a c heterodimerizer  (TaKaRa)

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: The Kalirin-7 GEF domain induces a concentration-dependent increase in baseline spine volume. A, Construct and experimental schedule. Two delivery methods were used: lipofection ( B, C ) and AAV ( D, E ). B, Example confocal images of dendrites expressing mVenus-FKBP-K7GEF via plasmid lipofection. mScarlet signal is shown as a volume marker. Scale bars, 1 µm. C, Distribution of dendritic spine volumes under plasmid lipofection conditions, plotted as a function of binned spine mVenus intensity. Spine volume was estimated by deconvolution (see Methods). mVenus intensity was measured for each spine, normalized by the estimated spine volume, and averaged across spines within each dendrite. The mean mVenus intensity per unit volume at 50 ng/mL lipofection was defined as 10 a.u./μm³. Six lipofection concentrations (2–500 ng/mL) are shown in different colors. No A/C heterodimerizer was applied. Kruskal-Wallis test ( H = 81, p = 5.5 × 10 -16 ) followed by Mann-Whitney test against neurons expressing cell-filling mScarlet alone (N (spines/dendrites) = 69/6). -10 0 a.u./μm 3 , N = 85/10, U = 2853, p = 0.77; 10 0 -10 0.5 a.u./μm 3 , N = 53/5, U = 1698, p = 0.50; 10 0.5 -10 1 a.u./μm3, N = 34/5, U = 588, p = 4.1 × 10 -5 ; 10 1 -10 1.5 a.u./μm3, N = 33/5, U = 310, p = 3.1 × 10 -9 ; 10 1.5 a.u./μm3, N = 17/3, U = 96, p = 1.0 × 10 -7 . D, Example confocal images of dendrites expressing mVenus-FKBP-K7GEF via AAV transduction. Sparsely expressed mScarlet, by using a double-floxed inverted open (DIO) reading frame system in conjunction with low Cre expression, is shown as a volume marker. Scale bars, 1 µm. E, Distribution of dendritic spine volumes under AAV transduction conditions, plotted as a function of binned spine mVenus intensity as in ( C ). Four different AAV concentrations (3.0 × 10^8-1.0 × 10^10 GC/mL) are shown in different colors. Kruskal-Wallis test ( H = 55, p = 2.7 × 10 -11 ) followed by Mann-Whitney test against neurons expressing cell-filling mScarlet alone. -10 0 a.u./μm 3 , N (spines/dendrites) = 60/5, U = 1946, p = 0.56; 10 0 -10 0.5 a.u./μm 3 , N = 37/5, U = 1256, p = 0.89; 10 0.5 -10 1 a.u./μm3, N = 52/7, U = 751, p = 4.8 × 10 -8 ; 10 1 -10 1.5 a.u./μm3, N = 11/2, U = 95, p = 7.2 × 10 -5 , F, Representative images of highly branched dendritic spine (yellow arrow heads). Scale bars, 1 µm. G, Fraction of highly branched spines per dendrite expressing mVenus–FKBP–K7GEF via lipofection (grey) or AAV (red). Statistical analysis was performed using Mann-Whitney test with Bonferroni correction. Lipofection -10 0.5 a.u./μm 3 (N (dendrites) = 15) vs Cell-fill only (N = 7), U = 51, p = 0.91; AAV -10 0.5 a.u./μm 3 (N = 10) vs Cell-fill only, U = 34, p = 0.82; Lipofection 10 0.5 -a.u./μm 3 (N = 13) vs Cell-fill only, U = 0.0, p = 3.4 × 10 -4 ; AAV 10 0.5 -a.u./μm 3 (N = 9) vs Cell-fill only, U = 2.5, p = 1.6 × 10 -3 ; -10 0.5 a.u./μm 3 lipofection vs AAV, U = 60, p = 0.65; 10 0.5 -a.u./μm 3 lipofection vs AAV, U = 68, p = 0.90. * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Concentration Assay, Construct, Expressing, Plasmid Preparation, Marker, MANN-WHITNEY, Transduction

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: AAV-based validation confirms that IRESv10-linked uniSynK reliably induces spine enlargement with minimal unintended morphological abnormalities under baseline conditions A, Schematic of uniSynK (single construct linked via IRESv10) and split SynK (two constructs). Constructs were delivered using AAV. B, (Left) Representative confocal images of dendrites expressing split SynK. For visualization purpose only, higher concentrations were used while maintaining the same delivery ratio (PSDΔ1,2-FRB-mTq, 1 × 10¹¹ GC/mL; mVenus-FKBP-K7GEF, 2 × 10⁹ GC/mL). Scale bars, 50 µm. (Right) Representative confocal images of dendrites expressing uniSynK before (−5 min) and after (35 min) application of A/C heterodimerizer. mScarlet signal is shown as a volume marker. Scale bars, 1 µm. C, Distribution of the mVenus-to-mTq expression ratio in spines for the split construct. Ratios were calculated for individual spines and averaged per cell for plotting. Cells were classified into three groups based on these ratios: within the IRESv10 range defined under 3 μg/mL plasmid lipofection (including both mTq and mVenus; ; group 2), above this range (group 1), and below this range (group 3). D, Cumulative distributions of dendritic spine volumes under four conditions: uniSynK and split SynK groups 1–3. N (spines/dendrites/neurons) = 227/29/16 (IRESv10), 27/4/2 (group 1), 175/26/12 (group 2) and 57/8/4 (group 3). Statistical comparisons were performed using the Kolmogorov–Smirnov (K–S) test against the uniSynK condition with Bonferroni correction.Group 1, D = 0.44, p = 6.4 × 10 -5 ; Group2, D = 0.13, p = 0.062; Group3, D = 0.15, p = 0.26. E, F, Averaged time courses ( E ) and summary quantification ( F ) of changes in spine volume from neurons expressing uniSynK and split SynK groups 1–3. Data in ( E ) are shown as the mean ± s.e.m. Values in ( F ) represent the average between 20 and 100 min after A/C heterodimerizer administration. N (dendrites/neurons) = 29/16 (uniSynK, IRESv10), 4/2 (split SynK group1), 26/12 (split SynK group2) and 8/4 (split SynK group 3). The median (horizontal line), quartiles (boxes), and range within 1.5 times the interquartile range (whiskers) are denoted. Statistical comparisons were made using Mann–Whitney test against the uniSynK condition with Bonferroni correction. Group 1, U = 61, p = 0.98; Group 2, U = 443, p = 0.52; Group 3, U = 198, p = 8.6 × 10 -3 . * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Biomarker Discovery, Construct, Expressing, Marker, Plasmid Preparation, MANN-WHITNEY

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: Removal of the Kalirin-7 C-terminal region and addition of mTq does not induce significant changes in spine volume distribution or intrinsic dynamics. A, Cumulative distribution of dendritic spine volumes under three conditions. K7GEF-Cterm (original), N = 92; K7GEF only, N = 89; K7GEF only-w/ mTq, N = 172 spines. Statistical comparisons were made using the Kolmogorov-Smirnov (K-S) test with Bonferroni correction. K7GEF-Cterm vs K7GEF only, D = 0.069, p = 0.97; K7GEF-Cterm vs K7GEF only-w/ mTq, D = 0.12, p = 0.37. B , Time courses of the estimated absolute volume (see Methods) of spines before and after A/C heterodimerizer application, along with their averages (blue line) and s.e.m. (blue shade) for a representative neuron expressing PSDΔ1,2-FRB-mTq and mVenus-FKBP-K7GEF. C, D, Intrinsic spine fluctuation of neurons expressing PSDΔ1,2-FRB-mTq and mVenus-FKBP-K7GEF, before and after A/C heterodimerizer administration, each calculated per 20-min interval. Each plotted point represents the mean ( C ) and the s.d. ( D ) of spine-head volume changes in 24 pooled spines with similar baseline volumes. Error bars represent s.e.m. values ( C ) and the 95% confidence intervals of the estimated s.d. ( D ). Data are fitted by a zero ( C ) and to the 2/3 power of the baseline spine volume ( D ). * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Expressing

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: Combining two SynK components using IRESv10 induces dendritic spine enlargement across a broad expression range without detectable structural abnormalities under plasmid lipofection. A, Schematic of a single-vector construct combining two SynK components via IRES variants (IRESv). Three variants with different translation efficiencies were used: IRESwt, IRESv10 and IRESv12. B, Representative confocal images of dendrites expressing each IRES construct, delivered via plasmid lipofection: IRESwt (left), IRESv10 (middle) and IRESv12 (right). Scale bars, 1 μm. C, Distributions of mTq and mVenus intensities in dendritic spines under six conditions (v12, v10, and wt at 1 and 3 μg/mL) before adding A/C heterodimerizer (−5 min). Signals were measured per spine and averaged per dendrite. Gray shading indicates mVenus concentrations that induce unintended baseline changes in spine volume ( ; 10 0.5 a.u./μm 3 ). Only dendrites with background-subtracted signal intensity ≥ 0 are shown. Mean ± s.e.m. for each condition are indicated by crosshairs. Linear regression lines are shown for each IRES variant. D, Distributions of mTq intensities in dendritic spines under six conditions (v12, v10, and wt at 1 and 3 μg/mL lipofection). Only dendrites with background-subtracted signal intensity ≥ 0 are shown. N (dendrites/neurons) = 8/6 (wt, 3 μg/mL), 19/11 (v10, 3 μg/mL), 11/6 (v12, 3 μg/mL), 9/7 (wt, 1 μg/mL), 13/9 (v10, 1 μg/mL) and 14/8 (v12, 1 μg/mL). Statistical comparisons were made using the Kruskal-Wallis test (1 μg/mL, H = 0.78, p = 0.68; 3 μg/mL, H = 1.8, p = 0.41). E, Expression ratio of mVenus to mTq for the three IRES variants. Signals were measured per spine and averaged per neuron. Statistical comparison between v10 (3 μg/mL) and v10 (1 μg/mL) was performed using the Mann–Whitney test ( U = 35, p = 0.29). F, The concentration range at which SynK constructs linked via each IRES variant induce A/C-dependent spine enlargement without affecting baseline spine volume. (Top) Averaged dendritic spine volume before A/C heterodimerizer addition, plotted for each IRES variant across expression levels measured by spine mTq intensity. Data were binned in doubling intervals, with the first bin defined as ≤50 a.u., and the mean ± s.e.m. for each bin is shown. Statistical comparisons were performed using the Mann–Whitney test, comparing each variant to cell-fill–only neurons within each bin, with Bonferroni correction. For wt, - 50 a.u./μm³, N (spines) = 36, U= 1469, p = 0.13; 50-100 a.u./μm³, N = 43, U = 1853, p = 0.027; 100 - 200 a.u./μm³, N = 44, U = 2086, p = 8.3 × 10 -4 ; 200 - 400 a.u./μm³, N = 19, U = 954, p = 2.5 × 10 -3 . For v10, - 50 a.u./μm³, N = 37, U = 1586, p = 0.041; 50-100 a.u./μm³, N = 69, U = 2775, p = 0.094; 100 – 200 a.u./μm³, N = 107, U = 4303, p = 0.064; 200 - 400 a.u./μm³, N = 51, U = 1779, p = 0.92; 400 – 600 a.u./μm³, N = 12, U = 457, p = 0.57 . For v12, - 50 a.u./μm³, N = 49, U = 1851, p = 0.38; 50-100 a.u./μm³, N = 54, U = 1806, p = 0.77; 100 - 200 a.u./μm³, N = 79, U = 2844, p = 0.65; 200 – 400 a.u./μm³, N = 53, U = 1905, p = 0.70; 400 - 600 a.u./μm³, N = 18, U = 646, p = 0.80. (Middle) Percentage change in dendritic spine volume following A/C heterodimerizer administration, plotted in the same manner. Statistical comparisons were performed using the Mann–Whitney test, comparing each variant to IRESv10 within each bin, with Bonferroni correction. For wt, - 50 a.u./μm³, U = 659, p = 0.94; 50-100 a.u./μm³, U = 1352, p = 0.43; 100 - 200 a.u./μm³, U = 2723, p = 0.13; 200 – 400 a.u./μm³, U = 563, p = 0.30. For v12, - 50 a.u./μm³, U = 872, p = 0.77; 50-100 a.u./μm³, U = 1718, p = 0.46; 100 - 200 a.u./μm³, U = 3927, p = 0.41; 200 - 400 a.u./μm³, U = 595, p = 8.8 × 10 -7 ; 400 - 600 a.u./μm³, U = 33, p = 1.6 × 10 -3 . (Bottom) For each bin, the mean spine volume before A/C and the mean change in spine volume were calculated. For each IRES variant, bins exhibiting ≥15% increase in spine volume without a significant increase in baseline dendritic spine volume were identified, and the corresponding spine mTq concentration range (minimum to maximum) is indicated by markers. G, Representative confocal image of a dendrite expressing PSDΔ1,2–FRB–mTq–IRESv10–mVenus–FKBP–K7GEF before (−5 min) and after (35 min) application of A/C heterodimerizer. mScarlet signal is shown as a volume marker. Scale bars, 1 µm. H,I, Averaged time courses ( G ) and summary quantification ( H ) of changes in dendritic spine mVenus concentration in quantified as the mVenus-to–mScarlet ratio (top) and spine volume (bottom) from neurons expressing SynK constructs linked via IRESwt, IRESv10, or IRESv12, as well as an IRESv10 GEF-dead mutant (dGEF), following lipofection at 3 μg/mL. mVenus signals for IRESv12 were not plotted due to unreliable detection. Data in ( G ) are shown as the mean ± s.e.m. Values in ( H ) represent the average between 40 and 120 min after A/C heterodimerizer administration. N (dendrites/neurons) =8/6 (wt), 19/11 (v10), 11/6 (v12), 8/5 (v10 dGEF), and 3/2 (cell-fill only). The median (horizontal line), quartiles (boxes), and range within 1.5 times the interquartile range (whiskers) are denoted. Statistical comparisons were made using the Kruskal-Wallis test (top, H = 3.7, p = 0.16; bottom, H = 16, p = 2.5 × 10 -3 ) followed by Mann–Whitney test against neurons expressing IRESv10 with Bonferroni correction (bottom, wt, U = 76, p = 1.0; v12, U = 164, p = 0.011; v10-dGEF, U = 126, p = 6.5 × 10 -3 , Cell-fill only, U = 23, p = 1.3 × 10 -3 ). J, Ratio of highly branched spines per dendrite under 3 μg/mL plasmid lipofection. Statistical comparisons were performed using the Kruskal–Wallis test ( H = 11, p = 9.7 × 10 -3 ), followed by Mann–Whitney tests against neurons expressing cell-filling mScarlet alone (without SynK). IRESwt, U = 5, p = 8.0 × 10 -3 ; IRESv10, U = 62, p = 0.80; IRESv12, U = 31, p = 0.51. K, Spine density in neurons expressing SynK linked via IRESv10 following 3 μg/mL lipofection, before and after A/C heterodimerizer administration, quantified per 10 μm of dendrite. N (dendrites/neurons) =19/11 (v10). Wilcoxon signed-rank test ( U = 12, p =0.71). L, M, Intrinsic spine fluctuation of neurons expressing PSDΔ1,2–FRB–mTq–IRESv10–mVenus–FKBP–K7GEF, before and after A/C heterodimerizer administration, each calculated per 20-min interval. Each plotted point represents the mean ( L ) and the s.d. ( M ) of spine-head volume changes in 21 pooled spines with similar baseline volumes. Error bars represent s.e.m. values ( L ) and the 95% confidence intervals of the estimated s.d. ( M ). Data are fitted by a zero ( L ) and to the 2/3 power of the baseline spine volume ( M ). * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Expressing, Plasmid Preparation, Construct, Variant Assay, Comparison, MANN-WHITNEY, Concentration Assay, Marker, Mutagenesis

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: C-terminal truncation of Kalirin-7 preserves spine enlargement in SynK. A, Schematic of the original SynK construct. B, Representative confocal image of a dendrite expressing PSDΔ1,2–FRB (3 μg/mL) and mVenus–FKBP fused to Kalirin-7 GEF and C-terminal regions (0.05 μg/mL) before (−5 min) and after (35 min) application of A/C heterodimerizer. mScarlet signal is shown as a volume marker. Scale bar, 1 µm. C, Time courses of changes in the mVenus signal in dendritic spines (top) and spine volume (bottom), including their averages (bold line) and s.e.m (shade) for a representative neuron expressing PSDΔ1,2–FRB and mVenus–FKBP–K7GEF–Cterm. Values are plotted as a percentage change from the baseline average. D, Plasmid construct of SynK lacking the Kalirin-7 C-terminal region. E, Representative confocal image of a dendrite expressing PSDΔ1,2–FRB (3 μg/mL) and mVenus–FKBP–K7GEF (w/o Cterm, 0.05 μg/mL). Scale bar, 1 µm. F, Time course of mVenus and mScarlet signals in dendritic spines for a representative neuron expressing PSDΔ1,2–FRB and mVenus–FKBP–K7GEF. G, H, Averaged time courses ( G ) and summary ( H ) of changes in mVenus signal in dendritic spines (top) and spine volume (bottom) from neurons expressing SynK with either K7GEF–C-terminal region (original) or K7GEF alone. Data in ( G ) are shown as the mean ± s.e.m. Values in ( H ) represent the average between 40 and 120 min after A/C heterodimerizer administration. N (dendrites/neurons) = 12/7 (K7GEF-Cterm) and 11/4 (K7GEF only). The median (horizontal line), quartiles (boxes), and range within 1.5 times the interquartile range (whiskers) are denoted. Statistical comparisons were made using the Mann–Whitney test (top: U = 53, p = 0.44; bottom: U = 58, p = 0.64). * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Construct, Expressing, Marker, Plasmid Preparation, MANN-WHITNEY

Journal: bioRxiv

Article Title: uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

doi: 10.64898/2026.05.02.720486

Figure Lengend Snippet: mTq tagging of PSDΔ1,2-FRB does not interfere with SynK function. A, Schematic of the SynK construct with mTq fused to PSDΔ1,2–FRB and lacking the Kalirin-7 C-terminal region. B, Representative confocal images of a dendrite expressing PSDΔ1,2-FRB-mTq and mVenus-FKBP-K7GEF before (−5 min) and after (35 min) application of A/C heterodimerizer. Images of mScarlet signal are shown as a volume marker. Scale bar, 1 µm. C , Time courses of changes in the mVenus signal in dendritic spines (top) and spine volume (bottom), including their averages (bold line) and s.e.m (shade) for a representative neuron expressing PSDΔ1,2–FRB–mTq and mVenus–FKBP–K7GEF–Cterm. Values are plotted as a percentage change from the baseline average. D, E, Averaged time courses ( D ) and summary ( E ) of changes in mVenus signal in dendritic spines (top) and spine volume (bottom) from neurons expressing SynK with mTq tagging of PSDΔ1,2-FRB. Data without mTq are reproduced from and shown as dashed lines ( D ) and hollow box plots ( E ) for comparison. Data in ( D ) are shown as the mean ± s.e.m. Values in ( E ) represent the average between 40 and 120 min after A/C heterodimerizer administration. N (dendrites/neurons) = 16/9 (w/ mTq) and 11/4 (w/o mTq). The median (horizontal line), quartiles (boxes), and range within 1.5 times the interquartile range (whiskers) are denoted. Statistical comparisons were made using the Mann–Whitney test (top: U = 91, p = 0.90; bottom: U = 106, p = 0.39). * p < 0.05; ** p < 0.01; n.s., not significant.

Article Snippet: Immediately before application, a portion of the remaining medium was mixed with the A/C heterodimerizer (Takara Bio; 0.5 mM stock in ethanol) and then returned to the dish to achieve a final concentration of 4 μM.

Techniques: Construct, Expressing, Marker, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A) Schematic depiction of induced proximity between eGFP-FRB*-β-catenin and HA- and FKBP12 F36V - tagged Open Reading Frames (ORF) using the small molecules Rapamycin or the Rapalog A/C Heterodimeriser. B, C) Rapamycin or A/C Heterodimeriser induced dimerisation of eGFP-FRB*-β-catenin and CSNK1D- or SIAH2-FKBP12 F36V leads to β-catenin degradation in DLD-1 colorectal cancer cells. Double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) were infected with virus expressing the indicated -FKBP12 F36V -HA fusion proteins, selected with puromycin for 2 days and then treated with Rapamycin (0.5 µM), A/C Heterodimeriser (1 µM) or equivalent volumes of DMSO for 24 h. Then, eGFP-FRB*-β-catenin abundance was quantified by flow cytometry. B) Histograms of eGFP intensity of one representative biological replicate are shown normalised to mode. C) Median fluorescence intensity of eGFP in n = 4 or 7 biological replicates with mean and standard deviation. A minimum of 8,800 cells per condition were recorded in the final gate. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test comparing treatment groups to DMSO. * Padj ≤ 0.05, *** Padj ≤ 0.0001, ns = not significant. D) Double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) expressing CSNK1D-FKBP12 F36V exhibit a strong growth defect when treated with Rapamycin (0.5 µM) or A/C Heterodimeriser (1 µM) over the course of 14 days. Medium with drugs was refreshed every 2 days. Confluence was analysed using an Incucyte time-lapse microscope. Representative of n = 3 biological replicates. FKBP12 - 12-kDa FK506-Binding Protein; FRB* - FKBP12-Rapamycin Binding Domain, T2098L; a.u. - arbitrary unit

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: Infection, Virus, Expressing, Flow Cytometry, Fluorescence, Standard Deviation, Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A) Experimental outline of bulk RNA-sequencing (RNAseq) experiment: double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) were infected with virus expressing CSNK1D-FKBP12 F36V -HA and selected with puromycin for 2 days. Then, cells were treated with A/C Heterodimeriser (1 µM) or equivalent volumes of EtOH for 8 h or 16 h before RNA isolation for differential gene expression analysis. B) Principal component analysis (PCA) plot confirmed that variance between RNAseq samples of three biological replicates stemmed mainly from type and time of treatment. C) 8 h and 16 h treatment with the A/C Heterodimeriser induced a robust downregulation of Wnt/β-catenin target genes. Heatmap of scaled expression of genes associated with the β-catenin dependent Wnt signalling pathway in control (EtOH treated) or A/C Heterodimeriser treated samples. D, E) Volcano plots depicting differentially expressed genes at 8 h or 16 h after A/C vs. EtOH control treatment. Significantly regulated genes are shown in dark grey with Padj ≤ 0.01 and fold change > 2. F) Gene Ontology (GO) biological processes (BP) differentially downregulated in A/C Heterodimersier vs. EtOH control treated samples at 8 h and 16 h included ‘Canonical Wnt signalling pathway’ as a driver term. G) Enrichment plots depict depletion of genes contained in the human gene set ‘HALLMARK_WNT_BETA_CATENIN_SIGNALING’ (M5895) in A/C Heterodimeriser vs. EtOH control treated samples at 8 h and 16 h. ES - Enrichment score; NES – normalised enrichment score; FKBP12 - 12-kDa FK506-Binding Protein; FRB* - FKBP12-Rapamycin Binding Domain, T2098L

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: RNA Sequencing, RNA sequencing, Infection, Virus, Expressing, Isolation, Gene Expression, Control, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A, B) CSNK1D-dependent degradation of β-catenin is kinase activity and Ubiquitin-Proteasome System (UPS) dependent. DLD-1 CTNNB1 eBFP2/eGFP cells (clone #28) were infected with virus expressing the indicated -vhhGFP fusion proteins, selected with puromycin for 2 days and then analysed or further treated. A) Kinase-activity dead mutants of CSNK1D do not degrade eBFP2/eGFP-β-catenin. Representative Western Blot of n = 3 biological replicates. B) Inhibitors of the UPS rescue CSNK1D-dependent degradation of β-catenin and lead to accumulation of T41/S45 phosphorylated β-catenin. After selection, cells were treated with DMSO (0.1 %), the proteasome inhibitor MG132 (10 µM), the E1 inhibitor TAK-243 (1 µM), the inhibitor of neddylation MLN-4924 (10 µM) or Bafilomycin (1 µM) for 18 h before harvest and analysis by Western Blot. C, D, E, F) Genome-wide CRISPR/Cas9 knock-out fitness screen identifies regulators of CSNK1D-dependent β-catenin degradation. Highlighted are genes involved in cell-cell contacts (green), transcriptional regulation (light green), neddylation (pink), the SCF β-TrCP E3 ubiquitin ligase complex (red, FBXW11 is also known as BTRC2), and others (black). C) Schematic of positive selection screen in double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) with stable expression of CSNK1D-FKBP12 F36V -HA. After puromycin selection, cells were split into three replicates per condition and treated with A/C Heterodimeriser (1 µM) or equivalent volumes of EtOH. Media and compounds were refreshed every 2 days. D) Rank order plot depicting the gene level summary of sgRNAs that regulate CSNK1D-dependent β-catenin degradation identified at Endpoint (T16). Black dots represent regulators with FDR < 0.01. E) overall sgRNA distribution and individual sgRNA enrichment at T16 of the highlighted genes, summarised in F) . G, H, I) GSK3 and AXIN are dispensable for CSNK1D-induced degradation of β-catenin. G) HEK293 Flp-In T-REx GFP-CTNNB1 cells were transfected with CRISPR ribonucleoproteins (RNPs) to generate GSK3A/GSK3B or AXIN1/AXIN2 double knock-out (KO) cell lines. H) Western blot confirms gene knock-out. I) HEK293 Flp-In T-REx cells stably expressing eGFP-β-catenin under a TET-inducible promoter were transfected with the indicated - vhhGFP fusion proteins and eGFP expression was measured by flow cytometry as read-out for β-catenin abundance. Individual data points of four or five independent experiments are shown with mean and standard deviation. A minimum of 4,500 cells per condition were recorded in the final gate. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test comparing all -vhhGFP groups to NLuc-vhhGFP. * Padj ≤ 0.05, ** Padj ≤ 0.0001, ns = not significant. For Western blots: * signal from previous staining; kDa = Kilodalton; GAPDH served as loading control; RLuc - Renilla luciferase; NLuc - NanoLuc luciferase; vhhGFP - nanobody binding to eGFP and eBFP2; a.u. - arbitrary unit

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: Activity Assay, Ubiquitin Proteomics, Infection, Virus, Expressing, Western Blot, Selection, Genome Wide, CRISPR, Knock-Out, Transfection, Stable Transfection, Flow Cytometry, Standard Deviation, Staining, Control, Luciferase, Binding Assay