells were assayed in -well assay format in Assay Medium at, cells/well. Following overnight incubation, -point serial dilutions of compounds were applied to the wells (. % final DMSO) for ~ h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the compound treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Note that high concentrations of Puromycin and Celastrol exhibited significant cytotoxicity and were excluded from the analysis. Figure B Activator Panel Dose Response ( h stim) Log [nm] compound -AAG Puromycin Celastrol Arachidonic acid Bortezemib/Velcade Prostaglandin PGA HSE-bla HeLa cells were assayed in -well assay format in Assay Medium at, cells/well. Following overnight incubation, -point serial dilutions of inhibitor Quercetin were applied to the wells and incubated for ~ h prior to stimulating them with nm -AAG for ~ h (. % final DMSO) and then loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the inhibitor treated wells from the / ratios obtained with the untreated control wells (n = for each data point). The highest concentrations of Quercetin exhibited significant cytotoxicity and were excluded from the analysis. C Heat Shock Response Figure C Heat Shock Response vs. -AAG. Unstim -AAG ^C HSE-bla HeLa cells were assayed in -well assay format in Assay Medium at, cells/well. Immediately following cell HSE-bla HeLa cells were assayed in -well as say format in plating, -point serial dilutions of compounds were applied to Assay Medium at, cells/ well on two different plates. the wells (. % final DMSO) for ~ h prior to loading the wells Following overnight incubation at C, Assay Medium containing with LiveBLAzer -FRET B/G Substrate (µm final concentration DMSO (to simulate compound addition with final.% DMSO) of CCF-AM) for hours. Emission values at nm and nm was applied to first plate, which was then subjected to heat shock were obtained using a standard fluorescence plate reader. at C for hour followed by a hour incubation at C before s were calculated by dividing the / ratios loading with substrate. To the second plate, Assay Medium of the compound treated wells from the / ratios obtained containing DMSO was applied to the unstimulated control wells with the untreated control wells (n = for each data point). Note while nm final -AAG was applied to the stimulated wells that high concentrations of Puromycin and Celastrol exhibited (. % final DMSO) followed by a hour incubation at C prior significant cytotoxicity and were excluded from the analysis. to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the heat shock treated wells or the compound treated wells from the / ratios obtained with the unstimulated control wells (n = for each data point). Below are shown representative fluorescent images. Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
nm/nm Stealth TM RNAi Testing Figure A RNAi panel, -AAG stimulated.......... NT unstim NT stim mock MedGC b-lac HSF- HSF- HSF- HSF- HSF- HSF- HSF- HSF- HSF- reader and the / ratios were plotted (n = for each data point). Below are shown representative fluorescent images. Assay-ready Cryopreserved Cells Testing Figure A -AAG activator dose response curve.... -AAG (cryo) -AAG (dividing) HSE-bla HeLa cells were plated in -well format in Growth Medium at, cells/well and reverse transfected using using Lipofectamine TM RNAiMAX Transfection Reagent and nm of Stealth RNAi duplexes against HSF, HSF, and HSF. Controls were as follows: nontransfected and unstimulated cells (NT unstim), nontransfected and stimulated cells (NT stim), mock transfected (no RNAi duplex), Stealth RNAi Negative Control Med GC, and Beta-lactamase positive control RNAi duplex. At ~ hours post-transfection, a nm final concentration of - AAG was applied to the wells and the plate was incubated for an additional hours prior to loading with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Fluorescence emission values at nm and nm were obtained using a standard fluorescence plate reader and the / ratios were plotted (n = for each data point). Below are shown representative fluorescent images. nm/nm Figure B RNAi panel, heat shock stimulated........ NT mock MedGC b-lac HSF- HSF- HSE-bla HeLa cells were plated in -well format in Growth Medium at, cells/well and reverse transfected using using Lipofectamine TM RNAiMAX Transfection Reagent and nm of Stealth RNAi duplexes against HSF, HSF, and HSF. Controls were as follows: nontransfected cells (NT), mock transfected (no RNAi duplex), Stealth RNAi Negative Control Med GC, and Beta- CCF-AM) for hours. Fluorescence emission values at nm lactamase positive control RNAi duplex. At ~ hours posttransfection, the plate was subjected to heat shock at C for hour followed by a hour incubation at C prior to loading with LiveBLAzer -FRET B/G Substrate (µm final concentration of and nm were obtained using a standard fluorescence plate HSF- HSF- HSF- HSF- HSF- HSF- HSF-. Log [nm] -AAG Cryopreserved HSE-bla HeLa cells were thawed and plated in -well assay format in Assay Medium at, cells/well in comparison to an actively dividing culture. Following overnight incubation, serial dilutions of -AAG were applied to the wells (. % final DMSO) for ~ h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Figure B Quercetin inhibitor dose response curve..... - - - Log [um] Quercetin Quercetin (cryo) Quercetin (dividing) Cryopreserved HSE-bla HeLa cells were thawed and plated in -well assay format in Assay Medium at, cells/well in comparison to an actively dividing culture. Following overnight incubation, -point serial dilutions of Quercetin were applied to the wells and incubated for ~ h prior to stimulating them with nm -AAG for ~ h (. % final DMSO) and then loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the inhibitor treated wells from the / ratios obtained with the untreated control wells (n = for each data point). The highest concentrations of Quercetin exhibited significant cytotoxicity and were excluded from the analysis. Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
Cell Culture and Maintenance Thaw cells in Growth Medium without selection (Blasticidin) and culture them in Growth Medium with selection. Pass or feed cells - times a week and maintain them in a C/% CO incubator. Maintain cells between % and % confluence. Note: We recommend passing cells for three passages after thawing before using them in the beta- assay. For more detailed cell growth and maintenance directions, please refer to lactamase protocol. Table Cell Culture and Maintenance Component Growth Medium ( ) Growth Medium (+) Assay Medium Freeze Medium DMEM with GlutaMAX TM ml ml ml Dialyzed FBS (dfbs) Do not substitute! ml ml. ml HEPES ( M). ml. ml. ml NEAA (x) ml ml ml Pen/Strep (x) ml ml ml Blasticidin µg/ml Recovery Cell Culture Freezing Medium % Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
Assay Performance with Variable Cell Number Figure -AAG dose response with varying cell plating density Log [nm] -AAG K K K K K HSE-bla HeLa cells were plated onto a -well assay plate in Assay Medium at varying cell densities. Following overnight incubation, serial dilutions of the Hsp inhibitor -AAG were applied to the wells (. % final DMSO) for h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Assay Performance with variable DMSO concentration Figure -AAG dose response with.,.,. and % DMSO.. % DMSO.% DMSO.% DMSO. % DMSO Log [nm] -AAG HSE-bla HeLa cells were plated onto a -well assay plate in Assay Medium at, cells/well. Following overnight incubation, serial dilutions of the Hsp inhibitor -AAG were applied to the wells in the presence of varying final DMSO concentrations for h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Assay performance with Variable Substrate Loading Time Figure -AAG dose response with increasing loading times....... Log [nm] -AAG. h h. h h h HSE-bla HeLa cells were plated onto a -well assay plate in Assay Medium at, cells/well. Following overnight incubation, serial dilutions of the Hsp inhibitor -AAG were applied to the wells (. % final DMSO) for h prior to loading the wells with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for and reading the plate at varying time points. Emission values at nm and nm were obtained using a standard fluorescence plate reader. Response Ratios were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Assay performance with Variable Stimulation Time Figure -AAG dose response with varying stimulation times h h h Log [nm] -AAG HSE-bla HeLa cells were plated onto a -well assay plate in Assay Medium at, cells/well. Serial dilutions of the Hsp inhibitor -AAG were applied to the wells immediately (for h) or for h or h following O/N incubation in Assay Medium. Wells were loaded with LiveBLAzer -FRET B/G Substrate (µm final concentration of CCF-AM) for hours. Emission values at nm and nm were obtained using a standard fluorescence plate reader. s were calculated by dividing the / ratios of the -AAG treated wells from the / ratios obtained with the untreated control wells (n = for each data point). Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor
References. Westerheide SD and Morimoto RI. () Heat shock response modulators as therapeutic tools for diseases of protein conformation. Journal of Biological Chemistry :-.. Davenport EL, Morgan GJ, and Davies FE. () Untangling the unfolded protein response. Cell Cycle :-. Tel: E-mail: tech_service@invitrogen.com www.invitrogen.com/cellsensor