DOI: 10.5061/dryad.z34tmpgsx
title: Data from: Diethylcarbamazine elicits calcium signals in HEK293 cells by activation of heterologously expressed <em>Brugia malayi</em> TRP-2b channels

description: Diethylcarbamazine is a classic anthelmintic that is used for the
 prevention and treatment of lymphatic filariasis. The mode of action of
 diethylcarbamazine is still not well understood, with the consensus that
 it acts on the host immune system, rather than directly acting on the
 adult parasite. Recent studies have found that diethylcarbamazine acts on
 the muscle of adult female Brugia malayi, generating temporary spastic
 paralysis mainly through the Transient Potential Receptor C (TRPC)
 orthologue TRP-2. Activation of TRP-2 leads to inward currents in the
 muscle, an increase in intracellular calcium, and subsequent muscle
 contraction. These studies have demonstrated that Brugia malayi TRP-2 is
 activated by diethylcarbamazine. In this study, we heterologously
 expressed the Brugia malayi TRP-2b channel in the Human Embryonic Kidney
 (HEK) 293 cell line. Application of diethylcarbamazine to Bma-trp-2
 b-transfected HEK293 cells leads to larger and more frequent increases in
 intracellular calcium compared to non-transfected cells. This increase can
 be inhibited using the TRPC-specific antagonist SKF96365. Our study shows
 that diethylcarbamazine’s action is dependent upon the Brugia malayi TRP-2
 channel and may also, in addition, activate endogenous mammalian TRP
 channels.
description: <strong>Materials and Methods</strong>
 <em>HEK283 maintenance:</em> The
 Human Embryonic Kidney 293 (HEK293) cell line was used for all experiments
 in this study. HEK293 cells were maintained in a humidified growth
 incubator set to 37 °C in 5% CO2 following previous published methods.
 Cells were cultured in 10 mL Dulbecco’s Modified Eagle Medium (DMEM 1X)
 supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v)
 Penicillin-Streptomycin (Pen/Strep). Media was exchanged every 3 days.
 Cells were passaged for a maximum of 14 generations. Passaging was
 achieved by discarding the previous media and washing cells with 1X PBS
 warmed to 37 °C to remove cellular debris. Cells were treated with 2.5 mL
 of 0.25% trypsin-EDTA warmed to 37 °C, and incubated at 37 °C in 5%
 CO<sub>2</sub> for 2-3 minutes to detach cells. After
 incubation, 10 mL of warmed DMEM + 10% FBS + 1% Pen/Strep was added as the
 serum contains deactivators of trypsin. Cells were gently pipetted up and
 down to break clumps and to help detach cells remaining on the plate. 1 mL
 of cells were added to 37 °C warmed 10 mL fresh DMEM + 10% FBS + 1%
 Pen/Strep in new plates and placed in the incubator. Cells were frozen
 during every passage by adding 950 µL cell media to 50 µL 5% (v/v) DMSO
 and mixing gently before being stored at -80 °C.
   <em>Brugia supply and
 maintenance:</em> Live adult female
 <em>Brugia malayi</em> were shipped overnight from the
 NIH/NIAID Filariasis Research Reagent Resource Centre (FR3; College of
 Veterinary Medicine, University of Georgia, Athens, USA). <em>Brugia
 malayi</em> were maintained in non-phenol red Hyclone Roswell Park
 Memorial Institute (RPMI) 1640 media containing 10% heat-inactivated FBS
 and 1% penicillin-streptomycin. Parasites were separated individually into
 a 24 well microtiter plate containing 2 mL of the RPMI media. Parasites
 were held in an incubator set to 37 °C in 5%
 CO<sub>2</sub>. <em>Cloning and
 maintenance of Bma-trp-2b:</em> Adult
 <em>Brugia malayi</em> worms were snap-frozen in liquid
 nitrogen and crushed into fine powder in a 1.5 mL micro-centrifuge tube
 using Kimble Kontes Pellet Pestle (Fisher Scientific, Waltham, MA, USA).
 Total RNA was extracted using TRIzol Reagent (Life Technologies, Carlsbad,
 CA, USA) according to manufacturer’s instructions. cDNA was synthesized
 using SuperScript VILO Master Mix (Life Technologies, Carlsbad, CA, USA)
 from approximately 1 µg RNA as template. Full-length
 <em>Bma-trp-2b</em> (<em>Bm5246b.1</em>) was
 amplified using Platinum SuperFi Polymerase Master Mix (Thermo Fisher,
 Waltham, MA, USA) using the primers SSK160F and SSK160R (Table 1). Primers
 were made with the sequences flanking the expression vector pTarget
 (P2A::mCardinal), with SSK160F flanking the BamHI site and SSK160R
 flanking KpnI and the P2A site. Sequencing of the <em>pTarget
 (P2A::mCardinal)::Bma-trp-2b</em> plasmid is available in
 supplementary information (see Supplementary data online). The amplicon
 was analyzed using agarose gel electrophoresis and was purified using
 NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). The
 purified <em>trp-2b</em> cDNA was cloned into the pTarget
 vector using Infusion HD Cloning Kit (Takara Bio, San Jose, CA, USA) under
 the manufacturer’s guidelines. Upon cloning, the plasmids were
 sequence-verified and ready for transfection into HEK293 cells.
 The plasmid was added to Mix and Go! Competent JM109
 <em>Escherichia coli</em> bacteria (Zymo Research, Irvine, CA,
 USA) which were grown in LB media with 25 µg/ml ampicillin at 37 °C for
 16-18 hours. The plasmid was purified from bacterial lysates using
 Monarch® plasmid miniprep kit (New England Biolabs, Ipswich, MA, USA) and
 diluted in nuclease free water. The extracted plasmid concentrations were
 analyzed using an Implen NanoPhotometer® N120 (Implen, Munich, Germany).
 All plasmid stocks were stored at -20 °C.
 <em>Transfection of HEK293 cells:</em>
 For transfection, we followed techniques as previously described.
 Briefly, 500 µL of HEK293 cells were cultured in 5 ml of DMEM + 10% FBS
 and 1% Pen/Strep for 72 hours or until 90% confluence was achieved in a
 humidified growth incubator set to 37°C and 5% CO<sub>2</sub>.
 Transfection was achieved using cationic lipid-based reagent
 Lipofectamine® 3000 transfection kit (Invitrogen, Thermo Fisher
 Scientific, Waltham, MA, USA) according to manufacturer’s instructions.
 Before transfection, old media was discarded, and cells were washed with 5
 mL warm 1X PBS. Cells were incubated in OPTI-MEM® I (1X) Reduced Serum
 Medium + GlutaMAX <sup>TM</sup>-I for 40 minutes at 37°C in 5%
 CO<sub>2</sub>. Whilst cells were incubating, the transfection
 media was prepared by adding 5-8 µg of plasmid to a ratio of 3.75 µL
 lipofectamine per µL plasmid in 125 µL OPTI-MEM and by adding 2 µL P3000
 per µL of plasmid to 125 µL OPTI-MEM. All components were mixed and
 incubated for 15 minutes at room temperature in the dark to improve
 transfection quality. When added, the transfection solution was applied
 over the area of the cell plate and the sample was incubated for a minimum
 of 6 hours at 37 °C in 5% CO2. After the transfection period the OPTI-MEM
 solution was discarded and replaced with DMEM supplemented with 10% FBS
 and cells were placed in a humidified growth incubator set to 30 °C at 5%
 CO2 for 48 hours to promote expression but slow cell growth. Before
 experiments, cell health was assessed via light microscope.
 <em>HEK293 cDNA synthesis and RT-PCR detection of TRP
 channels:</em> To obtain cDNA we cultured HEK293
 cells either transfected with 5-8 µg of the
 <em>Bma-trp-2b</em> expression vector or without the vector at
 37 °C at 5% CO2 for 7 days. HEK293 cells were homogenized in 1 mL of
 TRIzol by gentle pipetting, followed by total RNA extraction according to
 the TRIzol Reagent protocol. One microgram (1 µg) of total RNA from each
 culture was used to generate cDNA by reverse transcription (RT) using
 SuperScript VILO<sup>TM</sup> Master Mix following the
 manufacturer's protocol. PCR was conducted to detect the presence of
 the known endogenous human TRPC channels TRPC1, TRPC3, TRPC4 and TRPC6 by
 targeting the cDNA of htrp-1, htrp-3, htrp-4 and htrp-6 using previously
 published primers that target the encoding regions of each gene (Table 1).
 To measure the expression of Bma-trp-2b cDNA, we screened using primers
 targeting the encoding region of trp-2b that we have previously used
 <sup>7</sup> (Table 1). <em>hβ-actin</em> was used
 as a reference gene. Negative controls included enzyme, water, and both
 forward and reverse primers for hβ-actin with no cDNA template. The
 cycling conditions for PCR were an initial denaturation for 2 mins at 95
 °C, followed by 35 cycles of 95 °C for 30 secs, 61 °C for 30 secs, 72 °C
 for 20 secs, and a final extension at 72 °C for 10 mins using GoTaq ® G2
 Hot Start Green Master Mix (Promega, Madison, WI, USA). PCR products were
 then separated on a 2% Agarose gel containing SYBR® Safe DNA Gel Stain,
 followed by visualization and images were captured using an Azure 600
 imaging system set to SYBR Safe (Excitation 472nm, Emission
 595nm). <em>Capturing cell
 images</em>: White and red fluorescent images
 were captured using Occular 2.0.1.496 imaging software (Digital Optics,
 Auckland, New Zealand). For white light images the exposure settings were
 10 ms with no binning Figs. 2A & B (left panels). Red fluorescence
 imaging was achieved using a Lambda LS Xenon bulb lightbox which delivered
 light via a fiber optic cable to the microscope (Sutter Instruments,
 Novato, CA, USA) which passed through a green/red filter box (EF-4 AT
 TRITC/CY3 LP; Excitation: 527.5 - 552.5nm, Emission 575nm Long Pass,
 Dichroic Mirror) (Chroma Technology, Bellows Falls, VT, USA). Fluorescent
 light emission was controlled by using the shutter. The exposure settings
 for the mCardinal fluorescent images were 500 ms with 2x binning Figs. 2 A
 & B (middle panels). For Fluo-3 fluorescence, images were captured
 using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA) with
 exposure settings at 250 ms with 2x binning under pseudo-color settings to
 illustrate calcium levels Figs. 1 A & B (right panels). All images
 were captured using a Photometrics Retiga R1 Camera (Teledyne
 Photometrics, Tucson, AZ, USA).  
 <em>Preparation and loading Fluo-3AM:</em>
 Before recording, DMEM + 10% FBS media is discarded, and cells
 are washed with warmed 1X PBS to remove cellular debris. Cell detachment
 was achieved by adding 500 µL of 0.25% trypsin-EDTA and incubating at 37
 °C in 5% CO<sup>2</sup> for 2-3 minutes. Next, 5 mL of DMEM +
 10% FBS was added to deactivate the trypsin, and cells were resuspended by
 gentle pipetting. Clean 12 mm circle coverslips coated with poly-D-lysine
 following methods previously described were placed in a clean Petri dish
 and 20 µL of cell media is added to each disc. To promote cell adhesion,
 DMEM + 10% FBS was added to cover the coverslip, and samples are incubated
 for a minimum of 2 hours before recording at 37 °C in 5% CO2~.
 After incubation the 12 mm glass cover slip was placed in a
 Warner RC26G laminar flow chamber (Warner Instruments, Holliston, MA,
 USA), immersed in HEK293 cell buffer (150mM NaCl, 5 mM KCl, 5 mM
 CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>, 15 mM
 HEPES and 10 mM glucose, pH 7.3) and cells were visualized under white
 light on a Nikon Eclipse TE3000 microscope fitted with a 20X/0.45 Nikon
 PlanFluor objective to ensure that they appeared healthy before Fluo-3AM
 loading (Figs.2A & B). Fluo-3AM (Sigma-Aldrich, St. Louis, MO,
 USA) loading was achieved by incubating the cells in HEK293 cell buffer
 containing 5 µM Fluo-3AM and 10% Pluronic F-127 (10% v/v) for 20 minutes
 with the recording chamber connected to a Dual Automatic Temperature
 Controller (Warner Instruments, Holliston, MA, USA) maintained at 36-37
 °C. After incubation, the Fluo-3AM solution was discarded, and the sample
 was incubated in HEK293 cell buffer for an additional 10 minutes at 36-37
 °C to promote calcium loading. Before performing calcium imaging, cell
 transfection of the <em>Bma-trp-2b</em> plasmid was verified
 under green light using a long pass green/red filter box. Transfected
 cells that were fluorescing red were selected for recording Fig. 2A. We
 observed no red fluorescence in non-transfected cells Fig. 2B. Fluo-3AM
 loading was confirmed under blue light using a blue/green filter box (EF-4
 AT EGFP/FITC/CY2/ALEXA FLUOR Band Pass; Excitation: 465-495nm, Emission:
 525-545nm, Dichroic Mirror) (Chroma Technology, Bellows Falls, VT, USA)
 and visualized under pseudo-color settings using MetaFluor 7.10.2 (Figs.
 2A & B). Any cell that did not show Fluo-3 fluorescence regardless
 of fluorescence under the red channel was discarded. We observed no
 significant difference in the levels of Fluo-3 fluorescence between
 <em>Bma-trp-2b</em> transfected and non-transfected cells
 (Fig. 2C).   <em>Measurement
 of Ca<sup>2+</sup> fluorescence:</em>
 All recordings were performed on a Nikon Eclipse TE3000
 microscope fitted with a 20X/0.45 Nikon PlanFluor objective, using a
 Photometrics Retiga R1 Camera. Light control was achieved using a Lambda
 10-2 with a shutter controller. Fluorescence was achieved using a Lambda
 LS Xenon bulb lightbox (Sutter Instruments, Novato, CA, USA) which
 delivered light via a fiber optic cable to the microscope which passed
 through a blue/green filter box. Fluorescent light emission was controlled
 by using the shutter. Minimal illumination exposure was used to prevent
 photobleaching. During recordings cells were
 continuously perfused with HEK293 cell buffer solutions. Cells were
 exposed to either 10 µM carbachol for one minute, 30 µM diethylcarbamazine
 (DEC) for five minutes, 30 µM arachidonic acid (AA) for five minutes, 10
 µM SKF96365 for three minutes (SKF), 10 µM SKF96365 + 30 µM DEC (SKF +
 DEC) for 5 minutes or 10 mM CaCl<sub>2,</sub> which was used
 as a positive control to determine cell viability. If a cell failed to
 respond to the 10 mM CaCl<sub>2</sub> control signal it was
 discarded from the data pool regardless of previous signals to our
 compounds. Every experiment in this study has a 100% cell response to 10
 mM CaCl<sub>2</sub>. A change in fluorescence ≥3% was
 classified as a positive response to a compound as the average spontaneous
 change in cells continuously perfused with HEK293 buffer was
 ~2.5%.   All solutions were
 delivered to the chamber under gravity feed through solenoid valves
 controlled using a VC-6 six-channel Valve Controller through an inline
 heater set at 37 °C (Warner Instruments, Holliston, MA, USA), at a rate of
 1.5mL/min. At the start of all experiments, cell samples were left under
 blue light for a minimum of 1 minute to promote settling and equilibration
 of the fluorescent signal and to monitor any cells that may detach from
 the cover slip before the application of compounds. All
 calcium signal recordings were acquired and analyzed using MetaFluor
 7.10.2 with exposure settings at 250 ms with 2x binning. Maximal percent
 calcium signal amplitudes (ΔF) were calculated using the equation F1-F0/F0
 x 100, where F1 is the fluorescent value and F0 is the baseline value. All
 F0 values were taken at the time point each compound was applied for every
 cell analyzed. All representative response traces are presented as the
 average percent change in calcium fluorescence with the standard error of
 the mean (±SEM) of all cells from a single recording. Traces were
 generated by converting the calcium signal profiles of all the cells from
 a single recording to percentages using the previously described ΔF/F0
 equation, with the time of stimulus application being F0 (0% for all
 cells) and 100% being the peak value for each cell. The mean percentage
 change in fluorescence and the SEM was calculated for each time point
 during the application of each compound. All baseline values were taken at
 the time point each compound was applied for every cell. Traces are
 represented as mean ±SEM.  
 <em>Statistical Analysis:</em>
 Statistical analysis of all data was done using GraphPad Prism
 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). To ensure
 reproducibility, we repeated our experiments: the numbers of cells, number
 of preparations, the concentrations, and durations of applications are
 provided in the legends of the figures. Analysis of calcium amplitudes and
 percentage of cells responding was done using paired or unpaired student
 <em>t</em>-tests with <em>P &lt; 0.05</em>
 being considered significant. The data are presented as mean ± SEM for
 each treatment.
 <em>Chemicals:</em> Source of
 chemicals; SKF96365 was purchased from Tocris. Carbachol was purchased
 from EMD Millipore. All other chemicals were supplied by Sigma
 Aldrich.
description: # Data from: Diethylcarbamazine elicits calcium signals in HEK293 cells by
 activation of heterologously expressed *Brugia malayi* TRP-2b channels
 Dataset DOI: [10.5061/dryad.z34tmpgsx](10.5061/dryad.z34tmpgsx) ##
 Description of the data and file structure HEK 293 cells were transfected
 with TRP-2 channels of the filarial parasite, *Brugia malayi,* that causes
 elephantiasis. The anthelmintic, diethylcarbamazine, is used for treatment
 and prophylaxis of elephantiasis, but its molecular and cellular mode of
 action is not clear. The data consists of calcium signal peak measurements
 and the % of cells responding to diethylcarbamazine in experiments
 designed to determine the mode of action of diethylcarbamazine. We show
 that diethylcarbamazine produces calcium signals in transfected cells but
 smaller calcium signals in non-transfected cells. We interpret the
 observations to show that diethylcarbamazine activates the *Brugia* TRP-2
 channels, which are calcium permeable and the effects of
 diethylcarbamazine are TRP-2 dependent. We also propose that
 diethylcarbamazine can have effects on endogenous TRPC HEK 293 channels
 and discuss the possibility of effects of diethylcarbamazine on host TRPC
 channels on immune cells ### Files and variables #### File:
 Figures_Packet_08-01-25.pptx **Description:** The file contains PowerPoint
 figures, Figs 1-6, of the manuscript and 3 supplementary figure files ####
 File: Fig_2C_histogram_data.prism **Description:** Average baseline
 fluorescence of Fluo-3 for every cell used from all recordings in this
 study for *Bma-trp-2b* transfected (white bar) and non-transfected
 (hatched bar) during the first minute of recording before any stimulus was
 applied. No significant difference was seen between the two cell
 populations. N.S. = not significant to *Bma-trp-2b* (*Bma-trp-2b* vs
 non-transfected, *P* = 0.5578 *t* = 0.5887, *df* = 76, unpaired *t*-test).
 *Bma-trp-2b n* = 1392 cells from 41 recordings, non-transfected *n* = 1576
 cells from 37 recordings. All values represented as means ± SEM. ####
 File: Fig_3E_histogram_data.prism **Description:** Total Ca2+ amplitudes
 in response to 30 µM DEC (yellow; + = 27.7% ± 2.7%, - = 1.3% ± 0.1%) and
 10 mM CaCl2 (grey; + = 62.3% ± 3.2%, - = 52.5% ± 1.7%) in *Bma-trp-2b*
 transfected (+; clear) and non-transfected (-; hatched) HEK293 cells. ***
 significantly different: *Bma-trp-2b* DEC vs non-transfected DEC, *P &lt;
 0.0001, t* = 16.78 *df* = 364, unpaired *t*-test. ** significantly
 different: *Bma-trp-2b* CaCl2 vs non-transfected CaCl2 *P 0.007, t* = 2.7
 *df* = 547, unpaired *t*-test. *Bma-trp-2b n* = 6 recordings, *DEC* =
 90/273 cells, CaCl2 = 273/273 cells. Non-transfected *n* = 7 recordings,
 DEC = 27/276 cells, CaCl2 = 276/276 cells. All values represented as means
 ± SEM. #### File: Fig_3F_Histogram_Data.prism **Description:** Mean
 percent of cells showing a Ca2+ response larger than the average
 spontaneous amplitudes in response to DEC (yellow; + = 32.7 ± 6.6%, - =
 5.43 ± 3.53%) and 10 mM CaCl2 (grey; + = 100%, - = 100%) in *Bma-trp-2b*
 transfected (+; clear) and non-transfected (-; hatched) HEK293 cells. Note
 100% of cells analyzed responded to 10 mM CaCl2. ** significantly
 different: *Bma-trp-2b* DEC vs non-transfected DEC *P 0.003, t* = 3.796
 *df* = 11, unpaired *t*-test. *Bma-trp-2b n* = 6 recordings.
 Non-transfected, *n* = 7 recordings. All values represented as means ±
 SEM. #### File: Fig_4E_Histogram_Data.prism **Description:** Total Ca2+
 amplitudes in response to 10 µM carb (pink; + = 22.6% ± 0.8%, - = 15.4% ±
 0.4%) and 30 µM DEC (yellow; + = 31.9% ± 2.2%, - = 10.4% ± 0.5%) in
 *Bma-trp-2b* transfected (+; clear) and non-transfected (-; hatched)
 HEK293 cells. *** significantly different to *Bma-trp-2b* carb
 (*Bma-trp-2b* carb vs non-transfected carb *P = &lt;0.0001*, t = 8.115
 *df* = 749, unpaired *t*-test). *** significantly different to
 *Bma-trp-2b* DEC (*Bma-trp-2b* DEC vs non-transfected DEC *P* = *&lt;
 0.0001, t* = 5.467, *df* = 526, unpaired *t*-test). *Bma-trp-2b n* = 9
 total recordings, carb = 336/449 cells, DEC = 405/449 cells.
 Non-transfected *n* = 10 total recordings, carb = 415/557 cells, DEC =
 123/557 cells. All values represented as means ± SEM. #### File:
 Fig_4F_Histogram_Data.prism **Description:** Mean percent of cells showing
 a Ca2+ response larger than the average spontaneous amplitudes in response
 to 10 µM carb (pink; + = 75.7% ± 6%, - = 74.5% ± 7.3%) and to 30 µM DEC
 (yellow; + = 90.2% ± 2.3%, - = 22% ± 6.2%) in *Bma-trp-2b* transfected (+;
 clear) and non-transfected (-; hatched) HEK293 cells. 100% of cells
 analyzed responded to 10 mM CaCl2. N.S. not significantly different to
 *Bma-trp-2b* carb (*Bma-trp-2b* carb vs non-transfected carb *P 0.9042, t*
 = 0.1121 *df* = 17, unpaired *t*-test). *** significantly different to
 *Bma-trp-2b* DEC (*Bma-trp-2b* DEC vs non-transfected DEC *P &lt; 0.0001,
 t* = 9.804, *df* = 17, unpaired *t*-test). *Bma-trp-2b n* = 9 recordings.
 Non-transfected *n* = 10 recordings. All values represented as means ±
 SEM. #### File: Fig_5C_Histogram_Data.prism **Description:** Total Ca2+
 amplitudes in response to 10 µM carbachol (pink; + = 26% ± 1.2%, - = 20.6%
 ± 1.1%) and to 30 µM AA (green; + = 16% ± 0.7%, - = 9.7% ± 0.4%) in
 *Bma-trp-2b* transfected (+; clear) and non-transfected (-; hatched)
 HEK293 cells. ** significantly different to *Bma-trp-2b* carbachol
 (*Bma-trp-2b* carbachol vs non-transfected carbachol *P 0.001, t* = 3.30
 *df* = 529, unpaired *t*-test). *** significantly different to
 *Bma-trp-2b* AA (*Bma-trp-2b* AA vs non-transfected AA *P &lt; 0.0001, t*
 = 6.115, *df* = 454, unpaired *t*-test). *Bma-trp-2b* n *= 14 total
 recordings,* *carbachol = 284/425 cells, arachidonic acid* *n* = 303/425
 cells. Non-transfected *n* *= 10 total recordings, carbachol* = 247/444
 cells, arachidonic acid = 153/444 cells. All values represented as means ±
 SEM. #### File: Fig_5D_Histogram_Data.prism **Description:** Mean percent
 of cells showing a Ca2+ responses larger than the average spontaneous
 amplitudes in response to 10 µM carbachol (pink; + = 66.9% ± 4.5%, - =
 55.7% ± 7.7%) and to 30 µM AA (green; + = 71.3% ± 4.1%, - = 34.4% ± 5.1%)
 in *Bma-trp-2b* transfected (+; clear) and non-transfected (-; hatched)
 HEK293 cells. N.S. not significantly different to *Bma-trp-2b* carbachol
 (*Bma-trp-2b* carbachol vs non-transfected carbachol, *P 0.1994, t* =
 1.323 *df* = 22, unpaired *t*-test). *** significantly different to
 *Bma-trp-2b* AA (*Bma-trp-2b* AA vs non-transfected AA, *P &lt; 0.0001, t*
 = 5.641, *df* = 22, unpaired *t*-test). *Bma-trp-2b n* = 14 total
 recordings. Non-transfected *n* = 10 total recordings. All values
 represented as means ± SEM. #### File: Fig_6C_Histogram_Data.prism
 **Description:** TTotal Ca2+ amplitudes in response to 10 µM carbachol
 (pink; transfected = 17.1% ± 0.9%, non-transfected 10.5% ± 0.5%), 10 µM
 SKF + 30 µM DEC (red; transfected = 1.5% ± 1.8%, non-transfected 1% ±
 0.1%) and 30 µM DEC (yellow; transfected = 4% ± 0.3%, non-transfected 1.3%
 ± 0.1%) in *Bma-trp-2b* transfected (clear) and non-transfected (hatched)
 HEK293 cells. *** significantly different to SKF + DEC (*Bma-trp-2b* SKF +
 DEC vs *Bma-trp-2b* DEC *P &lt; 0.0001, t* = 8.929, *df* = 243, paired
 *t*-test). *Bma-trp-2b n* = 11 total recordings, carbachol = 164/244
 cells, SKF + DEC = 27/244 cells, DEC = 120/244 cells. Non-transfected *n*
 = 10 total recordings carbachol = 164/293, SKF + DEC = 7/293 cells, DEC n
 = 22/293 cells. All values represented as means ± SEM #### File:
 Fig_6_D_Histogram_Data.prism **Description:** Mean percent of cells
 showing a Ca2+ response larger than the average spontaneous amplitudes in
 response to 10 µM carbachol (pink; transfected = 65.6% ± 8.1%,
 non-transfected 56.4% ± 5.6%), 10 µM SKF + 30 µM DEC (red; transfected =
 11.1% ± 2.1%, non-transfected 2.4% ± 0.7%) and 30 µM DEC (yellow;
 transfected = 49.2% ± 10.3%, non-transfected 7.5% ± 5.1%) in *Bma-trp-2b*
 transfected (clear) and non-transfected (hatched) HEK293 cells. Note 100%
 of cells analyzed responded to 10 mM CaCl2. ** significantly different to
 SKF + DEC (*Bma-trp-2b* SKF + DEC vs *Bma-trp-2b* DEC *P = 0.0055*, t =
 3.527, *df* = 10, paired *t*-test). N.S. not significantly different to
 non-transfected *SKF + DEC *(non-transfected *SKF + DEC* vs
 non-transfected DEC *P = 0.326*, t = 1.039, *df* = 9, paired *t*-test).
 *Bma-trp-2b n* = 11 total recordings. Non-transfected *n* = 10 total
 recordings. All values represented as means ± SEM. #### File:
 Fig_S2C_Histogram_Data.prism **Description:** Maximal amplitudes of the
 CaCl2 amplitudes in *Bma-trp-2b* transfected HEK293 cells, 43.9% (± 1%)
 (grey bar) and non-transfected HEK293 cells, 65.2% (± 1.8%) (grey hatched
 bar). *Bma-trp-2b* transfected HEK293 #### File:
 Fig_S3C_Histogram_Data.prism **Description:** Maximal amplitudes of the
 CaCl2 amplitudes in *Bma-trp-2b* transfected HEK293 cells, 36.5% (± 1.1%)
 (grey bar) and non-transfected HEK293 cells, 46.6% (± 1.4%) (grey hatched
 bar). *Bma-trp-2b* transfected HEK293 *n* = 425, non-transfected HEK293
 #### File: Fig_S4G_Histogram_Data.prism **Description:** Maximal
 amplitudes of the SKF amplitudes in *Bma-trp-2b* transfected, 2.1% (±
 0.2%) (white bar) and non-transfected, 1.2% (± 0.1%) (hatched bar) and
 CaCl2 amplitudes in *Bma-trp-2b* transfected HEK293 cells, 28.1% (± 1%)
 (grey bar) and non-transfected HEK293 cells, 25.1% (± 0.7%) (grey hatched
 bar). *Bma-trp-2b* #### File: Williams_et_al.docx **Description:** The
 manuscript describing effects of the anthelmintic, diethylcarbamazine, on
 TRP-2 transfected HEK293 cells. We conclude the mode of action of
 diethylcarbamazine is dependent on the presence of TRP-2 channels of the
 filarial nematode parasite and discuss the mode of action of this drug. ##
 Code/software Prizm Graphpad (10.5.0) is required to view the data files
 and PowerPoint is required to view the Figures. ## Access information
 Other publicly accessible locations of the data: * NA Data was derived
 from the following sources: * NA