Journal: PLoS ONE

Article Title: The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study

doi: 10.1371/journal.pone.0158354

Figure Lengend Snippet: The CTCs were identified and enumerated via a) positive nuclear staining (Hoechst), b) positive cytokeratin staining, and c) negative CD45 staining. d) Overlay of all images showing size and morphological characteristics.

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin (BSA) (PAA) in PBS for 1 h. Primary antibodies, including anti-pan-cytokeratin-fluorescein isothiocyanate (FITC) (CK8, CK18 and CK19, Abcam) and anti-CD45- allophycocyanin (APC, Invitrogen), were added for 1 h. The wire was then rinsed 3 times with 3 ml of PBS, and the nuclei were counterstained with Hoechst 33258 (Invitrogen).

Techniques: Staining

Journal: Oncotarget

Article Title: Transdifferentiation of human male germline stem cells to hepatocytes in vivo via the transplantation under renal capsules

doi: 10.18632/oncotarget.14713

Figure Lengend Snippet: ( A – D ) Immunohistochemistry with DAB staining revealed the expression of AAT and ALB in the grafts derived from human SSC line plus mouse liver mesenchymal cells (A, B) and from human SSC line without liver mesenchymal cells (C, D). Notes: Black arrow in figure A indicated cell cords with tight cell-cell interactions, while green arrow denoted liver plate-like structure. Scale bars in A–D = 10 μm. ( E , F ) Immunohistochemistry showed the coexpression CK18 and eGFP (E) as well as CYP1A2 and eGFP (F), and the expression of HumNuc ( G ) in the grafts derived from human SSC line and mouse liver mesenchymal cells. Notes: asterisks denoted HumNuc-negative cells. Replacement with primary antibody with PBS ( H ) was used as a negative control. Scale bars in E and F = 20 μm; and scale bars in G and H = 40 μm.

Article Snippet: The primary antibodies were CK18 (Santa Cruz), CYP1A2 (Santa Cruz), human nuclear antigen (HumNuc) (Millipore), GPR125 (Abcam), and VASA (Abcam), and incubated in a humidified chamber overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Expressing, Derivative Assay, Negative Control

Journal: Oncotarget

Article Title: Transdifferentiation of human male germline stem cells to hepatocytes in vivo via the transplantation under renal capsules

doi: 10.18632/oncotarget.14713

Figure Lengend Snippet: The antibodies used for immunostaining and Western blots

Article Snippet: The primary antibodies were CK18 (Santa Cruz), CYP1A2 (Santa Cruz), human nuclear antigen (HumNuc) (Millipore), GPR125 (Abcam), and VASA (Abcam), and incubated in a humidified chamber overnight at 4°C.

Techniques: Immunostaining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System

doi: 10.3390/ijms17040584

Figure Lengend Snippet: Histological and immunohistochemical analysis of primary human liver cells cultured in bioreactors with or without diclofenac application: cell material from untreated control bioreactors (control BR) ( A , D , G ); 300 µM bioreactors (300 µM BR) ( B , E , H ); or 1000 µM bioreactors (1000 µM BR) ( C , F , I ) was investigated at the end of the experimental phase on Day 10 of culture. The upper row ( A – C ) shows hematoxylin and eosin staining of cultures; the middle row ( D – F ) shows CK18 ( green ) and vimentin ( red ); and the lower row ( G – I ) shows BCRP ( green ) and CYP2C9 ( red ) immunoreactivity. Counterstaining of nuclei is shown in blue (scale bar = 50 µM).

Article Snippet: Monoclonal mouse antibodies against CK18 (ab668, Abcam, Cambridge, UK) or BCRP (ab3380, Abcam), and polyclonal rabbit antibodies against vimentin (sc-5565, Santa Cruz, Santa Cruz, CA, USA) or CYP2C9 (PAP091, Nosan, Yokohama, Japan) were used as primary antibodies.

Techniques: Immunohistochemical staining, Cell Culture, Control, Staining

Journal: Oncotarget

Article Title: Glucocorticoid receptor antagonism promotes apoptosis in solid tumor cells

doi: 10.18632/oncotarget.27989

Figure Lengend Snippet: ( A ) In the MIA PaCa-2 model, efficacy of the combination of paclitaxel + relacorilant was significantly better than paclitaxel alone (non-parametric T -test P < 0.0001). ( B ) In the MIA PaCa-2 model, efficacy of paclitaxel + gemcitabine + relacorilant was better than paclitaxel + gemcitabine alone (non-parametric T -test P = 0.0005). ( C ) In the HeLa model, efficacy of paclitaxel + relacorilant was significantly better than paclitaxel alone (non-parametric T -test P < 0.0001). ( D ) In the CC6279 model, efficacy of paclitaxel + relacorilant was significantly better than paclitaxel alone (non-parametric T -test P < 0.0001). Relacorilant alone had no significant effect on tumor growth. Error bars represent the standard error; all studies 10 animals/group. ( E ) Tumor cells were labeled using cytokeratin 18 immunohistochemistry (top). In serial sections, apoptotic caspase activity (cleaved caspase 3, bottom) and proliferation (Ki67, not shown) were assessed. ( F ) Relacorilant increased the cleaved caspase intensity and prevalence (H-score) within the tumor cells compared to paclitaxel alone. *** Mann-Whitney, P < 0.0001. Abbreviation: IHC, immunohistochemistry.

Article Snippet: Primary antibodies included CK18 (EPR17347, Abcam), Ki67 (SP6, Abcam or 12202S, Cell Signaling Technologies), and cleaved-caspase-3 (9664s, Cell Signaling Technologies) and were all of rabbit origin.

Techniques: Labeling, Immunohistochemistry, Activity Assay, MANN-WHITNEY

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: CK18 KD promotes the proliferation and attenuates the apoptosis of HeLa cells. (A) CK18 mRNA expression in HeLa cells after transient transfection with CK18 -specific shRNAs or control vector, as determined by qRT-PCR (left); CK18 protein expression was decreased by shRNA1, as revealed by western blotting (right). GAPDH was used as an internal control. The control samples and shCK18 samples were from the same gel in the western blotting experiment, but not side by side. (B) An MTT assay revealed that transfection with the CK18 -shRNA1 upregulated cell proliferation in HeLa cells; (C) Plots and quantification of flow cytometric analysis of apoptosis following CK18 KD in HeLa cells. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01. CK18, cytokeratin 18; Knockdown, KD; shCK18, CK18 knockdown by shRNA in HeLa cells; Ctrl, HeLa cells transfected with scrambled plasmid as the control.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, MTT Assay, Standard Deviation, Knockdown, shRNA

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: Differential gene expression in response to CK18 KD. (A) Quantification of CK18 expression using RNA-seq data, FPKM values were calculated as explained in Materials and methods. (B) Volcano plot of the CK18-regulated genes using edgeR software (filtering criteria, FC ≥2 and FDR<0.05) treating RNA-seq data from two samples in each group as experimental replicates. Red indicates upregulated genes and blue indicates downregulated genes. (C) Heat map of all the 593 differentially expressed genes in the control and CK18 shRNA-treated samples. (D) Overlap of two sets of DEGs. One set was identified as described in B. The other set was identified by treating one CK18 shRNA-treated and one control RNA-seq data as a simple pair using edgeR software; the resulting two pairs of DEGs were then overlapped to obtain the common simple-pair DEGs. CK18, cytokeratin 18; FPKM, fragments per kilobase of transcript per million fragments mapped; DEGs, differentially expressed genes.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Gene Expression, Expressing, RNA Sequencing, Software, Control, shRNA

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: Functional enrichment analysis of DEGs. GO and KEGG analysis were performed on DEGs obtained using the two different strategies . (A) Top representatives of GO biological process terms and KEGG pathways up- and downregulated by CK18 of the DEGs in CK18-knockdown and control groups (named as replicates). (B) Top representatives of GO biological process terms and KEGG pathways up- and downregulated by CK18 of the DEGs overlapping in the two replicate groups (named as simple pair). DEGs, differentially expressed genes; CK18, cytokeratin 18.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Functional Assay, Knockdown, Control

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: Identification and functional analysis of CK18-regulated splicing events. (A) Classification of different types of alternative splicing events regulated by CK18 protein. (B) Analysis of the overlap between CK18-regulated genes (DEGs from the two different analyses and RASGs. (C) Top ten GO biological processes and KEGG functional pathways enriched by alternative gene splicing. CK18, cytokeratin 18; DEGs, differentially expressed genes; RASG, alternative splicing genes.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Functional Assay, Alternative Splicing

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: (A-C) Validation of CK18-regulated genes (DEGs) by quantitative PCR and (C) western blotting in (A and C) HeLa cells, and in (B) HepG2 cells. (B) The effectiveness of three different CK18 -specific shRNAs (left) and the effect of multiple CK18 -specific shRNAs on the expression of four CK18-regulated genes (right) are presented. (C) Validation of the effect of CK18 -specific shRNA1 on the protein level (left) and mRNA level (right) of CASP8. Experimental data are presented as the mean ± standard deviation of at least three experiments. *P<0.05 and **P<0.01. CK18, cytokeratin 18; DEGs, differentially expressed genes.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation

Journal: Oncology Reports

Article Title: Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic-related genes and pathways in HeLa cells

doi: 10.3892/or.2019.7166

Figure Lengend Snippet: Validation of CK18-regulated ASEs. (A) Validation of an ASE of FAS in HeLa and HepG2 cells. (B) Validation of ASEs of CTNNB1 in HeLa and HepG2 cells. Left panels reveal the IGV-sashimi plots for alternative splicing changes that occurred in HeLa cells in response to CK18 knockdown. The annotated transcripts for the gene are presented below. Right top panels reveal the schematic diagrams depicting the structures of alternative splicing events, AS1 (shown in red) and AS2 (shown in green); exon sequences are denoted by boxes, and intron sequences by the horizontal lines. Right bottom panels reveal the RNA-seq and RT-qPCR quantification of alternative splicing events. The altered ratio of ASEs in RNA-seq were calculated using the formula: AS1 junction reads/AS1 junction reads+AS2 junction reads; while the altered ratio of AS events in qPCR were calculated using the formula: AS1 transcripts level/AS2 transcripts level. CK18, cytokeratin 18; ASEs, alternative splicing events. *P<0.05 and **P<0.01.

Article Snippet: The membrane was incubated with blocking solution (1X TBS; 0.05% Tween-20; 5% non-fat milk) at room temperature for 1 h and incubated overnight with primary antibodies raised against CK18 (dilution 1:1,000; cat. no. A0389), CASP8 (dilution 1:1,000; cat. no. A0215) and GAPDH (dilution 1:1,000; cat. no. AC027) (all from ABclonal, Wuhan, China).

Techniques: Biomarker Discovery, Alternative Splicing, Knockdown, RNA Sequencing, Quantitative RT-PCR