human hcc derived lines hepg2  (DSMZ)

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