Fig 1: C-terminal alternative exons of TCF7L2 in transcripts expressed in total human brain and pancreatic islets and cellular localisation of TCF7L2 splicing form detected by assay ex13-13b (TCF7L2-NE). a Results of PCR amplification with primers ex10 Forw and ex13b Rev of TCF7L2 (ESM Table 1) in cDNA from total human brain and pancreatic islets. cDNA prepared from 10 ng total RNA was used for all PCR reactions. The PCRs were performed with Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) and the PCR products were resolved on a 2% (wt/vol.) agarose gel. Four types of PCR fragments (forms A–D) were observed based on the combination of alternative exons 12, 13, 13a and 13b. b Schematic representation of C-terminal exons of TCF7L2. Constitutive exons are represented by black rectangles and alternative exons by white rectangles. Black triangles mark alternative translation stops. Arrows indicate positions of PCR primers. Forms A and B use a stop codon within exon 13b and forms C and D use a stop codon within exon 13a. In the forms with alternative exons 13a and 13b (C and D) two in-frame stop codons are separated only by 37 bp. Expression of these forms (assay ex13a-13b) was very low in brain and islets and was not studied further. c Confocal imaging of cellular localisation of the recombinant TCF7L2-NE in human PANC-1 cell line (pancreatic cancer). Upper and lower panels represent images of PANC-1 cells transfected with the TCF7L2-NE Halo-tag expression construct (GenBank FJ010169) [3]. Non-transfected cells serve as controls for specificity of detection. For the expression construct a full-length cDNA for TCF7L2-NE splicing form was cloned into a pFC8A expression vector with a C-terminal Halo-tag (Promega, Madison, WI, USA). The endotoxin-free plasmid was prepared with a EndoFree plasmid Maxi kit (Qiagen, Gaithersburg, MD, USA). For transfection, human PANC-1 (pancreatic cancer) cells were plated on chamber slides (Thermo Fisher Scientific, Rochester, NY, USA) and transfected next day with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, the cells were fixed and incubated with primary rabbit anti-HaloTag antibodies (Promega) and with mouse anti-α-tubulin antibodies ab7291-100 (Abcam, Cambridge, MA, USA). Secondary donkey anti-rabbit and anti-mouse antibodies labelled with Alexa fluor 594 and 488 were used for imaging (Invitrogen). The anti-fade ProLong Gold mounting media with DAPI (Invitrogen) was used to mount the cover slides. The imaging was performed with a confocal microscope LSM 510 Meta (Carl Zeiss Microimaging, Thornwood, NY, USA) with ×63 magnification
Fig 2: Seeded aggregation of reporter ChFP-α-syn by exogenous α-syn assemblies assessed by increased resistance to proteolysis.Western blot analysis of the ChFP-α-syn resistance to proteinase K in lysates from Neuro2A cells exposed for 24 h to 0.3 nM α-syn fibrils, equivalent to 2.5 μM monomeric α-syn (a), 300 nM large GA-cross-linked α-syn oligomers, equivalent to 5 μM monomeric α-syn (b), or 5 μM monomeric α-syn (c). The lysates (40 μl corresponding to ~80000 cells), were incubated in the presence of the indicated concentrations of proteinase K for 20 min at 37 °C. The proteolytic reactions were stopped by addition of 1 mM PMSF and immediate denaturation in Laemmli buffer for 5 min at 95 °C. The samples were analyzed on 12% Tris-Glycine SDS-PAGE. ChFP–α-syn (a–c) was probed with mouse monoclonal anti-α-syn antibody (BD Biosciences Cat #610787). The immunoreactivity of α-tubulin (mouse monoclonal antibody DM1A, Abcam Cat #ab7291) in the initial lysate was used as a loading control (d). ChFP-α-syn assemblies seeded by α-syn fibrils resisted 0.1 μg/ml proteinase K (a). ChFP-α-syn from cells exposed to monomeric α-syn was fully degraded by 0.01 μg/ml proteinase K (c). ChFP-α-syn originating from cells exposed to large GA-cross-linked oligomers resisted 0.01 μg/ml and was fully degraded by 0.05 μg/ml proteinase K (b).
Fig 3: ARID1A levels are significantly reduced in ARID1A knockout cells. (A) Subcellular fractions generated from HEK-293T ARID1A knockout (white) and wild-type (black) cell lines were tested for ARID1A using our established ELISA protocol with the primary antibody ab182560 (Abcam, UK). Student’s t-test was performed to determine p-values. ns p > 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; (B) Western blot analysis was performed as described in Materials and Methods, and primary antibody ab182560 was used against ARID1A, while ab7291 was used against alpha-Tubulin. For ARID1A knockout (KO) and wild-type (WT) analysis, lyophilized cell pellets ab257250 (Abcam, UK) were used where each lane contains 6 µL of the reconstituted pellet according to the manufacturer’s instructions. The molecular weight markers (MW) at 250 kDa and 50 kDa indicate presumable ARID1A (242 kDa heavy protein) and alpha-Tubulin (50 kDa heavy protein), respectively. (C) Western blot analysis was performed as described in Materials and Methods using ab182560 and ab7291 as primary antibodies. HEK-293T cells (ab278824, Abcam, UK) of both ARID1A WT and KO were fractionated in cytosol (CYT), membrane (M), free nucleus (FN), and chromatin (CHR). Analysis focused on a band at nearly 242 kDa, in comparison to a molecular weight marker (right lane), which might be specific for ARID1A (arrow); (D,E) show intensity ratios measured in (B,C) using ImageJ [30] as well as p-values obtained by Student’s t-test, respectively.
Fig 4: ARID1A expression in tumor lysate using Western blot. (A) The lysate of the tumor tissue (Case no. 37) is fractionated into cytosol (CYT), membrane (M), free nuclear (FN), and chromatin (CHR) fractions. Western blot analysis was performed according to our previous study [30]. Equal amounts of protein (30 µg) from tissue homogenates were separated by SDS-PAGE (4569033, Bio-Rad Laboratories, Hercules, CA, USA) and transferred to 0.2 µm nitrocellulose membranes (1620112, Bio-Rad Laboratories, USA) for 60 min at 100 V. Membranes were blocked overnight at 4 °C and incubated with primary antibodies against ARID1A (Ab182560, 1:2000, Abcam, UK) or α-tubulin (Ab7291, 1:2200, Abcam) as loading control, followed by HRP-conjugated secondary antibodies: K4003 (Dako, Glostrup, Denmark, diluted 1:500 in TBS-T) for ARID1A or anti-mouse antibody (K400111, Dako, Denmark, diluted 1:500 in TBS-T) for alpha-Tubulin, respectively. Signals were detected using ECL (RPN2235, VWR, Darmstadt, Germany) and visualized on hyperfilms (28-9068-37, VWR, Germany). Molecular weight markers were included in each run. Comparison with a molecular weight marker (MW, left lane) suggests the size of the intense band to be approximately 242 kDa, which matches full-length ARID1A (black arrow) or alpha-tubulin (50 kDa, blue arrow). An unspecific reaction at 16 kDa was observed, suggestive of MNase, as discussed in [30]. (B) Intensity measurement was performed using ImageJ, version 1.54p [34]. (C) Corresponding ELISA results of ARID1A levels measured in the lysates used in (A). The original Western blots are shown in Supplementary Figure S1.
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