Figure 5.
PEG10 confers growth advantage and HDACi/Pi resistance via a KLF2/NF-κB axis in CTCL cells. (A) Spearman correlation analysis of gene expression from control (Ctrl) as well as PEG10-silenced (shPEG10) HH cells with MF-LCT samples. The 2 dots on each line represent the correlation coefficients between each MF-LCT sample with a Ctrl as well as a shPEG10 sample, respectively. Paired P = 5 x 10−8. (B) Volcano plot of upregulated (red) and downregulated (blue) DEGs (fold change ≥2 or ≤−2 and P < .05) between PEG10-suppressed (shPEG10) and control (Ctrl) HH cells and KLF2 gene pointed out in green color. (C) KLF2 mRNA and protein levels of PEG10-suppressed (shPEG10-1, shPEG10-2) HH cells, RF1b-overexpressed (RF1b) Hut78 cells, and their control cells (Ctrl; vector). (D) Coimmunoprecipitation of nuclear extracts from control (Ctrl) and KLF2-suppressed (shKLF2) Hut78 cells using a PACF antibody (lane 2 and 5) or normal rabbit IgG (lane 3 and 6) with protein A/G agarose. PCAF, p65, and KLF2 were identified by western blot. Lanes 1 and 4 show input control, respectively. Schematic model of KLF2-mediated NF-κB activity inhibition is shown on the right panel. (E,F) NF-κB luciferase activity of KLF2-silenced (shKLF2) Hut78 cells and control cells (Ctrl); (E) RF1b-overexpressed (RF1b) Hut78 cells and control cells (vector); (F) via NF-κB luciferase reporter assay. (G) Median tumor volume of xenograft tumors (n = 5, each) after subcutaneously injecting PEG10-suppressed (shPEG10) HH cells, PEG10 and KLF2-suppressed (shPEG10 + shKLF2) HH cells were measured over time. (H,I) Specific apoptosis induced by 1 μM SAHA (H) and 12.5 nM bortezomib (I) for 48 hours, respectively, in different transduced HH cells. Ctrl indicates scrambled shRNA controls; shPEG10 and shKLF2 indicate shRNA sequences targeting PEG10 and KLF2, respectively. (J) RIP assay was executed in cell lysates from PEG10-RF1b overexpressed Hut78 cells using anti-Flag or anti-IgG, then the enrichment of KLF2 was detected by RT-PCR and qRT-PCR. (K) RNA stability assays were performed in PEG10-RF1b overexpressed Hut78 cells and control cells using Actinomycin D to disrupt RNA synthesis, and the degradation levels of the KLF2 mRNA were measured over 0, 20, 40, and 60 minutes. Data are represented as the mean standard deviation. *P < .05; **P < .01; ***P < .001; ns, no significance.

PEG10 confers growth advantage and HDACi/Pi resistance via a KLF2/NF-κB axis in CTCL cells. (A) Spearman correlation analysis of gene expression from control (Ctrl) as well as PEG10-silenced (shPEG10) HH cells with MF-LCT samples. The 2 dots on each line represent the correlation coefficients between each MF-LCT sample with a Ctrl as well as a shPEG10 sample, respectively. Paired P = 5 x 10−8. (B) Volcano plot of upregulated (red) and downregulated (blue) DEGs (fold change ≥2 or ≤−2 and P < .05) between PEG10-suppressed (shPEG10) and control (Ctrl) HH cells and KLF2 gene pointed out in green color. (C) KLF2 mRNA and protein levels of PEG10-suppressed (shPEG10-1, shPEG10-2) HH cells, RF1b-overexpressed (RF1b) Hut78 cells, and their control cells (Ctrl; vector). (D) Coimmunoprecipitation of nuclear extracts from control (Ctrl) and KLF2-suppressed (shKLF2) Hut78 cells using a PACF antibody (lane 2 and 5) or normal rabbit IgG (lane 3 and 6) with protein A/G agarose. PCAF, p65, and KLF2 were identified by western blot. Lanes 1 and 4 show input control, respectively. Schematic model of KLF2-mediated NF-κB activity inhibition is shown on the right panel. (E,F) NF-κB luciferase activity of KLF2-silenced (shKLF2) Hut78 cells and control cells (Ctrl); (E) RF1b-overexpressed (RF1b) Hut78 cells and control cells (vector); (F) via NF-κB luciferase reporter assay. (G) Median tumor volume of xenograft tumors (n = 5, each) after subcutaneously injecting PEG10-suppressed (shPEG10) HH cells, PEG10 and KLF2-suppressed (shPEG10 + shKLF2) HH cells were measured over time. (H,I) Specific apoptosis induced by 1 μM SAHA (H) and 12.5 nM bortezomib (I) for 48 hours, respectively, in different transduced HH cells. Ctrl indicates scrambled shRNA controls; shPEG10 and shKLF2 indicate shRNA sequences targeting PEG10 and KLF2, respectively. (J) RIP assay was executed in cell lysates from PEG10-RF1b overexpressed Hut78 cells using anti-Flag or anti-IgG, then the enrichment of KLF2 was detected by RT-PCR and qRT-PCR. (K) RNA stability assays were performed in PEG10-RF1b overexpressed Hut78 cells and control cells using Actinomycin D to disrupt RNA synthesis, and the degradation levels of the KLF2 mRNA were measured over 0, 20, 40, and 60 minutes. Data are represented as the mean standard deviation. *P < .05; **P < .01; ***P < .001; ns, no significance.

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