Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a Morphology of HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with doxycycline treatment for 72 h. Scale bar = 50 µm. b Confocal immunofluorescence for E-cadherin (green) and DAPI (blue) in HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment for 96 h. Scale bar = 30 μm. c Cell viability of HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with (red) or without (blue) doxycycline treatment by CCK-8 assay. All data represent the mean ± SD ( n = 4, ** P < 0.01, *** P < 0.001, **** P < 0.0001). d Colony formation assay of HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment for 8 days. Dot plots show number and average size of colonies calculated by imageJ software. All data represent the mean ± SD ( n = 3, ** P < 0.01, *** P < 0.001). e Confocal immunofluorescence (left panel) for Ki-67 (red) and DAPI (blue) in HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment. Scale bar = 50 μm. Dot plots (right panel) show quantification of the frequency of Ki-67-positive cells in each vision. All data represent the mean ± SD ( n = 10, **** P < 0.0001). f SA-β gal staining (left panel) of HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment. Red arrows represent SA-β gal-positive cells. Scale bar = 50 μm. Dot plots (right panel) show quantification of the frequency of SA-β gal-positive cells in each vision. All data represent the mean ± SD ( n = 10, **** P < 0.0001).

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: Immunofluorescence, CCK-8 Assay, Colony Assay, Software, Staining

Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a Confocal immunofluorescence for SOX1 (red) and DAPI (blue) in HONE1 and CNE2 cells. Fluorescence images are respectively merged to display location of SOX1 (red). Scale bar = 4 µm. b Schematics of full length and mutated SOX1 proteins used in this study. c Morphology of HONE TRE-(X) and CNE2 TRE-(X) cells (X stand for vehicle, wild type SOX1 or mutant SOX1) under doxycycline treatment for 3 days. Scale bar = 50 µm. d Confocal immunofluorescence for SOX1 (red) and DAPI (blue) in HONE TRE-(X) and CNE2 TRE-(X) cells under doxycycline treatment for 3 days. Scale bar = 4 μm. e Western blot analysis of SOX1, KRT5, KRT13, and β-actin expression in HONE TRE-(X) and CNE2 TRE-(X) SOX1 cells under doxycycline treatment for 3 days. β-actin was used as a control. f SA-β gal staining (left panel) of HONE TRE-(X) and CNE2 TRE-(X) cells under doxycycline treatment for 7 days. Red arrows represent SA-β gal-positive cells. Scale bar = 50 μm. Dot plots (right panel) show quantification of the frequency of SA-β gal-positive cells in each vision. All data represent the mean ± SD ( n = 5, **** P < 0.0001).

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: Immunofluorescence, Fluorescence, Mutagenesis, Western Blot, Expressing, Control, Staining

Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a RNA-Seq analysis displays heat map of keratins gene family expressed in 42 Chinese NPC patients and 4 non-NPC tissues from GEO database (GSE68799). mRNA intensities were rlog transformed and are displayed as colors ranging from red to blue. Both rows and columns are clustered using correlation distance and average linkage. b RNA-Seq analysis shows heat map of keratin gene family expressed in HONE1 TRE-SOX1 (left) and CNE2 TRE-SOX1 (right) cells under doxycycline treatment for 4 days. mRNA intensities were rlog transformed and are displayed as colors ranging from red to blue. Both rows and columns are clustered using correlation distance and average linkage. c RT-PCR analysis of keratin genes in HONE1 TRE-SOX1 (upper) and CNE2 TRE-SOX1 (lower) cells with (red) or without (blue) doxycycline treatment for 48 h. Data are normalized by the amount of ACTB mRNA and represent mean ± s.e.m. ( n = 3, n.s.: P > 0.05, *** P < 0.001, **** P < 0.0001). d Western blot analysis of keratin proteins, SOX1, and β-actin in HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment for 96 h. β-actin was used as a loading control. s.e.: short exposure, l.e.: long exposure. e GSEA of SOX1 (Dox+) vs. control (Dox−) in HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells using ‘Hallmark G2M Checkpoint’, ‘Hallmark E2F Targets’, and ‘Hallmark mitotic spindle’ gene sets. NES normalized enrichment score, FDR false discovery rate.

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: RNA Sequencing, Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a Significantly up-regulated (upper) or down-regulated (lower) mRNAs (|fold-change | ≥2 and P < 0.05) in HONE1 TRE-SOX1 /CNE2 TRE-SOX1 (Dox+) compared to corresponding control groups (Dox−) were shown by venn diagrams. b GO analysis of the differently expressed genes. Blue column represents the total number of genes annotated with each GO term, while red is −log 10 of P value. c Western blot analysis of keratin proteins and β-actin of wild type HONE1 cultured with conditional-media (CM) of HONE1 TRE-SOX1 cell with (SOX1) or without (vec) doxycycline treatment for 48 h. β-actin was used as a loading control. d Differential feature plot for CM and cells of HONE1 TRE-SOX1 with or without doxycycline treatment by LC–MS untargeted metabolomics. Only features that are dysregulated ( P -value ≤ 0.05, fold change ≥ 1.5) are displayed. Upregulated features are shown in green, while downregulated features in red. The size of each bubble corresponds to the log fold change of that feature. The shade of the bubbles corresponds to the magnitude of the P -value (the darker the color, the smaller the P -value). Red arrows represent metabolites in retinoid pathway. e Summary of fold change, P -value, mass-to-charge ratio ( m / z ), and retention time (rt) of metabolites in retinoid pathway screened in d . f Western blot analysis of KRT5, KRT13, and β-actin of wild type HONE1 and CNE2 cells with or without RAce treatment for 72 h. β-actin was used as a loading control. g Colony formation assay of wild type HONE1 and CNE2 cells with vehicle, RA (10 μM), or RAce (10 μM) treatment for 8 days. h Cell viability of wild type HONE1 and CNE2 cells with (red) or without (blue) doxycycline treatment by CCK-8 assay. All data represent the mean ± SD ( n = 4, **** P < 0.0001).

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: Control, Western Blot, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Colony Assay, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a A brief overview of retinoic acid signaling pathway. Retinol transports to cells in a complex with CRBP through vitamin A receptor STRA6. In cytoplasm, retinol is oxidized and converted to RA. RA can complex with CRABP1/2 and transports to the nucleus. Following, RA forms a complex with RXR–RAR or RXR–PPARβ/δ heterodimers and binds to DNA of retinoic acid response element (RARE) or PPAR response element (PPRE), thereby activating transcription of target genes. b RT-PCR analysis of STAR6, CRABP1, CRABP2, RARA, RARB, RARG, RXRA, RXRB, and RXRG genes in HONE1 TRE-SOX1 (left) and CNE2 TRE-SOX1 (right) cells with (red) or without (blue) doxycycline treatment for 48 h. Data are normalized by the amount of ACTB mRNA, and compared to the corresponding value for cells without doxycycline treatment. Data represent mean ± s.e.m. ( n = 3, n.s.: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). c A brief overview of all-trans-retinoic acid metabolic pathway. In cells, all-trans-retinol can be converted to all-trans-retinal by alcohol dehydrogenase (ADH) or short-chain dehydrogenase/reductase (SDR), or to all-trans-retinyl esters by lecithin retinol acyltransferase (LRAT). All-trans-retinal can be further converted to all-trans-retinoic acid by aldehyde dehydrogenase (ALDH). Finally, all-trans-retinoic acid is metabolized to inactive retinoids by CYP26s or UGTs. d RT-PCR analysis of LRAT, CYP26A1, CYP26B1, CYP26C1, UGT1A (total), UGT1A1, UGT1A6, UGT1A9, UGT2B7, and UGT8 genes in HONE1 TRE-SOX1 (left) and CNE2 TRE-SOX1 (right) cells with (red) or without (blue) doxycycline treatment for 48 h. Data are normalized by the amount of ACTB mRNA and represent mean ± s.e.m. ( n = 3, n.s.: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). e Western blot analysis of SOX1, UGT2B7, KRT5, KRT13, and β-actin expression in HONE1 TRE-SOX1 , CNE2 TRE-SOX1 as well as UGT2B7 overexpressed HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells under doxycycline treatment for 4 days. β-actin was used as a control. f Cell viability of HONE1 TRE-SOX1 , CNE2 TRE-SOX1 as well as UGT2B7 overexpressed HONE1 TRE-SOX1 and CNE2 TRE-SOX1 cells with or without doxycycline treatment by CCK-8 assay. All data represent the mean ± SD ( n = 4, **** P < 0.0001). g SA-β gal staining (left panel) of HONE1 TRE-SOX1 and UGT2B7 overexpressed HONE1 TRE-SOX1 cells under doxycycline treatment for 7 days. Red arrows represent SA-β gal-positive cells. Scale bar = 50 μm. Dot plots (right panel) show quantification of the frequency of SA-β gal-positive cells in each vision. All data represent the mean ± SD ( n = 5, n.s.: P > 0.05, **** P < 0.0001).

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, CCK-8 Assay, Staining

Journal: Cell Death & Disease

Article Title: SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway

doi: 10.1038/s41419-020-2513-1

Figure Lengend Snippet: a Morphology of wild type HONE1 and CNE2 cells with or without T-96 treatment for 72 h. Scale bar = 50 µm. b Western blot analysis of KRT5, KRT13, and β-actin of wild type HONE1 and CNE2 cells with 0, 1, 5, 10, 20 μM T-96 treatment for 72 h. β-actin was used as a loading control. c Cell viability of wild type HONE1 and CNE2 cells with (red) or without (blue) T-96 treatment by CCK-8 assay. All data represent the mean ± SD ( n = 4, ** P < 0.01, *** P < 0.001, **** P < 0.0001). d SA-β gal staining (left panel) of wild type HONE1 and CNE2 cells with or without doxycycline treatment for 7 days. Red arrows represent SA-β gal-positive cells. Scale bar = 50 μm. Dot plots (right panel) show quantification of the frequency of SA-β gal-positive cells in each vision. All data represent the mean ± SD ( n = 4, *** P < 0.001, **** P < 0.0001). e Summary of the present study. Retinoic acid (RA) is balanced to low level concentration in SOX1 low NPC cells, while SOX1 induces RA accumulation within NPC cells, promoting cell differentiation (upper panel). For treatment strategy, targeting at UGT2B7 induces NPC cell differentiation, which is associated with RA metabolism.

Article Snippet: The following antibodies were used: V5-tag, p21 Waf1/Cip1, Rb, Phospho-Rb (Ser780), Phospho-mTOR (Ser2448), Phospho-p70 S6 Kinase (Thr389), Phospho-p70 S6 Kinase (Ser371), and Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 13202, 2947, 9309, 9307, 2971, 9205, 9208 and 2855, respectively), SOX1 (GeneTex EPR4766), KRT19 (Abcam EP1580Y), KRT13, mTOR, p70 S6 Kinase and 4E-BP1, (Epitomics 2713-1, 1612-1, 1494-1, and 1557-1, respectively), KRT5, CDK4, CDK6, c-Myc, PPARγ, and β-actin (Proteintech 66727-1-Ig, 11026-1-AP, 14052-1-AP, 10828-1-AP, 16643-1-AP, and 60008-1-Ig, respectively), UGT1A6 and UGT2B7 (Signalway Antibody 43176 and C32390, respectively).

Techniques: Western Blot, Control, CCK-8 Assay, Staining, Concentration Assay, Cell Differentiation