α actin

Total proteins were extracted from tissues using TX-100 buffer (1% Triton X-100, 50mM Tris pH8.0, 150mM NaCl, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche, Germany). Samples were homogenized in TX-100 buffer and the supernatants were collected by centrifugation at 16,000g for 10 minutes. Protein concentration was determined by modified Lowry assay (Bio-Rad DC protein assay). The lysates were then loaded on 4–20% Tris-glycine gel (Invitrogen, Carlsbad, CA, USA). Protein was transferred to polyvinylidene difluoride (PVDF) membranes and incubated with protein-free T20 blocking buffer (Pierce, Rockford, IL). The membrane was incubated with antibodies against α-DG (IIH6C4) and α-actin (Sigma, St. Louis, MO, USA) in 20mM Tris pH7.4, 150mM NaCl, 0.1% Tween20 at 1:2000 dilutions. Alpha-DG and α-actin antibodies were detected by HRP-Goat anti-mouse IgM (Invitrogen, Carlsbad, CA, USA) and Goat anti-rabbit IgG-HRP conjugate (Bio-Rad, Hercules, CA, USA) respectively. Blots were developed with ECL (PerkinElmer, Waltham, MA, USA) and the images were exposed and processed by a LAS-4000 imaging system (Fujifilm, Valhalla, NY, USA).

For EhADH immunodetection, we obtained rabbit polyclonal antibodies (α-EhADH) against a specific EhADH peptide (N-566 QCVINLLKEFDNTKNI 582-C) localized within the adherence domain. New Zealand male rabbits were immunized three times (each 2 weeks) with 300 μg of this peptide diluted in TiterMax® Gold Adjuvant liquid (Sigma). Other primary antibodies used were: mouse against EhADH (mAbAdh) (Arroyo and Orozco, 1987 (link)), α-actin (kindly donated by Dr. José Manuel Hernández from Department of Cellular Biology, CINVESTAV, Mexico), α-claudin-1 (Invitrogen), α-occludin (Invitrogen) and α-caveolin-1 (Santa Cruz Biotechnology); rabbit α-ZO-2 (Invitrogen), α-ZO-1 (Invitrogen), α-occludin (Invitrogen), α-α/β tubulin (Cell Signaling), and α-PCNA (Azuara-Liceaga et al., 2018 (link)); and goat α-clathrin (Santa Cruz Biotechnology) and α-GAPDH (Santa Cruz Biotechnology). For some experiments, mouse IgM isotype control (Thermo Fisher) was used. Secondary antibodies included: α-rabbit, α-mouse and α-goat HRP-labeled IgG (1:10,000) (Life technologies); and α-rabbit, α-mouse and α-goat FITC-, TRITC- and Cy5-labeled IgM, and IgG (1:100) (Zymed) antibodies.

Antibodies used in the study were α-HA (Roche Cat# 11867423001, RRID: AB_390918), α-NFAT3 [(Sigma-Aldrich Cat# HPA031641, RRID: AB_10600826), for Proximity Ligation Assay (PLA)], α-RERG (Sigma-Aldrich Cat# SAB1403408, RRID: AB_10737054, for PLA), α-pan cytokeratin (Sigma-Aldrich Cat# F3418, RRID: AB_259536); α-actin (Thermo Fisher Scientific Cat# MA5-15739, RRID: AB_10979409), α-NFAT3 (Thermo Fisher Scientific Cat# PA1-021, RRID: AB_2267268, for Western blot); α-RERG (Proteintech Cat# 10687-1-AP, RRID: AB_2253772, for Western blot); α-HA (Abcam Cat# ab18181, RRID: AB_444303, For PLA). ReadyTector α-Mouse-HorseRadish Peroxidase (HRP) #720 500 and α-Rabbit-HRP #730 500 were from Candor. Lipofectamine 2000 #11668019 from Thermofisher Scientific; Dharmafect 1 #T-2001-03 and siNFAT3 #D-009584-07, siRERG #L-0082004; siCtl #D-001810-10 and # D-001210-01 were from Horizon.

Protein extracts used for Western Blot were generated by transfecting 5 × 10⁶ HeLa cells with 10 µg of expression vector DNA and lysing cells with RIPA buffer after 48 hr. Lysates were passed through a 23-gauge needle, incubated 30 min on ice, then centrifuged at 10000 g and 4°C for 10 min to remove cell debris. Total protein concentrations were determined via Bradford assay (Pierce Coomassie Plus [Bradford] Assay Kit, Thermo Fisher). Proteins were separated by discontinuous SDS-PAGE and transferred onto nitrocellulose membranes (1 hr at 100 V). Membranes were stained with α-SB antibody (RRID:AB_622119, R and D Systems, 1:500, 2 hr) and α-goat-HRP (RRID:AB_258425, Sigma, 1:10000, 1 hr) or with α-actin (RRID:AB_2223496, Thermo Scientific, 1:5000, 2 hr) and α-mouse-HRP (RRID:AB_228313, Thermo Scientific, 1:10000, 1 hr) for the loading control. Membranes were visualized using ECL Prime Western Blotting reagents.

To confirm CRISISS-induced DNA damage by measuring the cellular DNA damage response, an assay was conducted to detect phosphorylated KAP1 protein after CRISISS induction. For this purpose, cells of the established monoclonal HeLa cell line harboring CRISISS with (arC9 Alu HeLa clone #1) and without (arC9 scaff HeLa clone #1) Alu-specific sgRNAs were induced with 200 nM 4-HT and 1 µg/mL DOX. Cells were harvested at timepoints 1 dpi, 2 dpi and 3 dpi alongside uninduced cells of both cell lines as controls and lysed in RIPA buffer for 30 min at 4 °C in a rotating shaker. Subsequently, a WB analysis was carried out in a similar way as described before with a few exceptions: (i) 5 µg of total protein was loaded onto the acrylamide gel per sample, (ii) a 10% polyacrylamide gel was used, and (iii) instead of milk, bovine serum albumin was used for the blocking buffer. The following antibodies were used: α-pKAP1 (phospho S824, Abcam, RRID: AB_70369) at a dilution of 1:1000 in blocking buffer, α-actin (Thermo Fisher, RRID: AB_2223496) at a dilution of 1:5000 in blocking buffer, α-rabbit-HRP (Thermo Fisher, RRID: AB_228378) at a dilution of 1:1500 in blocking buffer and anti-mouse-HRP (Thermo Fisher, RRID: AB_228313) at a dilution of 1:5000 in blocking buffer.

After drug treatment, H9c2 cells were seeded on coverslips and permeabilized with 0.5% Triton X-100 for 15 min at room temperature, blocked with 5% goat serum for 1 h at room temperature and incubated with appropriate primary antibodies, ANP or α-actin (Thermo Fisher Scientific, United States), overnight at 4°C. The samples were washed three times with TBST followed by incubation with Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (1:200, Cell signaling technology, United States) for 1 h at room temperature. Nuclei were stained with DAPI (Sigma, United States) for 2 min at room temperature. Images were acquired with an FV1000 confocal microscope (Olympus, Japan) and analyzed with ImageJ software.