Lactylation-driven nuclear RIG-I promoted by lactate transporter inhibitor suppresses DNA damage repair through inhibiting PARP1 activity
Description
In the present study, we reveal that lactylation regulates the nuclear translocation and function of RIG-I (DDX58) in lung adenocarcinoma (LUAD). We demonstrate that the acetyltransferase PCAF mediates RIG-I lactylation, while inhibition of lactate efflux by syrosingopine increases intracellular lactate levels, thereby promoting RIG-I lactylation and importin8-dependent nuclear translocation. Nuclear RIG-I interacts with PARP1 and suppresses its activity, leading to impaired DNA damage repair. Furthermore, syrosingopine treatment enhances the sensitivity of LUAD cells to PARP inhibitors and improves the therapeutic efficacy of olaparib in vivo. This dataset contains the raw Western blot (WB) data supporting our findings on RIG-I lactylation, nuclear localization, and its regulatory role in DNA damage repair.
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Steps to reproduce
Denaturing immunoprecipitation (denaturing IP) Cells were transfected with the indicated plasmids using PEI reagent. After treatment with the indicated drugs (if needed) or 48 h after transfection, cells were collected and washed with 4°C PBS buffer and lysed in NP-40 buffer (1% protease inhibitor added, MCE, HY-K0010) for 2 h, and then add 0.1% SDS (1/10 volume of total lysate in 10% SDS) at 95°C for 10 min for denaturation before the antibody added. Following cooling to 4°C, the supernatant was incubated with the indicated antibodies or control IgG for 4 h and mixed gently with 30 μL of Protein G Sepharose beads for 4 h. The samples were then centrifuged and resuspended with NP-40 three times at 4°C, denatured, separated by SDS–PAGE, and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% skim milk and incubated with specific primary and secondary antibodies. Protein signals were detected and quantitated using the Odyssey Infrared Imaging System and Odyssey V3.0 software (LI-COR Bio-sciences) or Tanon 4600 Series Fully Automatic Chemiluminescence Image Analysis System (using Tanon Femto-sig ECL). Co-immunoprecipitation (Co-IP) Cells were transfected with the indicated plasmids using PEI reagent. After treatment with the indicated drugs (if needed) or 48 h after transfection, cells were collected and washed with 4°C PBS buffer and lysed in NP-40 buffer (protease inhibitor added, MCE, HY-K0010) for 2 h. The supernatant was incubated with the indicated antibodies or control IgG for 4 h and mixed gently with 30 μL of Protein G Sepharose beads for 4 h. The samples were then centrifuged and resuspended with NP-40 three times at 4°C, denatured, separated by SDS–PAGE, and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% skim milk and incubated with specific primary and secondary antibodies. Protein signals were detected and quantitated using the Odyssey Infrared Imaging System and Odyssey V3.0 software (LI-COR Bio-sciences) or Tanon 4600 Series Fully Automatic Chemiluminescence Image Analysis System (using Tanon Femto-sig ECL). IP–mass spectrometry HEK293T cells were transfected with Flag-RIG-I for 48 h, lysed in NP-40 buffer, and sonicated using a JY92-II sonicator. The supernatant was first incubated overnight with an anti-Flag conjugated with beads (Sigma, A2220) after being denatured. Following centrifugation at 3,000 rpm (4°C for 5 min), the supernatants were completely removed, and 60 μL of Flag–peptide was added to competitively bind to the beads. RIG-I protein was released, precipitated using Trichloroacetic acid (TCA), and subjected to mass spectrometry analysis for lactylation.
Institutions
- Peking University