Journal: bioRxiv

Article Title: Lineage transcription factors co-regulate subtype-specific genes providing a roadmap for systematic identification of small cell lung cancer vulnerabilities

doi: 10.1101/2020.08.13.249029

Figure Lengend Snippet: (A) RNA-seq (log2 FPKM) showing expression of SCN3A and KCNB2 , in SCLC tumors (T) or cell lines (C) in the specified SCLC subtype or NSCLC. The line indicates the mean, and the asterisks indicate p-values for each sample compared to SCLC-A tumors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (B) UCSC Genome Browser tracks showing ASCL1, NKX2-1, PROX1 and H3K27ac ChIP-seq signals in NCI-H2107 cells around the genes encoding SCN3A and KCNB2 . Black bars above the tracks indicate computationally called binding sites. (C-E) Immunoblots showing SCN3A and KCNB2 protein in NCI-H2107 cells, 72 hours post-transfection with control siRNAs or siRNA targeted against either ASCL1, NKX2-1 , or PROX1 (C), or SCN3A (D) or KCNB2 (E). (F) WST-1 assay for cell viability from cells from (D,E). Each data point represents a biological replicate, and error bars indicate SEM. ANOVA with Bonferroni’s multiple comparisons test was used to determine significant differences relative to control. ns; not significant. (G) Quantification of colony formation assays in soft agar using varying doses of KCNB2 inhibitor Quinine shows increased sensitivity of SCLC-A NCI-H889, compared to SCLC-N NCI-H524, and SCLC-P NCI-H526. (H) Histograms showing SCN3A+ cells are detected in live SCLC-A but not SCLC-N or SCLC-P cell cultures using an SCN3A extracellular domain specific antibody (red). The background fluorescence with the secondary antibody only is shown (gray). (I,J) RT-qPCR for SCN3A (I) and ASCL1 (J) mRNA from FACs isolated SCN3A+ (red) or SCN3A-(black) cells from mixtures of SCLC-A with SCLC-P or – N. Each data point represents a biological sample, error bars = SD around mean, unpaired t-test, * p<0.05, ** p<0.01.

Article Snippet: Cells were incubated with SCN3A antibody (Alomone Labs, Anti-SCN3A (Nav1.3) (extracellular), Cat #: ASC-023, 4.25 µg/ml) for 30 min on ice, washed 3 times with ice cold FACs buffer, incubated with Alexa Fluor 568-conjugated secondary antibody (Thermofisher, Catalog # A10042, 1:400) for 30 min on ice, washed 3 times as above, and resuspended in ice cold FACs buffer.

Techniques: RNA Sequencing Assay, Expressing, ChIP-sequencing, Binding Assay, Western Blot, Transfection, WST-1 Assay, Fluorescence, Quantitative RT-PCR, Isolation

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Characterization of Icagen compound 68 as an Nav1.3 channel inhibitor. The Icagen patent compound ( structure in D , inset ) was applied to CHO cells stably expressing the hNav1.3 channel to determine its potency and state dependency. (A) Average macroscopic Nav1.3 currents evoked in 0.1% DMSO vehicle (multihole X-plates) from resting state (Peak 1, blue line ) and following a prepulse to ∼50% inactivation membrane potential (Peak 2, green line ) from N = 6 cells (±SEM for every sixth sweep) show acceptable current stability (Peak 2 rundown <1%/min) during the ∼20-min duration of the whole-cell experiment. Peak 2 shows ∼50% inactivation (amplitude) compared with Peak 1. Each dose application epoch of the IC 50 experiment is separated by a gap due to pipette scheduling across different wells on the QPlate and ends with application of 300 nM TTX to block remaining current. Arrows show ‘liquid addition’ timing of each compound application. (B) Repeat of the experiment in (A) , but with increasing concentrations of Icagen compound 68 applied to a single cell (indicated by arrows ) after the initial vehicle stabilization period, showing the dose- and state-dependent inhibition of inactivated Peak 2 current (●) compared with minimal effects on resting Peak 1 current (■). In this and all other QPatch raw data current–time (IT) plots, current amplitude (in picoampere or in this case nanoampere) is plotted on the y-axis and experimental time (in s) on the x-axis. Scale bar shows 5 nA on y-axis and 100 s on x-axis. (C) Example of inactivated-state Peak 2 currents (following a 5-s prepulse to ∼50% inactivating membrane potential) in a typical cell showing dose-dependent inhibition of inward currents (vehicle = blue traces , 10 nM = green , 30 nM = pink , 100 nM = red , and 300 nM = purple ); Cyan traces are in the presence of 300 nM TTX, showing complete block of all fast inward currents. In this and all other raw current data traces exported from QPatch software, current amplitude (in picoampere) is plotted on the y-axis and time (ms) on the x-axis. Scale bar shows 5 nA on the y-axis and 1 ms on the x-axis. (D) Plot of mean ( N = 4 cells, ±SEM) % inhibition data allows fitting of IC 50 curves (sigmoidal dose–response) for Icagen compound block of resting-state Nav1.3 currents (♦ Peak 1, estimated 18 μM) and inactivated state (▼ Peak 2, 56 nM with hillslope of 1.2). Inset : structure of Icagen compound 68. CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide; IC 50 , 50% inhibitory concentration; SEM, standard error of the mean; TTX, tetrodotoxin.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Stable Transfection, Expressing, Transferring, Blocking Assay, Inhibition, Software, Concentration Assay

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Summary of Nav1.x-Selective Pharmacology and ND7-23 Cell TTX-S Current Components

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Inhibition

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Potency of 4,9 anhydro TTX against human Nav1.6 channels. (A, B) Heterologous hNav1.6 currents (single-hole QPlates) are stable throughout a 20-min experiment in 0.1% DMSO vehicle when evoked from resting state (B) or ∼50% inactivating membrane potentials ( Peak 2, ■ in A) . Peak 2 currents were 40% inactivated at the end of the vehicle period in 0.1% DMSO control cells and 53% of the amplitude of Peak 1 currents during initial vehicle period in cells exposed to 4,9 anhydro TTX. Scale bar in (A) shows 500 pA on the y-axis and 100 s on the x-axis. TTX (300 nM) added at the end of every experiment completely inhibits inward currents (A, olive traces in B) . Scale bar in (B) shows 100 pA on the y-axis and 2 ms on the x-axis. Arrows in A ( and C) show timing of addition of vehicle or compound. (C, D) After a brief stabilization period in vehicle, addition of increasing concentrations of 4,9 anhydro TTX dose-dependently inhibits hNav1.6 currents from resting or inactivated potentials ( blue and green traces in C , respectively ) without altering their kinetics ( resting-state current, D) . Effects of 3 nM ( purple traces ), 10 nM ( pink ), 30 nM ( cyan ), and 100 nM 4,9 anhydro TTX ( red ) on resting current in a typical cell are shown in (D) as well as control currents in vehicle ( olive traces ) and after application of 300 nM TTX ( green ); as 300 nM TTX completely inhibits Nav1.6 currents (A–C) , we subtract these sweeps offline to remove capacitance artifacts remaining after Cm and Rs compensation. Scale bar in (D) shows 200 pA on the y-axis and 2 ms on the x-axis. (E) The IC 50 of 4,9 anhydro TTX block of resting-state (32.9 nM, ♦) and inactivated-state hNav1.6 channels (4.3 nM, ▼) was obtained by fitting a sigmoidal dose–response curve to the mean ± SEM ( N = 4 cells) % inhibition data (variable hillslope parameters of 0.59 and 0.94, respectively).

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Blocking Assay, Inhibition

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Potency and selectivity of 4,9 anhydro TTX against human Nav1.7 channels. (A) Resting-state currents of hNav1.7 channels expressed heterologously in HEK cells are stable in 0.1% DMSO vehicle on the QPatch and completely blocked by 300 nM TTX. Each set of colored symbols represents a different cell. Scale bar shows 200 pA on the y-axis and 200 s on the x-axis. Arrows ( also in B and D) indicate timing of vehicle or compound additions. (B, C) Exposure of hNav1.7-expressing cells to concentrations of 4,9 anhydro TTX that inhibit hNav1.6 channels has no effect on resting-state Peak 1 current amplitude (B) or kinetics (C) , but all inward currents are completely blocked by addition of 300 nM TTX at the end of the experiment ( purple traces in C) . (B) Shows the time course of Nav1.7 current amplitude in two cells exposed to 3–100 nM 4,9 anhydro TTX, followed by 300 nM TTX. Scale bar in (B) shows 200 pA on the y-axis and 200 s on the x-axis, and 200 pA and 2 ms in (C) . (D, E) Application of 30-fold higher concentrations of 4,9 anhydro TTX produces dose-dependent inhibition of resting-state Peak 1 currents. Mean resting-state Nav1.7 current amplitude (±SEM from N = 3 cells) plotted over time is shown in (D) , and raw data from a single cell in (E ; red traces = vehicle, green = 0.1 μM, purple = 0.3 μM, cyan = 1 μM, blue = 3 μM, and brown = 300 nM TTX ) . Three hundred nanomolars of TTX applied to each cell at the end of the experiment completely blocked all inward currents. Scale bar in (E) shows 500 pA on the y-axis and 2 ms on the x-axis (F) The dose–response curve of hNav1.7 channel inhibition by 4,9 anhydro TTX reveals an IC 50 of 1.62 μM (mean ± SEM from N = 3 cells). HEK, human embryonic kidney.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Expressing, Inhibition

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Inhibition of TTX-S currents in ND7-23 cells by selective Nav1.7 antagonist Protoxin-II. (A, B) TTX-S currents in ND7-23 cells are stable in 0.05% BSA vehicle. Resting-state ( raw current traces in A and ● in B) and inactivated-state currents ( ■ in B) are stable throughout the 20-min whole-cell recording, and inactivated-state Peak 2 current is ∼50% of Peak 1 amplitude; current is blocked by 300 nM TTX applied at the end of the experiment ( blue traces in A , purple arrow in B) . Scale bar in (A) is 500 pA on the y-axis and 2 ms on the x-axis, and 500 pA and 100 s in (B) . (C) Application of nanomolar concentrations of Protoxin-II (in 0.05% BSA to preserve peptide activity and prevent absorption to plastic or glass surfaces) dose-dependently inhibits resting-state Peak 1 current in an exemplar cell; vehicle = brown traces , 0.3 nM = purple , 1 nM = cyan , 3 nM = blue , 10 nM = purple ; 300 nM TTX = pink traces ). Scale bar is 500 pA on the y-axis and 1 ms on the x-axis. (D) Time course plot from another cell illustrates current stability in vehicle and slow dose-dependent block of resting-state (●) and inactivated-state currents (■) at low nanomolar concentrations (concentrations and applications indicated by arrows ) and almost complete block after a double addition of 10 nM Protoxin-II before application of a saturating dose of 300 nM TTX. Scale bar is 500 pA on the y-axis and 100 s on the x-axis. (E) Plot of average % inhibition (mean ± SEM from N = 6 cells) of resting-state (♦) and inactivated-state (▼) current against Protoxin-II concentration reveals a Peak 1 IC 50 of 7.24 nM and a Peak 2 IC 50 of 5.73 nM. Sigmoidal least squares fit with hillslopes of 1.5 and 1.6 for resting- and inactivated-state curves, respectively.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Inhibition, Activity Assay, Blocking Assay, Concentration Assay

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Effect of an Nav1.3 inhibitor on TTX-S currents in ND7-23 cells. (A) Current–time plot of average (mean ± SEM from N = 5 cells) resting-state Peak 1 ( blue ) and inactivated-state Peak 2 ( green ) TTX-S current amplitude recorded in ND7-23 cells in vehicle (0.1% DMSO) and increasing concentrations of Icagen compound 68 (application times and concentrations in μM indicated by arrows ), illustrating state-dependent preference for inhibition of inactivated-state currents. Note that Peak 2 currents were ∼50% inactivated in the vehicle stabilization period. All currents remaining after incubation in 10 μM of Icagen compound are blocked by 300 nM TTX. (B) Histogram illustrating mean % inhibition values for resting-state ( black open circles ) and inactivated-state currents ( gray open squares ) after exposure to indicated concentrations of Icagen compound 68, followed by 300 nM TTX; values for individual cells are shown by each symbol, together with mean ± SEM error bars ( N = 3 cells). Inhibition of TTX-S currents was always <50%, so data could not be fitted with a reliable IC 50 curve. TTX-S, tetrodotoxin-sensitive.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Inhibition, Incubation

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Inhibition of TTX-S currents in ND7-23 cells by selective Nav1.6 antagonist 4,9 anhydro TTX. (A) Current–time plot of resting-state Peak 1 TTX-S current recorded in a typical ND7-23 cell in vehicle (0.1% DMSO applications shown by gray arrows ) showing stable current amplitude and lack of any initial runup or rundown in these cells. All inward currents are blocked by application of 300 nM TTX at the end of the experiment ( purple arrow ). Scale bar is 200 pA on the y-axis and 200 s on the x-axis. (B) In another cell, the application (indicated by arrows ) of increasing concentrations of 4,9 anhydro TTX shown to significantly inhibit Nav1.6 channels has little effect on resting-state Peak 1 (●) or inactivated-state Peak 2 currents (■), producing a maximal inhibition of ∼20% at 100 nM before washout in vehicle, and then complete block by 300 nM TTX at the end of the experiment. Scale bar is 1 nA on the y-axis and 200 s on the x-axis. Inhibition of TTX-S currents by 4,9 anhydro TTX was always <50%, so data could not be fitted with a reliable IC 50 curve. (C) Exemplar resting-state Peak 1 TTX-S current traces in 0.1% DMSO vehicle exhibit stable amplitude and kinetics during an ∼20-min whole-cell experiment. As all inward currents are completely blocked by 300 nM TTX ( cyan traces ), the average of the last three sweeps in TTX has been subtracted from all other sweeps to remove capacitance transients. Scale bar is 500 pA on the y-axis and 1 ms on the x-axis. (D) Overlay of resting-state Peak 1 currents in another cell in vehicle ( red traces ) and after exposure to 10 nM ( green traces ), 30 nM ( purple traces ), and 100 nM 4,9 anhydro TTX ( blue traces ), followed by washoff in vehicle ( olive traces ), and then complete inhibition by 300 nM TTX ( orange traces ). Sweeps recorded in TTX have been subtracted to remove capacitance transients remaining after online Cm and Rs compensation. Scale bar is 500 pA on the y-axis and 1 ms on the x-axis.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Inhibition, Blocking Assay

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Individual components of TTX-S current in ND7-23 cell inhibited by selective concentrations of Nav1.3, Nav1.6, and Nav1.7 antagonists. Individual cell and mean % inhibition (±SEM) of resting-state ( filled circles ) and inactivated-state TTX-S currents ( open squares ) in the presence of the indicated μM concentrations of the Nav1.3 inhibitor ICA ( N = 5) and nanomolar concentrations of the Nav1.6 antagonist, 4,9 anhydro TTX ( N = 4), and Nav1.7 blocker, Protoxin-II ( N = 6), are shown from independent samples of ND7-23 cells. Note the moderate inactivated state preference of the Icagen compound and 4,9 anhydro TTX compared with equipotent inhibition of resting- and inactivated-state TTX-S currents by Protoxin-II. A concentration of 300 nM TTX added to each cell at the end of every experiment completely reduced the remaining resting- and inactivated-state currents (resting state 98.5% ± 0.64% of vehicle control amplitude ( N = 26). ICA, Icagen compound 68.

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Inhibition, Concentration Assay

Journal: Assay and Drug Development Technologies

Article Title: Characterization of Endogenous Sodium Channels in the ND7-23 Neuroblastoma Cell Line: Implications for Use as a Heterologous Ion Channel Expression System Suitable for Automated Patch Clamp Screening

doi: 10.1089/adt.2016.704

Figure Lengend Snippet: Additive effects of selective Nav1.6 and Nav1.7 antagonists on TTX-S currents in ND7-23 cells. (A) Example IT plot of the additive effects of exposing an ND7-23 cell to Nav1.6-selective concentrations of 4,9 anhydro TTX (10–100 nM), followed by a saturating, but Nav1.7-selective, concentration of Protoxin-II (10 nM). Arrows indicate timing and concentration of compound applications. Resting-state (●) and inactivated-state current amplitudes (■) are stable in vehicle control (0.05% BSA), moderately inhibited by 100 nM 4,9 anhydro TTX, but slowly and almost completely reduced by 10 nM Protoxin-II; all remaining inward currents are blocked by 300 nM TTX. Scale bar is 500 pA on the y-axis and 100 s on the x-axis. (B) Summary % inhibition data from N = 6 individual cells (mean ± SEM shown by bars ) for resting-state ( filled circles ) and inactivated-state currents ( open squares ) after additive application of selective concentrations of 4,9 anhydro TTX and Protoxin-II to each cell, followed by 300 nM TTX (which inhibited resting-state currents to 97.2% ± 0.6% of vehicle control amplitude).

Article Snippet: Similarly, the Icagen compound ICA-121431 is a close analog of Nav1.3 patent compound 68 and is an almost equipotent (∼20 nM IC 50 ) antagonist of Nav1.3 and Nav1.1, but application of a saturating concentration of 300 nM of Icagen compound 68 had negligible effects on resting- or inactivated-state TTX-S currents in ND7-23 cells ( ).

Techniques: Concentration Assay, Inhibition