Supplementary Figures 1 - 9 from Activation of <i>SOX2</i> Expression by BRD4-NUT Oncogenic Fusion Drives Neoplastic Transformation in NUT Midline Carcinoma (2023)

&lt;p&gt;PDF file - 994KB, Supplementary Figure Legends Supplementary Figure S1. HCC2429 cells grow into stem cell-like spheres in ESC medium. (A) Morphology of HCC2429 cells cultured in normal culture medium (RPMI1640 with 10% FBS). (B and C) Morphology of HCC2429 spheres formed after switching to ESC medium. HCC2429 cells were seeded and allowed to attach to plates in normal culture medium. After 8 h, media were changed to full ESC medium. Photos were taken after one day of culture in full ESC medium. (D-F) HCC2429 cells grow into stem cell-like spheres in ESC medium. The micrograph was taken after five days of culture in basic ESC medium. Bar, 50 microm. Supplementary Figure S2. Alkaline phosphatase activity is detected in some HCC2429 cells. HCC2429 cells were fixed and stained using Leukocyte Alkaline Phosphatase Kit (86R-1KT, Sigma) according to the manufacturer's instructions. Bar, 50 microm. Supplementary Figure S3. JQ1+ treatment reduces SOX2 expression in a dose-dependent manner. HCC2429 cells were treated with DMSO control (labeled as 0) or JQ1+ at the indicated concentrations. Cells were harvested at 24 h and lysed for western blots with the indicated antibodies. Supplementary Figure S4. BRD4-NUT knockdown inhibits SOX2 expression in HCC2429. (A) HCC2429 cells were transfected with control (CO, D-001210-01, Dharmacon) or NUT-02 (D-022211-02, Dharmacon) siRNA, and harvested on day 1(1 d) and 2 (2 d). Whole cell lysates were immunoblotted with the indicated antibodies. BRD4 N antibody recognizes BRD4 aa 156-284, and detects both BRD4 and BRD4-NUT. (B) HCC2429 cells transfected with control (CO) or NUT-02 siRNAs were harvested at 36 h. The mRNA levels of SOX2 and BRD4-NUT genes were measured by RT-qPCR and normalized to the GAPDH mRNA levels. Relative mRNA levels in control siRNA-treated cells were set as 100%. Values represent the average of three independent experiments with error bars indicating standard deviation. ***p &lt; 0.001. Supplementary Figure S5. The effects of SOX2 knockdown on HCC2429 proliferation in normal culture condition. (A) HCC2429 cells were transfected twice with control (CO) or SOX2-01 siRNA. Cells were fixed on day 2 after the second transfection and immuno-stained with Ki-67 and SOX2 antibodies and DAPI. Bar, 10 microm. (B) The percentage of cells with positive Ki-67 signal as detected in (A) was calculated from more than 200 cells. Values represent the average of three independent experiments with error bars indicating standard deviation. ***p &lt; 0.001. (C) HCC2429 cells were transfected twice with control siRNA (CO) or SOX2-01 siRNA. At 24 h after the second transfection, 20,000 cells were seeded for a proliferation assay. Cell numbers were counted on day 1-4 after seeding. Values represent the average of three independent experiments with error bars indicating standard deviation. (D) HCC2429 cells transfected with control siRNA (CO) or SOX2-01 siRNA were harvested at 60 h post-transfection, fixed and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry using a BD FACSCalibur. Data were analyzed using the Watson model in FlowJo software. Supplementary Figure S6. Exogenous SOX2 rescues the cellular differentiation and growth inhibition induced by JQ1+. (A) Inducible HCC2429 SOX2 stable cells were treated with 100 nM JQ1- or JQ1+, together with or without 0.1 microg/ml Dox. Cells were harvested on day 5 and lysed for western blotting with the indicated antibodies. Asterisk indicates Dox-induced Xpress-tagged SOX2. Diamond indicates endogenous SOX2. This experiment was repeated more than three times with similar results. (B) Inducible HCC2429 control stable and SOX2 stable cells were treated with 100 nM JQ1- or JQ1+, together with or without 0.1 microg/ml Dox. Cells were fixed at 24 h after adding drugs, immuno-stained with β-tubulin antibody (C4585, Sigma) and counterstained with DAPI. Bar, 20 microm. (C) Cells treated as in (A) were fixed at 24 h and immuno-stained with Ki-67 antibody and DAPI. Bar, 20 microm. (D) The percentage of cells with positive Ki-67 signal as detected in (C) was quantified and calculated from more than 200 cells. Values represent the average of three independent experiments with error bars indicating standard deviation. **p &lt; 0.01. Supplementary Figure S7. BRD4-NUT fusion isoforms in different NMC cell lines. (A, B) PCR amplification of the BRD4-NUT chimeric transcripts from various NMC cells by RT-PCR using the indicated exon specific primers. (C) Sequencing results of the partial fusion transcript PCR products from (A) or (B) showed that NUT is fused in frame to BRD4 at different breakpoints in different NMC cell lines. (D) Schematic representation of the three BRD4-NUT fusion isoforms in NMCs. Isoform I found in HCC2429 was reported as the "Standard" BRD4-NUT fusion (10). Isoform III was reported as a "Novel" BRD4-NUT fusion isoform (11). Isoform II was first discovered in this study. Supplementary Figure S8. SOX2 is important for preventing cellular differentiation and maintaining sphere growth in other NMC cell lines. (A) 10-15 cells were transfected twice with control (CO), SOX2-01 (D-0011778-01, Dharmacon), or SOX2-02 (D-0011778-02, Dharmacon) siRNA. Cells were harvested on day 3 (3 d) and 4 (4 d) after the second transfection. Whole cell lysates were immunoblotted with the indicated antibodies. (B) SOX2-01 siRNA induces efficient knockdown in 10-15 cells. (C) The 10-15 cells transfected with control (CO) or SOX2-01 siRNA were seeded for sphere assay at 8 h post-transfection. The percentage of cells forming spheres was calculated after counting spheres formed from 480 seeded cells on day 7. Values represent the average of three independent experiments with error bars indicating standard deviation. ***p &lt; 0.001. (D) SOX2-01 siRNA induces efficient knockdown in Ty-82 cells. (E) Ty-82 cells transfected with control siRNA (CO) or SOX2-01 siRNA were seeded for the sphere assay at 8 h post-transfection. The percentage of seeded cells forming spheres was calculated after counting spheres from 240 seeded cells on day 10 and presented as in (C). **p &lt; 0.01. Supplementary Figure S9. p300 colocalizes with BRD4-NUT in punctate chromatin foci and also associates with the SOX2 gene in HCC2429. (A) HCC2429 cells were treated with DMSO or 40 microM C646 for 1 h. Cells were fixed and immuno-stained with p300 and NUT antibodies. Cells were also counterstained with DAPI. Bar, 10 microm. (B) HCC2429 cells were subjected to the ChIP assay with normal rabbit IgG (NR, as a negative control), NUT or p300 (sc-585, Santa Cruz) antibodies. ChIP samples were analyzed by qPCR using the same primers as in Fig. 3. Values represent the average of three independent experiments with error bars indicating standard deviation.&lt;/p&gt;