![PEG10 regulates cell size, facilitates tumor-forming ability and HDACi/Pi resistance in CTCL cells. (A) Suppression of PEG10 protein expression in HH cells by lentiviral transduction with 2 independent shRNA sequences (shPEG10-1, shPEG10-2) and overexpression of different PEG10 isoforms in Hut78 and Myla cells by lentiviral transduction with vectors expressing RF1b, RF1b/2, RF1b/2+CNF isoforms, respectively. HH cells transduced with scrambled shRNA (Ctrl) as well as Hut78 and Myla cells transduced with empty vectors (vector) served as controls. (B) Representative flow cytometry profiles of cell size (forward scatter [FSC], height on a linear scale) among control (Ctrl) and PEG10-suppressed (shPEG10) HH cells. Mean FSC signal intensity shown on the right panel. (C) Representative confocal images of successfully transfected GFP+ (green) control (Ctrl) and PEG10-suppressed (shPEG10) HH cells. Average cell area (pixel2) was calculated by Image J (right panel). (D) The number of colonies formed in the CFC assay among PEG10-suppressed (shPEG10-1, shPEG10-2) HH cells and control cells (Ctrl). (E) The number of colonies formed in the CFC assay among Hut78 cells with overexpression of RF1b, RFb1/2, RF1b/2+CNF, and control cells (vector). (F,G) Macroscopic pictures of xenograft mice and tumors after subcutaneously injecting control (Ctrl) and PEG10-suppressed (shPEG10) HH cells (F), as well as control (vector) and RF1b-overexpressed (RF1b) Hut78 cells (G) (n = 3, each). Median tumor volume was measured over time (right panels). (H,I) MTS-based cell viability assay of PEG10-suppressed (shPEG10) and control (Ctrl) HH cells under exposure to increasing dose of romidepsin (H), bortezomib (I) for 48 hours. (J,K) RF1b-overexpressed (RF1b) Hut78 cells and control cells (vector) were exposed to an increasing dose of romidepsin (J), bortezomib (K). Cell viability was evaluated after 48 hours. (L,M) Cleavage of caspase-3 was detected in HH cells with PEG10 silencing (shPEG10) and control cells (Ctrl) after treatment with different-dose SAHA (L), bortezomib (M) for 72 hours via western blot analysis. Data are represented as the mean standard deviation. *P < .05; **P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/139/4/10.1182_blood.2021012091/3/bloodbld2021012091f4a.png?Expires=1720285211&Signature=DIHXs5~9JDbakYp4uwSLbI7ph2qXhQeQXKAzE5vcr0j2VtdLYV2zwtr5B4KyjXmlX84wIDUyynXEd5RBVpU5BbTsGu1kBj~mIaElFm37kOfhepTyt~7LqrivFNc01JHqrgKu0fkx-QS5826TSYCgJlxvscFXAP60vMToG-0VlX~CU3-WXktP9QZ2s7Ie3T0MbPqV~RoxEQo0jkfFgkJ~sK0~2Iq-WTGMWbDbYBPVkfWuI-C19zLtxoW7amDA1QGd9NMvBnWaiyrNjsJJ5Gsn-HgXDm7MmkIEk84tAgUSHh3fbPrinW26MNk22Uk0YZsVRmA3k67u~Amb--nW8QqCvw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PEG10 regulates cell size, facilitates tumor-forming ability and HDACi/Pi resistance in CTCL cells. (A) Suppression of PEG10 protein expression in HH cells by lentiviral transduction with 2 independent shRNA sequences (shPEG10-1, shPEG10-2) and overexpression of different PEG10 isoforms in Hut78 and Myla cells by lentiviral transduction with vectors expressing RF1b, RF1b/2, RF1b/2+CNF isoforms, respectively. HH cells transduced with scrambled shRNA (Ctrl) as well as Hut78 and Myla cells transduced with empty vectors (vector) served as controls. (B) Representative flow cytometry profiles of cell size (forward scatter [FSC], height on a linear scale) among control (Ctrl) and PEG10-suppressed (shPEG10) HH cells. Mean FSC signal intensity shown on the right panel. (C) Representative confocal images of successfully transfected GFP+ (green) control (Ctrl) and PEG10-suppressed (shPEG10) HH cells. Average cell area (pixel2) was calculated by Image J (right panel). (D) The number of colonies formed in the CFC assay among PEG10-suppressed (shPEG10-1, shPEG10-2) HH cells and control cells (Ctrl). (E) The number of colonies formed in the CFC assay among Hut78 cells with overexpression of RF1b, RFb1/2, RF1b/2+CNF, and control cells (vector). (F,G) Macroscopic pictures of xenograft mice and tumors after subcutaneously injecting control (Ctrl) and PEG10-suppressed (shPEG10) HH cells (F), as well as control (vector) and RF1b-overexpressed (RF1b) Hut78 cells (G) (n = 3, each). Median tumor volume was measured over time (right panels). (H,I) MTS-based cell viability assay of PEG10-suppressed (shPEG10) and control (Ctrl) HH cells under exposure to increasing dose of romidepsin (H), bortezomib (I) for 48 hours. (J,K) RF1b-overexpressed (RF1b) Hut78 cells and control cells (vector) were exposed to an increasing dose of romidepsin (J), bortezomib (K). Cell viability was evaluated after 48 hours. (L,M) Cleavage of caspase-3 was detected in HH cells with PEG10 silencing (shPEG10) and control cells (Ctrl) after treatment with different-dose SAHA (L), bortezomib (M) for 72 hours via western blot analysis. Data are represented as the mean standard deviation. *P < .05; **P < .01.