Additional file 5 of USP13 drives lung squamous cell carcinoma by switching lung club cell lineage plasticity
Creators: name: Kwon, Juntae
name: Zhang, Jinmin
name: Mok, Boram
name: Allsup, Samuel
name: Kim, Chul
name: Toretsky, Jeffrey
name: Han, Cecil
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 5: Fig. S1. (A) Genomic alterations of USP family members in lung squamous cell carcinoma determined by cBioPortal analysis of TCGA databases (n=469). (B) Illustration of samples with 3q26 amplification in 469 LUSC patients. The 3q distal regions have been magnified to show the position of USP13, SOX2, and TP63 genes. (C) Correlation between copy number variation of USP13 and mRNA expression of squamous markers according to KRAS mutation in LUAD. Squamous markers include keratin 16 (KRT16), keratin 17 (KRT17), uroplakin 1B (UPK1B), and arachidonate lipoxygenase 3 (ALOXE3). X-axis is copy number variation of USP13 and the y-axis is log2(x+1) transformed RSEM normalized count. Error bars indicate mean ± SEM. Two-tailed unpaired t-tests, *p &lt; 0.05, **p &lt; 0.01. Fig. S2. (A) Schematic of KrasLSL-G12D/+; Trp53flox/flox (KP) and KrasLSL-G12D/+; Trp53flox/flox; Usp13LSL/LSL (KPU) alleles. (B) KP tumors were characterized by glandular formation (representative LUAD feature). The bottom is a high magnification of the top. Scale bar = 100 μm. (C) Different histological features in KPU tumors. (a) KPU tumors showed squamous characters such as keratin material (asterisk) and intercellular bridge (arrow). A small portion of the tumor exhibited small cell lung carcinoma (SCLC) (b) or carcinosarcoma-like histology (c).(a’), (b’) and (c’) are higher magnifications of the boxed area in (a), (b), and (c), respectively. Scale bar = 100 μm (D) H&amp;E staining of KPU mouse lung from Figure 2C (left). Black and red lines indicate LUAD and LUSC lesions, respectively. Scale bar = 5 mm. Representative images for H&amp;E, USP13, NKX2-1, SPC, and SOX2 IHC stains of the LUAD component in KPU tumors (right). Scale bar = 50 μm. (E) Schematic of KrasLSL-G12D/+ (K) and KrasLSL-G12D/+; Usp13LSL/LSL (KU) alleles. (F) Quantifying individual tumor number and area in K and KU mice at 55-58 weeks post-Ad-CMV-Cre infection (n= 3 and n= 5, respectively). Error bars indicate mean ± SEM. Two-tailed unpaired t-tests, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.01. (G) Representative images for H&amp;E, USP13, NKX2-1, SPC, SOX2 IHC stains of K and KU tumors. Scale bar = 50 μm. Fig. S3. (A) Heatmap showing the expression of marker genes for LUAD, LUSC, and SCLC in TCGA LUAD and LUSC samples. (B) Heatmap visualization of differentially expressed genes (DEGs) (log2FC &gt; 3.5, p &lt; 0.01, and q &lt; 0.01) between KP and KPU tumor samples. (C) Enrichment plots for hallmark xenobiotic metabolism, KRAS signaling down, and peroxisome for KP and KPU tumors. (D) GSEA analysis for hallmark G2M checkpoint, E2F targets, and MYC targets version 1 for LUAD and LUSC. (E) IPA analysis shows the top ten canonical pathways enriched in LUSC compared with LUAD. Each bar's color indicates predicted pathway activation or inhibition; Orange, positive z-score (activation); white, zero z-score; blue, negative z-score (inhibition); gray, no pattern. (F) Upstream regulator analysis exhibits transcription factors' predicted activation status in human LUSC. A positive z-score indicates activation and a negative z-score indicates inhibition. Fig. S4. (A) Correlation between USP13 and MYC mRNA expression (left), USP13 and SOX2 mRNA expression (center), and MYC and SOX2 mRNA expression (right) in LUSC. Data was obtained from cBioportal (TCGA, PanCancer Atlas) (n= 469). mRNA Expression is log2(x+1) transformed RSEM normalized count. (B) Correlation between USP13 and MYC mRNA expression in LUAD. Data was obtained from cBioportal (TCGA, PanCancer Atlas) (n= 507). (C-E) Representative IHC-staining images of USP13 (C), MYC (D), and SOX2 (E) in LUAD (left) and LUSC (right). The red boxed areas on the left images were magnified and shown on the right. Black scale bar = 100 μm, Greyscale bar = 50 μm. (F) Correlation between USP13 and MYC in the cytoplasm (left) and nucleus (right) of NSCLC patient samples. (G) Correlation between USP13 and SOX2 expression (left) and MYC and SOX2 expression (right) in LUSC. Data was obtained from Figure 4B (n= 130). IHC scores are analyzed using a two-tailed Pearson correlation coefficient. (H) Low magnification images of Figure 4F. Scale bar = 200 μm. Fig. S5. (A) Quantification of MYC expression levels relative to the vehicle is shown (left). qPCR of USP13 and MYC mRNA expression in HEK293T cells with or without USP13 overexpression. mRNA level was normalized to β-actin. Data are shown as means ± SD. ns, not significant, **p &lt; 0.01, and ****p &lt; 0.0001 (unpaired two-tailed t-test). (B) USP13-mediated deubiquitylation acts upon the Lys48 (K48) ubiquitination of MYC. 293T cells were transfected with FLAG-MYC, HA-Ub (WT or K48R), and Myc-tagged USP13. Fig. S6. (A) Section of wild-type mouse lung stained with anti-SPC (green) and anti-CC10 (red). Scale bar = 50 μm. (B) Section of wild-type mouse lung stained with anti-NKX2-1 (green) and anti-SOX2 (red). Scale bar = 50 μm. (C) Representative hematoxylin and eosin (H&amp;E) staining of KPU lung infected by Ad5-SPC-Cre or Ad5-CC10-Cre virus. Dotted lines indicate tumor nodules. Scale bar = 5 mm. Fig. S7. (A) Experiment design: KP or KPU mice received a single intratracheal injection of celltype- restricted Ad-SPC-Cre virus. The experiment was terminated 11-12 weeks post-viral infection. (B) Representative hematoxylin and eosin (H&amp;E) staining of KP and KPU lung at 12 weeks post-Ad-SPC-Cre infection. Dotted line indicates a tumor nodule. Scale bar = 5 mm. (C) Quantification of individual tumor number (left) and area (right) in KP and KPU mice at 11-12 weeks post-Ad-SPC-Cre virus infection (n= 5 mice/group). (D) Representative images for H&amp;E, USP13, NKX2-1, SPC, and SOX2 IHC stains of the KP and KPU tumors. Scale bar = 50 μm. (E) Representative NKX2-1 and SOX2 staining images from lung tumors in Ad-CC10-Cre infectedKP (left) and KPU (right) mice. Scale bar = 50 μm. (F) Quantification of lesions with NKX2-1 and SOX2 expression patterns from (E). (G) SPC and CC10 expression during cancer progression inAd-SPC-Cre infected KP and KPU lungs. Hyperplasia (arrow) and carcinoma in situ (CIS) (dotted line) are indicated. Scale bar = 50 μm. In (C) and (F), error bars indicate mean ± SEM. Two-tailed unpaired t-tests, *p &lt; 0.05, **p &lt; 0.01. Fig. S8. (A) Representative images for IHC staining of MYC in the KP and KPU tumors post- Ad5-SPC-Cre (left) or Ad5-CC10-Cre (right) infection. Scale bar = 50 μm (B) IHC quantification for MYC in tumors. Error bars indicate mean ± SEM. Two-tailed unpaired t-test, ns = not significant, ****p &lt; 0.0001. (C and D) Representative images of KP (C) and KPU (D) tumors with low, intermediate, and high MYC expression. Scale bar = 50 μm. (E) Cellular localization of MYC during tumor progression in KP (left) and KPU (right) lungs following Ad5-CC10-Cre infection. The bottom figures are higher magnifications of the boxed area in the top figures. Arrows indicate the cellular location of MYC protein; Black, cytoplasm; Green, whole cell; Orange, nucleus; Cyan, whole cell with nuclear enrichment. Black scale bar = 50 μm, white scale bar = 25 μm.