Journal: Cell Death & Disease

Article Title: CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

doi: 10.1038/s41419-021-04295-2

Figure Lengend Snippet: A Expression levels of cpap mRNA in liver tissues from WT mice treated with 100 mg/kg DEN (left, n = 6) for 48 h and with 25 mg/kg DEN (left, n = 7) for 11 months were determined by RT-qPCR and normalized to actin mRNA. Liver tissues from untreated mice (-) were used as the negative control. B Human hepatocytes treated with 25 ng/ml IL-6 or 10 ng/ml TNF-α for 24 h were collected for WB analysis (left) and RT-qPCR (right). Tubulin was used as the loading control for WB analysis; the expression level of CPAP mRNA was normalized by actin mRNA. C Human hepatocytes were treated with 25 ng/ml IL-6 (upper) or 10 ng/ml TNF-α (lower) for the indicated time periods. Total cell lysates were collected and subjected to WB analysis. Tubulin was used as the loading control. The quantitative results are shown. D Human hepatocytes were transiently transfected with GFP-CPAP and treated with 25 ng/ml IL-6 or 10 ng/ml TNF-α for 4 h and then with 200 μg/ml cycloheximide (CHX) for an additional 1 h. Total cell lysates were collected for WB analysis using anti-GFP antibodies. Tubulin was used as the loading control. The quantitative results are shown. E In situ PLA was performed in IL-6 or TNF-α-treated human hepatocytes using anti-CPAP and anti-SUMO-1 antibodies. PLA signals were detected by fluorescence microscopy as distinct bright red puncta at 400X magnification. F Human hepatocytes transfected with GFP-CPAP or GFP-CPAP SUMO-deficient mutant (GFP-CPAP/DM) were treated with (+) or without (-) TNF-α (left) or IL-6 (right) for 8 h. Total cell lysates were collected and subjected to WB analysis using anti-GFP antibodies. GAPDH was used as the loading control. The quantitative results are shown.

Article Snippet: Human hepatocytes (HHs) were purchased from ScienCell (HHs, Cat#5200, ScienCell Research Laboratories, Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Negative Control, Control, Transfection, In Situ, Fluorescence, Microscopy, Mutagenesis

Journal: Cell Death & Disease

Article Title: CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

doi: 10.1038/s41419-021-04295-2

Figure Lengend Snippet: A , B Human hepatocytes were transfected with GFP and GFP-CPAP, and then WB analysis was then performed using the indicated antibodies ( A ). The quantitative results are shown. The transcriptional activity of STAT3 and NF-κB was assessed by a reporter assay in GFP and GFP-CPAP-expressing cells ( B ). C Human hepatocytes transfected with CPAP shRNA, pSUPER-SP1 (SP1) and pSUPER-SP5 (SP5) , were collected for WB analysis as described above. pSUPER-NS2 (NS2) is the siRNA vector control. The quantitative results are shown. D , E The mRNA expression levels in hepatocytes transfected with HA-CPAP ( D ) and CPAP shRNA (SP1 and SP5) ( E ) were analyzed by RT-qPCR. F – I CPAP (F and H) and TNF-α (G and I) mRNA expression levels in adjacent normal liver tissues (NT) from HCC patients based on the lymphocytic inflammation status ( F – G : normal, n = 20, inflammation, n = 10) and the Ishak fibrosis score ( H – I : n = 32; 0: no fibrosis, 1: portal fibrosis (some), 2: portal fibrosis (most), 3: bridging fibrosis (occasional), 4: bridging fibrosis (marked), 5: incomplete cirrhosis, 6: cirrhosis) in the TCGA-LIHC dataset. The gene expression levels were normalized by FPKM normalization. J , K Correlation of CPAP and TNF-α mRNA levels in adjacent normal liver tissues (NT) with lymphocytic inflammation from TCGA dataset ( J ; n = 10) and the NCKUH HCC cohort ( K ; n = 38, Supplementary Table 1) was identified by the Pearson correlation coefficient. The data are presented as the mean ± SEM values. * p < 0.05, ** p < 0.01. Information of the Ishak fibrosis scores (0-6) and adjacent normal tissue inflammation grades (lymphocytic infiltration: slight, n = 20; mild+severe, n = 10) in the TCGA-LIHC dataset was obtained form the UCSC Xena browser (Supplementary Tables and ).

Article Snippet: Human hepatocytes (HHs) were purchased from ScienCell (HHs, Cat#5200, ScienCell Research Laboratories, Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions.

Techniques: Transfection, Activity Assay, Reporter Assay, Expressing, shRNA, Plasmid Preparation, Control, Quantitative RT-PCR, Gene Expression

Journal: Cell Death & Disease

Article Title: CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

doi: 10.1038/s41419-021-04295-2

Figure Lengend Snippet: A – C The expression levels of CCL-16 , MBL2 , and F2 mRNAs in human hepatocytes treated without (NC) or with IL-6 or TNF-α were determined by RT-qPCR. D – I The expression levels of CCL-16 , MBL2 , and F2 mRNAs in adjacent normal liver tissues (NT) of HCC patients from the TCGA-LIHC dataset with different Ishak fibrosis scores ( D – F ) and lymphocytic inflammation statuses ( G – I ) are shown. J – L Correlations between CCL-16 , MBL2 , and F2 mRNA levels and the TNF-α mRNA level in adjacent normal liver tissues with lymphocytic inflammation ( n = 10) from the TCGA-LIHC dataset were identified by the Pearson correlation coefficients. M The correlation between TNF-α and CCL-16 mRNA expression levels in the adjacent normal liver tissues of HCC patients ( n = 38) from the NCKUH cohort was identified by the Pearson correlation coefficient. * p < 0.05, ** p < 0.01.

Article Snippet: Human hepatocytes (HHs) were purchased from ScienCell (HHs, Cat#5200, ScienCell Research Laboratories, Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

doi: 10.1038/s41419-021-04295-2

Figure Lengend Snippet: A Human hepatocytes were transfected with HA and HA-CPAP (left), or with GFP and GFP-CPAP (right), and the expression of CCL-16 was then evaluated by RT-qPCR analysis (left) and WB analysis (right) under normal culture (NC) condition or after treatment with 10 ng/ml TNF-α for 24 h. B Human hepatocytes transfected with CPAP shRNA (SP1 and SP5) were collected to evaluate the expression of CCL-16 as described above. pSUPER-NS2 (NS2) is the siRNA vector control. The protein expression levels of CCL-16 are displayed as ratios and are shown below the blot image. C Conditioned media from GFP- and GFP-CPAP-transfected hepatocytes were collected to detect secreted CCL-16 by WB analysis. Total cell lysates were collected to determine the expression of GFP and GFP-CPAP. GAPDH was used as the loading control. The data are presented as the mean ± SEM values. * p < 0.05, ** p < 0.01. D , E The mRNA expression levels of CCL-16 and CPAP in adjacent normal liver tissues with lymphocytic inflammation were measured to determine their correlation by Pearson correlation analysis. TCGA-LICH dataset ( D , n = 10): R = 0.7909, p = 0.0064; NCKUH cohort ( E , n-38): R = 0.3264, p = 0.0455. F – G The levels of secreted CCL-16 protein, CPAP mRNA and TNF-α mRNA were determined in serum and adjacent normal liver tissues from the NCKUH cohort ( n = 16). The correlations between CCL-16 mRNA and CPAP mRNA levels ( F ) and between CCL-16 mRNA and TNF-α mRNA levels ( G ) were assessed by the Pearson correlation coefficients. H The correlation between CCL-16 mRNA and CPAP mRNA in the nontumoral part of HCC from the NCKUH cohort ( n = 43).

Article Snippet: Human hepatocytes (HHs) were purchased from ScienCell (HHs, Cat#5200, ScienCell Research Laboratories, Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions.

Techniques: Transfection, Expressing, Quantitative RT-PCR, shRNA, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

doi: 10.1038/s41419-021-04295-2

Figure Lengend Snippet: Upon viral infection or hepatic injury, human hepatocytes are under the stimulation of inflammatory cytokines, which increases CPAP protein stability through SUMO-1 modification. SUMO-1-modified CPAP enhances the expression of inflammatory cytokines, including TNF-α, IL-8, and CCL-16, by promoting the transcriptional activities of STAT3 and NF-κB. This CPAP-mediated enhancement of cytokine expression contributes to the chronic inflammatory microenvironment surrounding hepatocytes.

Article Snippet: Human hepatocytes (HHs) were purchased from ScienCell (HHs, Cat#5200, ScienCell Research Laboratories, Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions.

Techniques: Infection, Modification, Expressing