Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Specific retention of TAT-ΔNS3/4A-FITC in NS3/4A-HCC36- and HCV-infected cells. HCC36 cells, NS3/4A-HCC36 cells, or JFH-1-infected HCC36 cells were incubated with 10 μM TAT-ΔNS3/4A-FITC in the absence or presence of 2 μM telaprevir (NS3/4A protease inhibitor) at 37°C for 1 h. The cells were washed with DMEM containing 10% serum three times per hour. After culturing for 1 or 8 h, the phase contrast and fluorescence of viable cells were observed under a fluorescence microscope. Scale bar: 100 μm.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Infection, Incubation, Protease Inhibitor, Fluorescence, Microscopy

Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Specific retention of TAT-ΔNS3/4A-FITC in NS3/4A-expressing tumors in vivo . Mice bearing established NS3/4A-HCC36 and HCC36 tumors were injected with 500 μM TAT-NS3/4A-FITC and sacrificed after 4 h. Sections of NS3/4A-HCC36 (upper panels) and HCC36 (lower panels) tumors were stained with 520 HCV protease Assay Kit (AnaSpec) to detect NS3/4A activity in tumor sections. FITC-derived fluorescence (green) and NS3/4A activity (red) were observed under a fluorescence microscope. Scale bar: 1 mm.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Expressing, In Vivo, Injection, Staining, Protease Assay, Activity Assay, Derivative Assay, Fluorescence, Microscopy

Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Specificity and half-life of TAT-ΔNS3/4A- 124 I-FITC. (A) NS3/4A-HCC36 and HCC36 cells were incubated with 37 kBq of TAT-ΔNS3/4A- 124 I-FITC in the presence or absence of 2 μM Telaprevir at 37°C for 1 h. The cells were washed with DMEM containing 10% serum three times per hour. The cells were collected by treatment with trypsin at the indicated times. The radioactivity of the cells was then measured with a gamma-counter. The CPM was normalized by protein concentration. (B) Kinetics of the TAT-ΔNS3/4A- 124 I-FITC in vivo , BALB/c mice were intravenously injected with TAT-ΔNS3/4A- 124 I-FITC, and the radioactivity in serum samples collected at the indicated times was measured using a gamma-counter. t 1/2 = 2.55 min. Error bar: standard error of triplicate determinations.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Incubation, Radioactivity, Protein Concentration, In Vivo, Injection

Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Micro-PET imaging of NS3/4A activity in vivo . (A) Mice bearing established NS3/4A-HCC36 (right hind leg) and HCC36 (left hind leg) tumors were injected with 3,700 kBq of TAT-ΔNS3/4A- 124 I-FITC. Coronal and transverse images were acquired at 2, 4, and 6 h after injection of the probe. (B) Mice bearing established NS3/4A-HCC36 (right hind leg) and HCC36 (left hind leg) tumors were intraperitoneally injected with telaprevir (20 mg/kg/day for 3 days) before intravenous injection of TAT-ΔNS3/4A- 124 I-FITC (3,700 kBq). Coronal images of tumor sections were acquired at 2, 4, and 6 h after injection of the probe.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Micro-PET, Imaging, Activity Assay, In Vivo, Injection

Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Biodistribution of TAT-ΔNS3/4A- 124 I-FITC in xenograft mice. Mice bearing established NS3/4A-HCC36 and HCC36 tumors were injected with 3,700 kBq of TAT-ΔNS3/4A- 124 I-FITC. Selected organs and tumors were removed from the mice after 2 (white column), 4 (black column), and 6 (gray column) h. The radioactivity of individual organs was measured using a gamma-counter and normalized for sample weights. The biodistribution of TAT-ΔNS3/4A- 124 I-FITC in selected organs was expressed as percentage injected dose/g tissue. Data represent mean ± SEM.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Injection, Radioactivity

Journal: Frontiers in Microbiology

Article Title: Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

doi: 10.3389/fmicb.2022.896588

Figure Lengend Snippet: Micro-PET Imaging of NS3/4A activity in orthotopic liver implantation model. The NS3/4A-HCC36 tumors or HCC36 tumors from the ectopic tumors were harvested and transplanted into the left liver lobe of SCID mice ( n = 3). After 2 weeks, the 3,700 kBq of TAT-ΔNS3/4A- 124 I-FITC was intravenously injected. (A) PET imaging was performed at 4 h after injection of the probe. (B) The accumulation of radioactivity in NS3/4A-HCC36 tumors and HCC36 tumors was measured using a gamma-counter at 2 (white column), 4 (black column), and 6 (gray column) h after probe injection. Data represent mean ± SEM. Student’s t -test analysis of data. Statistical analysis was compared NS3/4A-HCC36 with HCC36. P < 0.05 was considered statistically significant.

Article Snippet: Human hepatocellular carcinoma cells HCC36 (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s Minimal Essential Medium (Sigma-Aldrich, Burlington, MA, United States) supplemented with 10% heat-inactivated bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, Burlington, MA, United States) at 37°C in a humidified atmosphere of 5% CO2.

Techniques: Micro-PET, Imaging, Activity Assay, Injection, Radioactivity

Journal: Journal of Translational Medicine

Article Title: Targeting protein arginine methyltransferase 5 inhibits human hepatocellular carcinoma growth via the downregulation of beta-catenin

doi: 10.1186/s12967-015-0721-8

Figure Lengend Snippet: Silencing PRMT5 decreases human HCC cell growth in vitro. a HepG2 and Bel-7404 cells were transfected with PRMT5 siRNA (si-PRMT5) or scramble negative control siRNA (si-NC) and cell proliferation was analyzed. b Normal liver HL-7702 cells were transfected with PRMT5 siRNA (si-PRMT5) or scramble negative control siRNA (si-NC) and cell proliferation was analyzed. c The effect of si-PRMT5 on colony formation of HepG2 and HL-7702 cells. Representative results of colony formation of vehicle ( left ), si-NC ( middle ), and si-PRMT5 ( right ) HepG2 and HL-7702 cells. 5-fluorouracil (5-Fu) is used as a positive control. d Knockdown of PRMT5 led to G1 arrest. HepG2 cells were transfected with si-NC or si-PRMT5 and then subjected to cell cycle analysis. e and f Western blot analysis of whole-cell lysates derived from HepG2 cells transfected with si-NC or si-PRMT5 using antibodies against PRMT5, β-catenin, Cyclin D1, H4R3me2s or H3R8me2s

Article Snippet: Four human HCC cell lines (HepG2, Bel-7404, Bel-7402 and SMMC-7721), one rat hepatoma cell line CBRH-7919 and one normal liver cell line HL-7702 were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured under conditions recommended.

Techniques: In Vitro, Transfection, Negative Control, Positive Control, Knockdown, Cell Cycle Assay, Western Blot, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Targeting protein arginine methyltransferase 5 inhibits human hepatocellular carcinoma growth via the downregulation of beta-catenin

doi: 10.1186/s12967-015-0721-8

Figure Lengend Snippet: AMI-1 inhibits HCC cell growth in vitro and in vivo. a The effect of AMI-1 on the proliferation of human HCC cell lines. b The effect of AMI-1 on tumor formation in a nude mouse xenograft model. c The expression of Bax, Bcl-2 and H4R3me2s in HepG2 cells treated with AMI-1 for 72 h. d Densitometric analysis of band intensities. β-actin was loading control. Control refers to vehicle-treated group

Article Snippet: Four human HCC cell lines (HepG2, Bel-7404, Bel-7402 and SMMC-7721), one rat hepatoma cell line CBRH-7919 and one normal liver cell line HL-7702 were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured under conditions recommended.

Techniques: In Vitro, In Vivo, Expressing, Control

Journal: Journal of Translational Medicine

Article Title: Targeting protein arginine methyltransferase 5 inhibits human hepatocellular carcinoma growth via the downregulation of beta-catenin

doi: 10.1186/s12967-015-0721-8

Figure Lengend Snippet: AMI-1 promotes the apoptosis and decreases migratory activity of HCC cells. a HCC cells were treated with vehicle or AMI-I and then stained by Annexin V-fluorescein isothicyanate (FITC) and propidium iodide (PI), followed by flow cytometry analysis. b AMI-1 decreased migratory activity of HCC cells measured by Transwell assay. Representative photos of stained cells are shown. Original magnification ×200. Control refers to vehicle-treated group

Article Snippet: Four human HCC cell lines (HepG2, Bel-7404, Bel-7402 and SMMC-7721), one rat hepatoma cell line CBRH-7919 and one normal liver cell line HL-7702 were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured under conditions recommended.

Techniques: Activity Assay, Staining, Flow Cytometry, Transwell Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 1. Expression of APRIL and BCMA in HCC-derived cell lines. (A) APRIL and BCMA are expressed in HCC-derived cell lines. TACI im- munoreactivity is absent, whereas staining for BAFF and BAFFR is also observed. A typical IHC is presented, repeated three times, in different cell preparations, with similar results. HepG2 cells are presented in the left column and Hep3B in the right. Original magnification 3200 in all panels. APRIL, TACI, BAFF, and BAFFR were detected with Fast Red and BCMA with DAB (see Materials and Methods for details). (B) RT-PCR of ligands (APRIL, BAFF) and receptors (BCMA, BAFFR, TACI) in HepG2 and Hep3B cells. Positive controls (adipose tissue-derived mesenchymal cells for APRIL and BAFF and isolated human lymphocytes for BCMA, TACI, and BAFFR) are also included. (C) Flow cytometry detection of membrane-expressed BCMA and BAFFR and absence of TACI staining in HepG2 and Hep3B cells, identified by FITC-labeled anti-receptor Abs. Mean fluorescence intensity (MFI) is also reported for unstained (control) and stained cells.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Expressing, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Isolation, Flow Cytometry, Membrane, Labeling, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 2. APRIL reduces cell growth and induces a blockade in the G2/M phase of the cell cycle, acting through BCMA. (A) Dose and time effect of APRIL (200 ng/ml) on cell growth of HepG2 and Hep3B cell lines. Cells were incubated for 3 and 6 d; the medium was changed once, at day 3, and supplemented with fresh APRIL. Mean 6 SEM of three different assays is presented, performed in triplicates. (B) Addition of Fc-BCMA, a soluble form of the receptor binding APRIL, reverts the effect of APRIL (200 ng/ml) in both cell lines, whereas Fc-BCMA alone or nonspecific IgG had no effect. Mean 6 SEM of three independent assays in triplicate. Incubation time was 6 d. (C) Transfection of cells with shBCMA RNA reverts the effect of APRIL (200 ng/ ml) on cell growth, after 6 d of incubation. In contrast, addition of nontarget shRNA has no effect. Results (mean 6 SEM of three experiments in qua- druplicate) are normalized compared with control (non-APRIL or shRNA-treated) cells. (D) Effect of APRIL (200 ng/ml) on the cell cycle of HepG2 (upper row) and Hep3B cells (lower row). Cells were synchronized by serum starvation for 24 h and then treated with 200 ng/ml APRIL in full culture medium for 24 additional hours. Annexin/propidium iodine staining and FACS analysis were performed as described in Materials and Methods. Similar results were obtained in cells treated for 48 h with APRIL (not shown). This figure presents a typical example, as analyzed with the ModFit program, and the table below shows mean 6 SEM of three repetitions. *p , 0.05, **p , 0.01.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Incubation, Binding Assay, Transfection, shRNA, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 3. BCMA signaling in HCC-derived cells. (A) Addition of 200 ng/ml APRIL in HepG2 or Hep3B cells, transfected with the pNFkB-Luc vector, carrying NF-kB response elements in front of the firefly luciferase gene, does not modify the transcriptional activity of NF-kB, after a 24-h incubation. In contrast, an induction is obtained with TNF-a, used as a positive control. Results (mean 6 SEM of three different experiments, in five replicates each) are normalized per the Renilla reporter gene. (B) Western blot of different signaling mediators (JNK, Akt, p38 MAPK and ERK phosphorylation) during a time course of HepG2 (left panel) and Hep3B cells (right panel) treated with 200 ng/ml APRIL. The ligand induces JNK and Akt phosphorylation, whereas p38 MAPK and ERK remain unaltered. A typical blot is presented, repeated three times with similar results. (C) The PI3K/Akt pathway does not participate in the antiproliferative action of APRIL. Treatment of HepG2 and Hep3B cells with LY-294002 (LY, 40 mM), 30 min prior to the addition of APRIL (200 ng/ ml, a 6-d incubation, with one change of the medium at day 3 and addition of fresh APRIL and LY), does not block APRIL-induced cell growth arrest. Results (mean 6 SEM of three assays in triplicate) are expressed compared with control (nontreated) cells. *p , 0.05, **p , 0.01. Similar results, with the use of wortmannin as a PI3K/Akt inhibitor are shown in Supplemental Fig. 3A. (D) JNK2 is implicated in APRIL-induced growth arrest in HepG2 cells. Incubation of cells with shRNA against JNK2 reverts the antiproliferative effect of APRIL (200 ng/ml) after 6 d of incubation. Figure presents the mean 6 SEM of three different experiments in triplicate.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Incubation, Positive Control, Western Blot, Phospho-proteomics, Blocking Assay, Control, shRNA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 4. Transcriptional effects of APRIL. (A) APRIL (200 ng/ml) incubation of HepG2 cells induces a time-related increase of upregulated tran- scripts; downregulated transcripts remain almost constant after 6 h of incubation. (B) Heat plot of leading edge analysis (39) of genes involved in sig- nificantly modified pathways. This plot permits the identification of genes modified by APRIL, in the majority of identified pathways. (C) Common genes modified by APRIL in the pathways identified by gene set enrichment analysis were assayed by qRT-PCR after 2, 6, and 12 h of APRIL incubation (200 ng/ ml) in the presence or absence of shRNA against BCMA and nontarget shRNA. Mean 6 SEM of two independent experiments assayed in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Incubation, Quantitative RT-PCR, shRNA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 5. Validation of JNK implication in FOXO3A, Akt, and GADD45 activation by APRIL. (A) Effect of APRIL on FOXO3A activation. HepG2 (upper panel) and Hep3B (lower panel) cells were transfected with a construct in which FOXO3A interaction sites have been cloned upstream of the 59 end of the luciferase gene. They were then incubated with 200 ng/ml APRIL, in the absence or presence of LY-294002 (LY, 40 mM) or shRNA against BCMA, for 24 h. Protein was collected and used for the luciferase assay. Figure presents graphs normalized per control (nontreated) cells. (B) Effect of JNK1 and JNK2 inhibition on FOXO3A activation in HepG2 and Hep3B cells. Only shRNA against JNK2 blocks APRIL-induced FOXO3A activation (*p , 0.05 compared with scrambled shRNA-transfected plus APRIL-treated cells). (C) Time course of JNK (left panel) or Akt (right panel) phosphorylation after incubation of HepG2 cells with 200 ng/ml APRIL, in the absence (squares) or presence (circles) of 10 mM SP600125. Quantitation of Western blot results is presented as mean 6 SEM of three independent assays. Similar results were obtained with Hep3B cells (not shown). (D) GADD45 transcription after incubation of HepG2 cells with APRIL, 200 ng/ml, for 2, 6, and 12 h. GADD45 was assayed by qRT-PCR. Anti-BCMA shRNA inhibited APRIL-induced GADD45 expression. In all panels, the mean 6 SEM of at least two assays performed in triplicate is presented.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Biomarker Discovery, Activation Assay, Transfection, Construct, Clone Assay, Luciferase, Incubation, shRNA, Control, Inhibition, Phospho-proteomics, Quantitation Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: APRIL binding to BCMA activates a JNK2-FOXO3-GADD45 pathway and induces a G2/M cell growth arrest in liver cells.

doi: 10.4049/jimmunol.1102891

Figure Lengend Snippet: FIGURE 6. JNK2 mediates APRIL-induced phosphorylation of Akt. (A) Typical Western blots from HepG2 cells transfected with shRNA against JNK1 and JNK2 (48 h) and treated with APRIL (200 ng/ml) for 10 min. The experiment was performed three times with identical results. (B) Quantification of pAkt/total-Akt from Western blots performed in three different experiments. Only JNK2 inhibition could block APRIL/BCMA-induced Akt phosphorylation. (C and D) JNK1 knockdown led to preferential inhibition of the 45-kDa JNK1/2 isoforms, whereas JNK2 knockdown led to inhibition of the 54-kDa isoforms of these molecules, a phenomenon suggestive of a “predominant” isoform expression from these genes in HepG2 cells. Mean 6 SEM of three independent experiments. *p , 0.05, **p , 0.01 denote significance compared with scrambled shRNA-transfected cells, treated or not with 200 ng/ml APRIL.

Article Snippet: Human HCC-derived lines HepG2 and Hep3B were purchased from DSMZ (Braunschweig, Germany); they were cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% FBS (Invitrogen, Carlsbad, CA), at 37 ̊C, 5% CO2.

Techniques: Phospho-proteomics, Western Blot, Transfection, shRNA, Inhibition, Blocking Assay, Knockdown, Expressing