What was tested in figure 3H? Explain the results in 3H. In your explanation say
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What was tested in figure 3H? Explain the results in 3H. In your explanation say what was measured, what were the controls, what cells the researchers used, and if results fit the researchers hypothesis.
PDLIM2 Reconstitution Inhibits Oncogenic NF-B RelA and STAT3 Activation to Suppress KSHV Tumorigenesis—To define the molecular mechanism by which PDLIM2 down-regulation contributes to the pathogenesis of KSHV, we focused on RelA and STAT3, two downstream targets of PDLIM2. As expected, a high nuclear expression of RelA and STAT3 was detected in the BCBL-1-vector and BC-1-vector cells (Fig. 3, A and B). However, the nuclear expression of RelA and STAT3 was inhibited in the BCBL-1-PDLIM2 and BC-1-PDLIM2 cells. The decrease of nuclear RelA and STAT3 was due to proteasomal degradation because treatment of the proteasome inhibitor MG132 led to increase of RelA and STAT3 at the nuclear matrix but not the promyelocytic leukemia or SC-35 bodies of those PDLIM2 stable cell lines (Fig. 3A). Consistently, the transcription activities and downstream target genes of NF-B and STAT3, such as Bcl-xL, survivin, and cyclin D1, were also suppressed in those PDLIM2 stable cell lines (Fig. 3, C and D). It is noteworthy that Bcl-xL, survivin, and cyclin D1 are known drivers of cell survival and proliferation. To validate whether PDLIM2 suppresses KSHV tumorigenesis via termination of RelA and STAT3 activation, we utilized the PDLIM2 PDZ or LIM domain deletion mutants. In line with previous studies (33, 34, 38–40), the twomutants failed to prevent the nuclear expression of RelA and STAT3 in PEL cells (Fig. 3E). Importantly, the two mutants also failed to suppress the growth and colony formation of PEL cells (Fig. 3, F and G). In further support of this, knockdown of RelA or STAT3 resulted in significant growth inhibition of PEL cells (Fig. 3H). Altogether, our studies suggest that KSHV somehow represses PDLIM2 expression, leading to persistent RelA and STAT3 activation and subsequent tumorigenesis and tumor phenotype maintenance.
FIGURE 3. PDLIM2 reconstitution inhibits oncogenic NF-B RelA and STAT3 activation to suppress KSHV tumorigenesis. A, confocal microscopic assays showing decreased RelA and STAT3 nuclear expression in BCBL-1 cells reconstituted with PDLIM2, which can be reversed by proteasome inhibition. B, IB assays showing decreased RelA and STAT3 nuclear expression in the PEL cells reconstituted with PDLIM2. Lanes 1 and 3 represent PEL cells stably expressing an empty vector, while lanes 2 and 4 represent PEL cells stably expressing PDLIM2. C, gene reporter assays showing decreased transcriptional activity of NF-B in PEL cells reconstituted with PDLIM2. D, qPCR analysis showing PDLIM2 down-regulation of Bcl-xL, survivin, and cyclin D1 in BCBL-1 cells. E, generation of BCBL-1 cells stably expressing PDLIM2 mutants defective in RelA and STAT3 suppression. The expression levels of these PDLIM2 mutants as well as the nuclear expression levels of RelA and STAT3 in the stable cell lines were examined by IB assay. Lanes 1–4 represent BCBL-1 cells stably expressing empty vector, PDLIM2 WT,PDZ, and LIM mutants, respectively. F, cell counting assays showing that PDLIM2 mutants defective in RelA and STAT3 termination lose the ability to suppress the growth of BCBL-1 cells in culture. G, soft agar assays showing that PDLIM2 mutants defective in RelA and STAT3 termination lose the ability to suppress the anchorage-independent growth of BCBL-1 cells. H, cell counting assays showing that knockdown of RelA or STAT3 inhibits the growth of BCBL-1 cells. Data are mean S.D. *, p 0.05; **, p 0.01.
Vector PDLIM2 RelA PDLIM2+MG132 Merge Vector PDLIM2 STAT3 STAT3 PDLIM2+MG132 Merge RelA RelA 20S DAP STAT3 20S DAPI DAP ReIA PML Merge DAPI DAPI DAPI STAT3 PML Merge DAPI Merge Merge RelA SC-35Merge DAPI Merge Merge STAT3 SC-35 Merge D oVector 61.2 PDLIM2 Vector 1 2 34 120r PDLIM2 100 STAT3 RelA RelA Hsp90 Hsp90 BCBL-1 BC-1 Z BCBL-1 BC-1 BCBL-1 BCBL-1 2.0f-oshVector BCBL-1 ector 4Vector EAPDz Day0 1 2 3 4 5 Day 0 3 5 7Explanation / Answer
Kapasi sarcoma virus (KHSV) is the causative agent of human primary effusion lymphoma (PEL). In this manuscript, the researchers want to analyse whether PDLIM2 gene is repressed by KHSV virus and if yes, the mechanism of it. Their hypothesis is that increased expression of PDLIM2 suppressed the growth of KSHV virus via repressing RelA and Stat 3 genes as well as causing NfkB inactivation. As a result, there will be loss of tumorigenesis.
In Figure 3, the authors analyzed whether PDLIM2 suppressed the growth and division of the KHSV virus in PEL-1 cells. BCBL-1 is human primary effusion lymphoma-1 (PEL-1) cell lines. They also analyzed whether growth of virus is linked to increased expression of transcription factors RelA and Stat3.
The figure 3 H indicates that inhibition of RelA and Stat3 results in significant growth inhibition (p is 0.01) of BCBL-1 cells. The body cavity bases lymphoma cells are infected with human herpesvirus 8 (HHV-8), which is also known as Kapasi sarcoma virus (KHSV). KSHV causes proliferation of these BCBL-1 cells. In this experiment, these cells were infected with shRNA (short hairpin loop RNA) vector (plasmid) containing either RelA (rectangle points) or Stat3 (traingle points). ShRNA are artificial RNA molecules that have a short hairpin loops that silences (inhibits transcription) genes. They are processed by Drosha and Dicer and loaded on to RNA inducing silencing complex (RISC). They can bind to mRNA and cleave it to reduce gene expression. Transcription factors RelA and signal transducer and activator of transcription 3 (Stat3) are downstream targets of tumor suppressor gene PDZ-LIM domain-containing protein 2 (PDLIM2). The control cells (with virus) were treated with a plain vector (plasmid lacking the gene sequence of either RelA or Stat3; quandrangle points). Growth curve of these cells were analyzed for 0, 3, 5 and 7 days. A plot of cell numbers (X 106) on y-axis was plotted vs days (on X-axis). ShRNA to any gene should reduce the transcription of the relevant gene.
Previous studies in Figure 3 indicate that PDLIM2 suppresses the expression of RelA and Stat3 via a proteosomal degradation in virus infected PEL-1 cells (Fig 3A and 3B). Further, PDLIM2 also decreased NFKb activity in these virus infected cells. Results in Fig 3H indicate that there was a significant decline in growth of the cells that were treated with shRNA to RelA or Stat3 as compared control cells (treated with vector). This result indicates that RelA and Stat3 are required for the growth of the virus infected cell. Thus, the growth of virus infected cells is suppressed when RelA and Stat3 are absent. These results fit the hypothesis of the researchers that expression of RelA and Stat3 is important for the tumorigenic potential of KHSV virus. If these genes are inactivated, then the virus will be unable to grow in the BCBL-1 cells, causing decline in cell division of the lymphoma cells. The researchers have linked the loss of tumorigenicity to the increased expression of PDLIM2. KSHV virus reduces PDLIM2 expression causing enhanced RelA and Stat3 expression and is thus able to grow and divide.
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