Arid1a

A pellet of VOA1066 cells was lysed in urea buffer that contains 90% 8.8 M urea, 2% 5 M NaH₂PO₄, 8% 1M Tris pH 8.0 with freshly added 10x Protease and 100x phosphatase inhibitor. 30 ug of cell lysate was resolved on a 6% SDS-PAGE gel for protein detection as previously described [11 (link)]. Primary antibodies include ARID1A (Sigma, HPA005456, 1:1000), ARID1B (Abgent, AT1190a, 1:2000), SMARCA2 (Cell Signaling, #6889), SMARCA4 (Abcam, ab110641, 1:2000), SMARCB1 (#612110, BD), p-AKT (S473) (Cell Signaling, #9271), total AKT (Cell Signaling, #9272) and PTEN (Cell Signaling, #9552S) and Vinculin (Sigma, v9131, 1:50000).

The PDX tumors were collected, fixed with 10% formalin for 24 hours, washed with PBS and maintained in 70% ethanol at 4°C. The tissue samples were dehydrated in graded ethanol, xylene, and embedded in paraffin. Immunohistochemistry of paraffin embedded section (5 µm) was performed using DAKO Envision+System (Dako, Santa Clara, CA). The following primary antibodies were used: PAX8 (Proteintech, Rosemont, IL, #10336-1-AP, 1:1000), ARID1A (Sigma-Aldrich, #HPA005456, 1:500), napsinA (Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UK, #NCL-L-NapsinA, 1:400), racemase (Zeta Corporation, Arcadia, CA, # Z2001L, 1:50), Ki67 (Dako, #M7240, 1:100), p-Rb (Ser807/811) (Cell Signaling, #8516, 1:200), γH2AX (Ser139) (Cell Signaling, #9718, 1:500), and cleaved caspase-3 (Asp175) (Cell Signaling, #9664, 1:200). Each antibody was incubated for 40 minutes at room temperature. Antigen retrieval for all targets was performed using a pressure cooker in citrate buffer (pH = 6). Appropriate positive and negative (incubation with secondary antibody only) controls were stained in parallel for each round of staining. The percentage of positive cells and staining intensity were reviewed by pathologist and quantified by ImageJ.

Cells were washed twice in ice-cold PBS and scraped on ice in ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Protein lysates were collected after centrifuging the cells at 13,000 rpm for 10 min at 4 °C. For each sample, 25 μg of protein was loaded onto a SDS-PAGE gel. After transferring the protein to a nitrocellulose membrane, the membrane was incubated with primary antibodies against ARID1A (Sigma-Aldrich), BRG1 (Cell Signaling Technology, Danvers, MA, USA), SNF5 (Cell Signaling Technology), and vinculin (Cell Signaling Technology). Then, the membrane was incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). The bands on the membrane were visualized using enhanced chemiluminescence plus western blotting reagent (Amersham Biosciences, Little Chalfont, UK).

All parental tumor tissues and PDX tissues fixed in 10% neutral buffered formalin were processed routinely and embedded in paraffin wax. The sections (3 μm thick) were stained with hematoxylin and eosin (HE) and histopathologically evaluated. Paraffin-embedded sections were also used for immunohistochemistry analysis, and primary antibodies against human pancytokeratin (prediluted; clone AE1/AE3, Dako, Glostrup, Denmark), PAX8 (1:50; clone PAX8R1, Abcam, Cambridge, MA, USA), estrogen receptor (ER) (prediluted; clone SP1, Ventana Medical Systems, Tucson, AZ, USA), TP53 (prediluted; clone DO7, Dako), ARID1A (1:2000; rabbit polyclonal, Sigma, St. Louis, MO, USA), PAX2 (1:200 dilution; clone EPR8586, Abcam), PTEN (1:200; clone D4.3, Cell Signaling Technology, Beverly, MA, USA), PMS2 (prediluted; clone EP51, Dako), and MSH6 (prediluted; clone EP49, Dako) were used for all cases. Primary antibodies against human CD45 (prediluted; clone M0701, Dako) were optionally used for several cases.