cell lines hek293 american type culture collection  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv6.1 and variants influence total Kv2.1 expression. A , Kv2.1 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 and 1:3 ratios as indicated, for 72 h in HEK293 cells. The Kv2.1 control group was cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated using SDS-PAGE and probed with anti-Kv2.1 and β-actin (loading control) antibodies. All the lanes in the representative blot are from the same blot. B , total expression of Kv2.1 in each group was first normalized to their respective β-actin signal and plotted as normalized to the Kv2.1 control expression in each experiment (n = 5). A repeated measures ANOVA test (on raw density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to Kv2.1 alone and # indicates p < 0.05 relative to the Kv2.1:Kv6.1 condition).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Control, Plasmid Preparation, Transfection, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv6.1 expression is diminished by coexpression with Kv2.1. A , Kv6.1 and variants were coexpressed with or without Kv2.1 in a 1:1 ratio as indicated, for 72 h in HEK293 cells. The Kv6.1 and variant control groups were cotransfected with GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated by SDS-PAGE and probed using an anti-GFP antibody (Kv6.1 constructs are EGFP-tagged), using β-actin as a loading control. Protein ladder molecular weights are in kDa. B , total expression of Kv6.1 in each group was normalized to the β-actin loading control and is plotted as normalized to the WT Kv6.1 control (expressed alone) expression in the corresponding experiment (n = 4–6). A paired t test was used to compare the expression between experimental groups for Kv6.1 or each variant (∗ and ∗∗∗ indicate p values of p < 0.05 and p < 0.001, respectively).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Variant Assay, Control, Plasmid Preparation, Transfection, SDS Page, Construct

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: HALO-label detection of cell surface Kv2.1 expression. A , schematic of a HALO-Kv2.1: Kv6.1 assembly. Membrane-impermeant Halo dye JF635i (shown in red ) reacts with the HaloTag in the extracellular S1-S2 linker of Kv2.1 at the cell surface. B , cumulative probability of JF635i integrated intensity from individual cells in each transfection condition (five independent experiments). C , log of integrated intensity of the HaloJF635i normalized to the Kv2.1 alone group (max value, 1) and the dsRed alone group (min value, 0), for indicated combinations of Kv2.1 and Kv6.1 variants. A repeated measures ANOVA (on the raw median log JF635i integrated intensity in each experiment) followed by a Tukey’s post hoc test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to HALO-Kv2.1 alone and # indicates p < 0.05 relative to the HALO-Kv2.1:Kv6.1 WT condition). D , HEK293 cells were transfected with combinations of HALO-Kv2.1 and Kv6.1 (WT or variants) as indicated, labeled with membrane-impermeant Halo-JF635i, and cell-by-cell fluorescence intensity was quantified using high content microscopy. The scale bar (in white ) represents 100 μm. E , insets from panel D Halo-JF635i label for Kv2.1 alone and with Kv6.1 variants as indicated. F , exemplar sweeps of HALO-Kv2.1 and Kv6.1 with a −40 mV conditioning pulse to assess inactivation (as done in E ). G , voltage dependence of inactivation of HALO-Kv2.1 with Kv6.1 variants (as measured in F , n = 3–6 per group). H , current density at +30 mV for indicated subunit combinations (1:1 cDNA ratio) after transient transfection in LM cells. One-way ANOVA followed by Tukey post hoc test was used to compare experimental groups, with ∗∗∗ and ∗∗ indicating p < 0.001 and p < 0.01, respectively, n = 6 per group. The control groups in all experiments were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Membrane, Transfection, Labeling, Fluorescence, Microscopy, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Stoichiometric interplay affects expression of Kv6.1 and Kv2.1. A , HEK293 cells were transfected with a constant (200 ng) amount of Kv6.1 ( left ) or Kv6.1[W416C] ( right ), and increasing amounts of Kv2.1 (0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with varying amounts of soluble GFP to ensure constant plasmid DNA in the transfection mixture. B , Kv2.1 bands were normalized to the Kv2.1 signal from the 1:0.5 (WT Kv6.1:Kv2.1) ratio condition. C , Kv6.1 bands were normalized to the Kv6.1 signal from the 1:0 (Kv6.1:Kv2.1) ratio condition. A paired t test was used to compare Kv6.1 versus Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel C ). Data in ( B , C ) are expressed as mean ± SEM from three independent experiments.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv2.1 coassembly underlies suppression of Kv6.1[W416C]. A , HEK293 cells were transfected with a constant (200 ng) amount of Kv2.1 and increasing amounts of WT Kv6.1 ( left ) or Kv6.1[W416C] ( right ), (Kv6.1 cDNA amounts: 0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with soluble GFP wherever applicable to ensure a constant amount of plasmid DNA in the transfection mixture. Protein ladder molecular weights are in kDa. B , Kv2.1 signal density was normalized to Kv2.1 signal from the 1:0 (Kv2.1:Kv6.1) ratio condition. A paired t test was used to compare Kv2.1 coexpressed with either Kv6.1 or Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel B ). Data are expressed as mean ± SEM from three independent experiments. C , Kv6.1 signal density was normalized to WT Kv6.1 signal from the 1:0.5 (Kv2.1:Kv6.1) ratio condition. Data are expressed as mean ± SEM from three independent experiments. D , schematic diagram indicating stability of Kv2.1 and Kv6.1[W416C] heteromers or homomers of each channel under either excess Kv2.1 or excess Kv6.1[W416C] compared to the other one.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Coassembly with Kv2.1 underlies Kv6.1 phosphorylation. A , Kv2.1 was coexpressed with Kv6.1 variants in HEK293 cells for 72 h. Lysates were treated with Lambda phosphatase (LP) as indicated, separated by SDS-PAGE and probed with an anti-Kv2.1 antibody (n = 3). B , Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells in 1:1 ratio. Lysates were treated with LP as indicated, separated by SDS-PAGE, and probed with an anti-GFP antibody (n = 3). Arrows denote shifted mobility of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.1. Mobility shifts are not seen for Kv6.1 (or variants) when expressed alone. C , Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells, in 3:1 (Kv2.1:Kv6.1) ratio. Lysates were treated with LP as indicated, separated by a 100 μM phos-tag SDS-PAGE gel, and probed with an anti-GFP antibody (n = 2 of 3:1 ratio and n = 4 for Kv2.1: Kv6.1 in 1:1 ratio, not shown here). Phosphorylated Kv6.1 and Kv6.1[L284P] bands are highlighted with arrows . A higher exposure is shown in the dotted box for clearer depiction of phosphorylated bands. Anti–β-actin was used as a loading control in panels A and B . The control groups (individual subunits expressed alone) were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in all experiments.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Phospho-proteomics, SDS Page, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Common features of Kv2.2 and Kv2.1 modulation by Kv6.1. A , current density at +30 mV was measured from indicated subunit combinations (1:1 cDNA ratio of Kv2.2 and Kv6.1 WT or Kv6.1[W416C]) after transient transfection in LM cells. Control groups were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture in all experiments. One-way ANOVA followed by Tukey post hoc test was used to compare groups (∗ indicates p < 0.05; ∗∗ indicates p < 0.001), n = 6. B , mOrange-Kv2.2 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 ratio for 72 h in HEK293 cells. Whole-cell lysates were separated on SDS-PAGE and probed with anti-Kv2.2 and Na + /K + -ATPase (loading control) antibodies. C , Kv2.2 signal in each group from panel B is plotted as normalized to the Kv2.2 control (channel expressed alone) (n = 3). A repeated measures ANOVA test (on raw Kv2.2 density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups (∗ indicates p < 0.05 relative to Kv2.2 alone). D , Kv6.1 or variants were expressed alone or with Kv2.2 (1:1 ratio), for 72 h in HEK293 cells. Whole-cell lysates were separated by phos-tag SDS-PAGE and probed using an anti-GFP (β-actin loading control (n = 3)). Arrows indicate the slower migration of phosphorylated forms of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.2 (representative blot from three experiments).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Transfection, Control, Plasmid Preparation, SDS Page, Migration

Journal: The Journal of Biological Chemistry

Article Title: Rewiring PTH receptor signaling: Hormone dimerization restores endosomal signaling lost in hypocalcemia-linked PTH mutant

doi: 10.1016/j.jbc.2025.110913

Figure Lengend Snippet: Progression of the active ligand–bound PTH 1 R conformations toward their inactive states by weighted ensemble (WE) simulations and FRET recordings in live cells. A and B , the structural features of the TM3–TM6 distance ( A ) and TM6 bending angle ( B ) adopted for visualizing the trajectories of receptor conformations from active toward inactive states. C – E , WE simulations show the progression of the active to inactive receptor conformations when bound by PTH WT ( C ), PTH R25C ( D ), and PTH dimer ( E ). The PTH R25C –PTH 1 R gradually progresses toward its inactive state. In contrast, the PTH dimer –PTH 1 R conformations undergo more constrained changes, indicating the active state’s preferential sampling (or retention). -ln(P) represents the free energy. F – H , the inset shows a schematic of the FRET-based PTH 1 R activation sensor (PTH 1 R CFP/YFP ) with YFP ( yellow ) fused to ICL3 and CFP ( blue ) attached to the receptor C-terminal tail. The graph shows averaged time courses of PTH 1 R activation by recording changes in the FRET ratio in HEK293 cells expressing PTH 1 R CFP/YFP . Cells were continuously perfused with control buffer or 1 μM agonist ( horizontal bar ). Means ± SD from N = 10 (PTH), N = 6 (PTH R25C ), and N = 5 (PTH dimer ).

Article Snippet: HEK293 (American Type Culture Collection) cells were grown in Dulbecco's modified Eagle's medium (DMEM) (low glucose; Life Technologies, 10567022) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, 10438-034), 100 units/ml penicillin, 100 μg/ml streptomycin (Life Technologies, 15140122) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Sampling, Activation Assay, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Rewiring PTH receptor signaling: Hormone dimerization restores endosomal signaling lost in hypocalcemia-linked PTH mutant

doi: 10.1016/j.jbc.2025.110913

Figure Lengend Snippet: PTH 1 R signaling. A , time courses and integrated responses (area under the curve) of ligand (100 nM) induced intracellular Ca 2+ elevation in HEK293 cells expressing recombinant PTH 1 R. Bars represent the mean values ± SD with n = 200 cells from three independent experiments. B and C , time courses ( B ) and integrated ( C ) responses of cAMP production after brief stimulation with 1 nM PTH, PTH R25C , or PTH dimer in HEK293 cells stably expressing recombinant PTH 1 R without (control) or with dynamin K44A expression (DynK 44A ). The percentage of cAMP responses is relative to the response in the presence of forskolin (FSK). Bars represent the mean values ± SD from n = 24 (PTH), n = 36 (PTH R25C ), n = 69 (PTH dimer ) cells in controls and n = 40 (PTH), n = 18 (PTH R25C ), n = 43 (PTH dimer ) cells in Dyn K44A from three independent experiments. D and E , schematic representing the experiment to measure internalization of PTH 1 R SEP ( E ). Time courses showing the internalization profiles for cells stimulated with PTH, PTH R25C , and PTH dimer . Mean values ± SD with n = 15 cells from three independent experiments ( F ). F and G , time courses in cAMP production ( F ) and corresponding integrated values ( G ) in MC3T3 cells expressing the native PTH 1 R. Means ± SD from n = 24 (PTH), n = 24 (PTH R25C ), n = 27 (PTH dimer ) cells from four independent experiments. Data are the mean ± SD with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and no significant (ns) by one-way ANOVA with Dunnett’s ( A ) and Tukey’s multiple comparison test ( G ).

Article Snippet: HEK293 (American Type Culture Collection) cells were grown in Dulbecco's modified Eagle's medium (DMEM) (low glucose; Life Technologies, 10567022) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, 10438-034), 100 units/ml penicillin, 100 μg/ml streptomycin (Life Technologies, 15140122) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Recombinant, Stable Transfection, Control, Comparison

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siControl) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). After 72 h, cells were analyzed by live imagining and protein lysate was harvested for western blot analysis. (A) Schematic of the TDP-43 loss of function Sensor (TS) system design. (B) Overview of the UNC13A-TS, CFTR-TS, and CUTS cassette design. (C) Representative live imaging of TS comparison (10X). (D) Mean intensity quantification of GFP signal intensity as shown in (C). (E-G) Western blot analysis of (E) UNC13A-TS, (F) CFTR-TS, and (G) CUTS developing again GFP and TDP-43 proteins. (H-J) Relative pixel quantification of GFP and TDP-43 band normalized to total protein (Ponceau S) for the indicated TS from E-G. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP signal; red = mCherry signal. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Comparison, Expressing, Control, Transfection, Western Blot, Imaging

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siControl) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). After 72 h, cells were analyzed by live imagining and protein lysate was harvested for western blot analysis. ( A) Mean intensity quantification of GFP signal intensity from live imaging, presented as fold change from mock. (B) Relative pixel quantification of GFP normalized to total protein (Ponceau S), presented as fold change from mock. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Comparison, Expressing, Control, Transfection, Western Blot, Imaging

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Low-dose siRNA TDP-43 (siTDP-43) treatment was performed in stable polyclonal HEK cells expressing CUTS. CUTS-expressing cells were reverse transfected with siRNA control (siControl) or siTDP43 in a dose-response curve (38 to 1200pM) in doxycycline supplemented media (1000ng/ml) for 72hr. (A) Representative immunofluorescence images of CUTS-expressing HEK cells under low doses of siRNA TDP-43 treatment. (60X). (B) Mean intensity quantification of GFP signal from (A) with normalization to the number mCherry positive cells. (C) Western blot of GFP and TDP-43 proteins from HEK cell lysate expressing CUTS under low doses of siRNA TDP-43. Ponceau S is shown as a loading control. (D) Pixel intensity quantification of the GFP and TDP-43 bands shown in (C), presented as fold-change from the mock-treated sample. (E) Schematic showing the position of qPCR primers, developed to detect CUTS cryptic exon inclusion (referred to as ’CUTS-CE’ and ’CUTS-J’). (F) Representative agarose gel showing qPCR product from melting curve detecting CUTS cryptic exon inclusion using the primers shown in (E). (G) qPCR quantification of the siTDP-43 dose curve presented as fold-change from the mock-treated sample. Purple text indicates the GFP fold change from the mock-treated sample. Red text indicates the percentage of total detectable TDP-43 knockdown. Linear regression analysis shown in (D) and (G) was performed on Log values. Fitting method = least squares regression. Green = GFP; red = mCherry. Scale bar = 50 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Control, Immunofluorescence, Western Blot, Agarose Gel Electrophoresis, Knockdown

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Stable HEK cells expressing CUTS were induced with doxycycline (1000 ng/mL) for 24 hours before transfection with the following plasmids: pCMV backbone, TDP-43 WT , TDP-43 ΔNLS , TDP-43 5FL , TDP-43 ΔNLS 5FL , or non-transfected. Following transfection, plasmids were expressed for 72 h, followed by live imaging and protein analysis. (A) Live-imaging of CUTS HEK cells expressing WT or mutant TDP-43 gene cassettes. (B) Representative western blot of exogenous and endogenous GFP and TDP-43. Ponceau S is shown as a loading control. (C) Relative GFP pixel intensity quantification of the band is shown in (B). Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Imaging, Mutagenesis, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: (A) Schematic of CUTS as an autoregulatory controller of TDP-43 expression (CUTS-TDP43). (B-C) TDP-43 siRNA (siTDP43) dose-response curve in stable polyclonal HEK cells expressing CUTS, CUTS-TDP43, or CUTS-TDP43 (codon optimized). The codon-optimized variation allows for continued expression during siTDP-43 treatment. HEK cells expressing the CUTS, CUTS-TDP-43 and CUTS-TDP-43 codon optimize system were reverse transfected with control siRNA (siControl) or siTDP43 in a dose-response curve (0.6nM-20nM) in a doxycycline (1000ng/ml) supplement media for 72hr. Cells were then used for live imaging or protein analysis. (B) Live imaging of the CUTS variants. (C) Immunoblot assay of GFP and TDP-43. Ponceau S is shown as a loading control. (D-E) CFTR minigene assay in stable CUTS or CUTS-TDP43 (codon optimized) expressing HEK cells. Cells were induced with doxycycline (1000 ng/mL) for 24 h before transfection with the CFTR minigene. Following an additional 24h of expression, cells were transfected with 20nM siControl or siTDP-43. Cells were harvested 48 h following siRNA transfection for RNA extraction and RT-PCR analysis. (D) PCR agarose gel of CFTR minigene. (E) PCR analysis of the ratio between the CFTR cryptic exon inclusion and the correctly spliced product from CFTR as shown in (D). Statistical significance was determined by student t-test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Control, Imaging, Western Blot, Mini Gene Assay, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis