Figures and data
CUTS is more precise than UNC13A-TS and CFTR-TS as a TDP-43 splicing sensor.
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.
Dose-response of CUTS and TDP-43 knockdown.
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.
CUTS enables detection of TDP-43 loss-of-function with ultra-low siRNA treament.
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.
Mislocalization or aberrant phase transitions induce TDP-43 LOF.
Stable HEK cells expressing CUTS were induced with doxycycline (1000 ng/mL) for 24 hours before transfection with the following plasmids: pCMV backbone, TDP-43WT, TDP-43ΔNLS, TDP-435FL, TDP-43ΔNLS 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.
TDP-43wt expression rescues loss-of-function in a HeLa TARDBP Knockout Cell Line.
Transient CUTS expression in Hela TDP-43 KO we induced with doxocycline (1000n/ml) for 24 hours before transfection of pCM backbone or TDP-43WT plasmids. Following transfection, plasmids were expressed for 72 h, followed by live imaging and protein analysis. (A) Live-imaging of Hela TDP-43 KO expressing CUTS in combination with TDP-43WT or pCMV backbone control. (B) Representative WB of exogenous and endogenous GFP and TDP-43. Ponceau S is shown as a loading control. (C) Relative GFP pixel intensity quantification of the protein bands shown in (B). (D) Agarose gel showing RT-PCR product from CUTS cryptic exon inclusion from wildtype or TDP-43 KO Hela cell lines using the CUTS-CE primers shown in Figure 2E. 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.
Autoregulatory rescue of TDP-43 loss-of-function by CUTS-TDP43 expression.
(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.
