Fig. 1

ZNF24 is an essential tumor suppressor in lung cancer. A Schematic of CRISPR-based screen for lung cancer suppressor genes. EKVX cells were infected with virus encoding Cas9, and selected with blasticidin for two weeks. Monoclone was picked to generate a stable cell line (EKVX-Cas9). EKVX-Cas9 were infected the sgRNA library with a multiplicity of infection of 0.3. Cells were selected with puromycin for 7 days and then subcutaneously transplanted into nude mice to grow tumor for two weeks. Genomic DNA were isolated from tumors and used as templates to amplify DNA fragment containing CRISPR sequence. PCR products were sequenced. B Impact of ZNF24 expression level on colony forming ability of A549i cells. A549i (1000 cells) were seeded in 6-well plates and treated with/without Dox for 2 weeks before quantification of colonies. Left: representative pictures. Right: statistics of colony number. C Effect of ZNF24 knockdown or re-expression on proliferation of EKVX cells. 1000 EKVX-shZNF24, sh/ + ZNF24 or shGFP cells were seeded in 96-well plates and cultured for 5 days. Cell viability was analyzed with CCK8. shGFP for control knockdown; shZNF24 for ZNF24 knockdown; sh/ + ZNF24 for ZNF24 re-expression in ZNF24 knockdown cells. D Impact of ZNF24 knockdown or re-expression on 2-D colony forming ability of EKVX cells. 1000 engineered EKVX cells were respectively inoculated in 6-well plates and cultured for 2 weeks before quantification for colonies. Left: representative pictures. Right: statistics of colony number. E Impact of ZNF24 expression level on soft-agar colony-forming ability of A549 and Hop62 cells. A549i, Hop62i (10,000 cells) cultured in top agar in the presence or absence of Dox (1 μg/mL) for 3–4 weeks before quantification for colonies. Scale Bar = 200 μm. left: representative images of colonies. right: statistics. F Impact of ZNF24 knockdown on soft-agar colony-forming ability of EKVX cells. 10,000 engineered EKVX cells were respectively inoculated in soft agar in 6 well-plates and cultured 3–4 weeks before quantification for colonies. Scale Bar = 500 μm. left: representative images of colonies. right: statistics. G Impact of ZNF24 expression on A549i xenografted tumors. A549i cells (2 × 10⁶) were inoculated into nude mice. Mice treated with Dox (n = 4) or control diet (n = 4) for 14 days. A representative image of the tumors at the end of the experiments is shown. Tumors were measured with a vernier caliper every 2 days. Tumor volume (mm³) is calculated by D × d²/2, where D is the longest diameter and d is the shortest diameter. Growth curve of the tumor is shown. H Impact of Knockdown the ZNF24 on EKVX xenografted tumors. The EKVX-shGFP and EKVX-shZNF24 (2 × 10⁶ cells) were inoculated into nude mice for tumor growth for 14 days. Tumor volume is recorded every 2 days. A representative image of the tumors at the end of the experiments is shown. I Impact of ZNF24 expression on autochthonous lung tumor formation in transgenic mice. TetO-Kras^G12D/CC10rtTA (designated KC) mice infected with virus for expressing control (KC-C) or ZNF24 (KC-Z) and were fed with Dox diet for 2 months. Tumor burdens were recorded through computed tomography (CT) scanning and histological sections. Scale Bar = 500 μm. J Statistics of tumor burden, tumor number, maximal tumor sizes (diameters) were measured. K Impact of ZNF24 expression on tumor formation in Lsl-Kras^G12D mice. The recombinant lenti-virus co-expressed Cre and CRISPR/Cas9 to infect Lsl-Kras^G12D by nasal inhalation. Comparison of lung tumor formation in K-sgZNF24 mice and K-sgTD mice six months after infection. Tumor burdens were recorded through Computed tomography (CT) scanning and histological sections. Scale Bar = 500 μm. L–M Statistics of tumor burdens, tumor numbers, maximal tumor sizes (diameters), Grade 1 (G1)- Grade 4 (G4) tumor of K were determined by tumor size and heterogeneity of nuclei of tumor cells. Bars are represented as means ± SEM of the indicated number (n) of repeats. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test