Tissue samples

All the tissue and plasma specimens (Shanghai, China, mean age ± SD, 60.05 ± 8.37 years; male: female ratio, 1.04:1) were recruited in Shanghai Chest Hospital (Shanghai, China) from March 2015 to December 2021. The diagnostic power was analyzed by receiver operating characteristic (ROC) curve analysis.

Immunoblotting (IB) and enzyme linked immunosorbent assay (ELISA)

Protein levels were analyzed using IB and ELISA following conventional protocols. For IB, the primary antibodies used were anti-YAP (Abcam, Cambridge, MA, USA, #ab52771), anti-GAPDH (CST, Boston, MA, USA, #5174 and #51332), anti-p-127-YAP (Abcam, #ab76252), anti-p-381-YAP (CST, #13619), anti-O-Glc-YAP (developed by Biolynx, Hangzhou, China), anti-HRS (Abcam, #ab72053), anti-PLD2 (CST, #13904), anti-RAB2B (Novus, Littleton, CO, USA, #NBP1-31631), anti-RAB27A (Abcam, #ab55667), anti-RAB27B (Abcam, #ab103418), anti-VAMP (Abcam, #ab36195), anti-ATG7 (Abcam, #ab52472), anti-SOX10 (Abcam, #ab227680), anti-Mycn (Abcam, #ab227822), anti-ALYREF (Abcam, #ab202894), anti-NSUN2 (Abcam, #ab259941), anti-DNMT2 (Abcam, #ab220175), anti-NSUN1 (Abcam, #ab270175), anti-NSUN3 (Abcam, #ab272616), anti-NSUN4 (Abcam, #ab235430), anti-NSUN5 (Abcam, #ab121633), anti-NSUN6 (Novus, #NBP1-92202), anti-NSUN7 (Biorbyt, Cambridge, UK, #orb258175), anti-YBX1 (Abcam, #ab255606), anti-EXOSC10 (Abcam, #ab94981), anti-SPI1 (Abcam, #ab227835), anti-CD63 (Abcam, #ab271286), anti-TSG101 (Abcam, #12501), anti-ALIX (Abcam, #ab275377), anti-CD9 (Abcam, #ab92726), and anti-calnexin (Abcam, #ab133615). For ELISA, YAP and NSUN2 levels were measured using kits from Yingxin Biotech Ltd. (Shanghai, China) as per manufacturer’s instructions.

Cell culture

Established A549, H1299, H1975, Wi-38, MRC-5 and BEAS-2B cell lines were purchased from Fuheng Biotechnology (Shanghai, China). H1975- and HCC827-based AZD9291 resistant cell lines were acquired from previous studies [5]. For 3D spheroid culture, basement membrane extract (BME) (Trevigen, Gaithersburg, MD, USA) was seeded in a 96-well plate at 50 μl/well and plated at 37 °C for 30 min. Then, cells with indicated genes overexpressed or knocked out were seeded on top of BME at a density of 1 × 105 cells per well. After formation of spheroids, they were treated with AZD9291 (MedChemExpress, Monmouth, NJ, USA) with or without GW4869 (Sigma, St Louis, MO, USA) for 24 h. Images were captured after staining with SYTOX green (Invitrogen, Carbsland, CA, USA) [37].

RNA-immunoprecipitation (RNA-IP)

For RIP assay, a Magna RIP Kit (Merck Millipore, Billerica, MA, USA) was used. Briefly, a total of 1 × 107 cells were lysed in complete RNA lysis buffer, then cell lysates were incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with 5 μg anti-m5C (Abcam, #ab10805), anti-DNMT2 (Abcam, #ab220175), anti-NSUN1 (Abcam, #ab270175), anti-ALYREF (Abcam, #ab202894), anti-NSUN2 (Abcam, #ab259941), anti-DNMT2 (Abcam, #ab220175), anti-NSUN3 (Abcam, #ab272616), anti-NSUN4 (Abcam, #ab235430), anti-NSUN5 (Abcam, #ab121633), anti-NSUN6 (Novus, #NBP1-92202), anti-NSUN7 (Biorbyt, #orb258175), anti-YBX1 (Abcam, #ab255606) antibodies or control IgG (CST #3900) overnight at 4 °C. Next, the beads were washed with washing buffer and incubated with proteinase K at 55 °C for 30 min to remove the proteins. The remaining RNA was extracted using Trizol (Ambion) and reverse-transcribed into complementary DNA using the PrimeScriptTM RT reagent kit (Takara). The SYBR premix Ex Taq (Takara) kit was used for real-time qPCR [31].

Regents and plasmids

For regents, ActD (MedChemExpress), CHX (Sigma), GW4869 (Sigma), AZD9291 (MedChemExpress). Plasmids DNMT2, NSUN1-7, ALYREF, YBX1, EXOSC10, Mycn, SOX10, SPI1, YAP-(-1k)-promoter, YAP-(-2k)-promoter, Mut4-, Mut5-, Mut6-YAP-3’ UTR overexpression plasmids were purchased from Biolink (Shanghai, China). miR-582-3p mimics, miR-582-3p inhibitors, WT-, Mut1-, Mut2- and Mut3-YAP-3′ UTR luciferase reporters, YAP knockout and YAP overexpression plasmids were acquired from previous studies [5, 7, 31]. Short hairpin RNAs (shRNAs) and lentiCRISPR v2-based constructs were used for knocking down and knocking out Mycn, SOX10, NSUN2 and ALYREF, respectively. The sequences for gRNAs and shRNAs were summarized in Table S1.

Quantitative RT-PCR (qPCR)

Total RNA was extracted using Trizol (Ambion, Carlsbad, CA, USA) and reverse-transcribed into complementary DNA using the PrimeScriptTM RT reagent kit (Takara, Dalian, China). The SYBR premix Ex Taq (Takara) kit was used for real-time qPCR. The primers are listed in Table S1.

Luciferase activity measurement

Luciferase activities were measured using a dual-luciferase kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Firefly reporters (including WT-, Mut1- Mut2-, Mut3-, Mut4- Mut5- and Mut6-YAP-3′ UTR, as well as YAP-(-1k)-promoter, YAP-(-2k)-promoter luciferase reporters) were co-transfected into lung cancer cells with Renilla plasmids using Lipofectamine™ 2000 transfection reagent (Invitrogen). After incubation for 48 h, the cells were harvested and then lysed in the passive lysis buffer from the kit. The fluorescence intensity of luciferase reporters was then examined and normalized to the Renilla luciferase activity.

Co-immunoprecipitation (co-IP)

co-IP was performed as described previously. Cell lysates were mixed with 50 ul protein A/G-magnetic beads (Novex, Oslo, Norway) and incubated at 4 °C overnight with the indicated antibodies. The beads were washed with Western/IP lysis buffer (Beyotime, Haimen, China), suspended in SDS-PAGE loading buffer and then detected by IB. The antibodies used for co-IP were: anti-YAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-101199).

RNA probe precipitation

Biotin-labeled probe was synthesized by Sangon Biotech (Shanghai, China). Cells were fixed using 4% formaldehyde, then lysed, sonicated, and centrifuged. Next, the supernatant was extracted as input, and the remaining amount was incubated with indicated probe and Dynabeads M-280 Streptavidin (Thermo Scientific, Waltham, MA, USA) overnight at room temperature. The next day, the mixture was washed and incubated with lysis buffer and proteinase K to reverse the cross-linking. Finally, the RNA mixture was extracted using Trizol and was detected via qPCR [5,6,7, 31]. Probe sequence was listed in Table S1.

Isolation and measurement of exosome

Exosome was isolated from cultured media of cells or plasma of human via three sequential centrifugation steps at 4 °C, (1) 15 min at 500 g to remove cells; (2) 30 min at 10,000 g to remove cell debris; and (3) ultracentrifugation at 110,000 g for 70 min to pellet exosome. The pellet was finally resuspended in PBS and centrifuged at 110,000 g for another 70 min to remove soluble and secreted proteins. The concentration and size of exosome were analyzed using nanoparticle tracking analysis (NTA) by a Nanosight NS 300 system (NanoSight Technology, Malvern, UK) [37].

Immunofluorescence (IF), immunohistochemistry (IHC),

IF and IHC were performed according to the conventional protocols. For IF, PKH67 (Sigma, MKCG5294) was probed for marking exosome packaged in H1299 cells. For IHC, the primary antibodies used in the study were anti-YAP (Abcam, #ab52771).

TEM analysis

Exosome was resuspended in 4% paraformaldehyde, and images were captured using the JEM1230 TEM (JEOL, Tokyo, Japan).

Measurements of cell viability, caspase 3/7 activity and anchorage-independent colony formation

Cell viability was detected using a CCK8 kit (Beyotime). Caspase3/7 activity was measured using Caspase 3/7 Glo luciferase reagent (Promega). As for anchorage-independent colony formation assay, LUAD cells were seeded in a six-well plate containing 0.3% agarose at a density of 6 × 103 cells per well. Two weeks later, the numbers of colonies were calculated under microscope.

Mouse experiments

A549 cells with indicated genes overexpressed or knocked out (initial 5 × 106) were subcutaneously injected into 6-week-old athymic nude mice (Jiesijie, Shanghai, China) with or without GW4869 treatment to acquire cell-derived xenograft mouse models. Fresh tissues in a size of 2 mm3 were subcutaneously implanted into six-week-old athymic nude mice to generate PDX mouse models. After successful passage, the PDX model could be used for further analysis. Tumor volumes were calculated as 0.5 × L × W2 (L indicating length while W indicating width) [5]. Histological sections were prepared using a microtome and subsequently stained with IHC or hematoxylin and eosin.

Statistical analysis

Tests used in this study included student’s t test, one-way, two-way ANOVA, χ2 test, and the Spearman rank-correlation analysis. A p < 0.05 was considered statistically significant.