Fig 2: USP13 elevates MYC expression in lung cancer, contributing to the elevation of LUSC features. A MYC expression during cancer progression in KP and KPU mice following Ad5-SPC-Cre (left) or CC10-Cre (right) infection. Hyperplasia (arrow) and carcinoma in situ (CIS) (dotted line) are indicated. Scale bar = 50 µm. B IHC quantification for the portion of MYC+ cells in the lesions. Each dot represents one tumor nodule. Error bars indicate mean ± SEM. Two-tailed unpaired t-tests, ns = not significant, ****p < 0.0001. C Western blot of USP13, MYC, SOX2, p40, and β-actin on mouse KP cell line expressing exogenous USP13 or MYC. D Western blot showing USP13, MYC, SOX2, p40, CK5, and β-actin in human LUAD cell lines expressing exogenous USP13 or MYC. E Western blot of USP13, MYC, SOX2, and β-actin in H441 cells with knockdown of MYC using siRNA followed by transfected with mock or USP13. F Western blot of USP13, MYC, SOX2, and β-actin in H441 cells treated with either DMSO or 10 μM of 10058-F4 (MYC inhibitor) for 24 h followed by transfection with mock or USP13. G Schematic showing the proposed model of how USP13 promotes LUSC tumorigenesis in club cells

Fig 3: USP13 overexpression drives LUSC development in the KP mouse model. A Schematic of the experimental procedure using KP and KPU mouse model. KP or KPU mice were infected with the Adeno-CMV-Cre virus (Ad5-CMV-Cre) via intratracheal administration. B Survival analysis of KP and KPU mice upon virus infection. The p-value was calculated using a log-rank test. C Representative hematoxylin and eosin (H&E) staining of KP and KPU lung at 12 weeks post-viral infection. Dotted lines indicate tumor nodules. Scale bar = 5 mm. D Quantification of individual tumor number and area in KP and KPU mice at 10-14 weeks post-Ad5-CMV-Cre virus infection (n=8 mice/group). E Representative images for H&E, USP13, NKX2-1, SPC, SOX2, and CK5 IHC stains of the KP and KPU tumors. The red boxed areas on the left images were magnified and shown on the right. Black scale bar = 50 µm, White scale bar = 20 µm. (F) Western blot showing the expression of USP13, NKX2-1, CK7, p63, and SOX2 in KP and KPU tumors. GAPDH is a loading control. G Quantification of tumor number and area for LUAD and LUSC components in KPU mice. n=8 for each group. H The ratio of NSCLC subtypes in each KPU mouse (n=8). In (D) and (G), error bars indicate mean ± SEM. Two-tailed unpaired t-tests, *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Verification of high C2 cluster cell percent correlates with poor patient prognosis. (a) Representative images for multiplexed immunofluorescence staining of C2 cluster cells in cervical cancerous tissues (CK5, green; TIMP1, cyan; EZH2, yellow; SF3B2, red; COPS8, violet). PanCK CK5 was used to mark cancer cells. DAPI was used to highlight all nuclei. Scale bars = 0.01 mm. (b) Overall survival analysis for cervical cancer patients (n = 85) with different proportions of C2 cluster cells in cancerous tissues. Differences between groups were evaluated using log rank test (p = 0.03) (cut-off value = 50%). (c) Disease free survival analysis for cervical cancer patients (n = 85) with different proportions of C2 cluster cells in cancerous tissues. Differences between groups were evaluated using log rank test (p = 0.03) (cut-off value = 50%). (d) Comparison of C2 cluster cell frequency between cancerous and matched paracancerous tissues from cervical cancer patients (p = 0.046) (rank-sum test). The proportion of C2 cluster cells was calculated using the ratio of cells co-expressing C2 maker genes (TIMP1, EZH2, SF3B2, COPS8, CK5) to the number of overall cells in the samples. (e) Comparison of C2 cluster cell frequency in cancerous tissues between relapsing patients and non-relapsing patients (p = 0.0806) (rank-sum test). (f) Comparison of C2 cluster cell frequency in cancerous tissues between patients with lymph node metastasis (LNM) and those with no lymph node metastasis (Non-LNM) (p = 0.0754) (rank-sum test). (g) Comparison of C2 cluster cell frequency in cancerous tissues between patients with squamous carcinoma and those with other pathological types (p = 0.955) (rank-sum test).

Fig 5: Impacts of BMPR2 mutation on airway lineage differentiation. (A) Schematic of the differentiation protocol from lung progenitors to alveolar and airway organoids, with representative brightfield images shown at indicated time points. Scale bar, 100 μm. (B) Characterization of alveolar organoids in PH group: alveolar type I cells (HOPX+), alveolar type II cells (SPA+, SPB+, SPC+), and lung epithelial cells (EPCAM+). Scale bar, 25 μm. (C) Characterization of airway organoids in PH group, including basal cells (KRT5+), club cells (SCGB3A2+), and goblet cells (MUC5AC+). Scale bar, 25 μm. (D) qRT-PCR analysis of marker expression in alveolar and airway organoids from PH and control groups. * p < 0.05, unpaired Student’s t-test.