u2os pml knockout  (ATCC)

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: DGATi and oleate treatment of U2OS cells promotes PML and CCTα association with the INM. (A) U2OS cells treated for 1 h with no addition (NA), oleate (500 µM), DGATi (10 µM) or oleate plus DGATi (10 µM) were immunostained for PML. DNA was visualized with Hoechst 33342 (bar, 10 µm). (B and C) cells were treated with oleate (B) or oleate plus DGATi (C) for up to 24 h, immunostained as described in panel A, and the percentage of cells positive for LAPS and PML patches was quantified. Each time point represents 30–50 cells from a representative experiment. (D) U2OS cells treated as described in panel A for 16 h were immunostained with antibodies against PML and CCTα. LDs were visualized with BODIPY 493/503 (bar, 10 µm). The nucleus is outlined, and regions of interest (ROI) are to the right (bar, 2 µm). (E, F, G and H) quantification of PML patches per cell (E), percentage of cells with PML patches (F), PML NBs per cell (G) and NE enrichment of CCTα (H). Results in E–H are the mean and SD from 9 to 12 fields of cells (6–10 cells/field) per treatment from three independent experiments. Statistical significance determined by two-way ANOVA with multiple comparisons. **** p < 0.0001, *** p < 0.001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Unsaturated fatty acids and DGATi promote PML patch formation and CCTα enrichment of the INM. (A) U2OS cells treated with no addition or 500 µM palmitate (16:0), stearate (18:0), oleate (18:1), linoleate (18:2), or α-linolenate (18:3) in the absence or presence of DGATi for 24 h were immunostained for PML and CCTα, and LDs visualized with BODIPY 493/503 (bar, 10 µm). The percentage of cells with LAPS (B) and PML patches (C), and NE enrichment of CCTα (D) were quantified. Results are the mean and SD 8–16 fields of cells ( n = 1057) from two experiments. Statistical significance determined using two-way ANOVA with multiple comparisons. **** p < 0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML patches are depleted of canonical PML NB-associated proteins. U2OS cells were treated with DGATi and oleate for 16 h and immunostained with antibodies against SUMO (A), SP100 (B) or DAXX (C). The nucleus was visualized with DAPI in the merged image. Small arrows point to PML patches and large arrows point to PML NBs (bar, 10 µm).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML patches are zones of nuclear lamina depletion. (A) Clover-PML U2OS cells treated with DGATi and oleate for treated for 24 h were immunostained for LMNA/C and DNA was visualized with Hoechst 33342 (bar, 5 µm). (B) Clover-PML U2OS cells treated as described above were immunostained for emerin (bar, 10 µm). Arrows indicate PML patches at regions of LMNA/C and emerin depletion.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML-II forms patches on the INM during inhibition of TAG synthesis. (A) U2OS PML KO cells transiently expressing GFP-tagged PML-I, PML-II, or PML-IV were treated with no addition (NA), oleate, or oleate and DGATi for 16 h and immunostained with antibodies against LMNA/C and CCTα (bar, 10 µm). The percentage of GFP-expressing cells ( n = 163) with (B) LAPS (identified as ring structures with associated GFP-PML and CCTα) and (C) PML patches were quantified. Results are the mean and SD of three independent experiments. Statistical significance assessed using Student's test. * p < 0.05, ** p < 0.01.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Inhibition, Expressing

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Stabilization of INM-associated CCTα in DGATi and oleate treated cells. (A) Lysates from U2OS cells treated with no addition (NA) or DGATi, with and without oleate for 16 h were immunoblotted for Lipin1, CCTα, and OSBP. (B) CCTα protein expression from panel A was quantified relative to no addition control (mean and SD of three experiments). (C) mRNA expression of PCYT1A in U2OS cells treated as described in panel A (mean and SD of four replicates from two experiments). (D) lysates of U2OS cells treated for 16 h with NA, DGATi (10 µM) in the presence of 0–500 µM oleate or tunicamycin (TM) were immunoblotted for CCTα, PML, eIF2α, pSer51 eIF2α (p-eIF2α) and OSBP (load control). (E) Immunoblots of lysates from cell treated with DGATi and oleate for 0 to 24 h. (F) U2OS cells were pretreated with oleate or oleate plus DGATi for 20 h followed by media without oleate or DGATi but supplemented with MG132 for 4 h. Cell lysates were immunoblotted for CCTα and OSBP. (G) Quantification of CCTα expression from panel H (mean and SD of four experiments) normalized to OSBP load control. Statistical significance was determined using Student's t test. * p <0.05, ** p <0.01, **** p <0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Expressing, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Temporal relationship between CCTα, PML and DAG content of the INM. (A) U2OS cells transiently expressing a nuclear-localized GFP-tagged DAG sensor and treated with oleate or oleate and DGATi for 24 h were immunostained for PML and LMNA/C (bar, 10 µm). A region of interest (ROI) from the merged images is shown (bar, 2 µm). (B, C, and D) Cells expressing the nGFP-DAG sensor were treated with oleate and DGATi for the indicted times and immunostained for PML. The NE enrichment of nGFP-DAG (B), NE enrichment of CCTα (panel C) and percentage of cells with PML patches (panel D) were quantified. (E, F, and G) Cells expressing nGFP-DAG sensor were pretreated with DGATi and oleate for 16 h followed by media with no addition. The NE enrichment of nGFP-DAG (E), NE enrichment of CCTα (F) and percentage of cells with PML patches (G) were quantified by confocal imaging at the indicated times. Results in panels B to G are the mean and SD from five fields of cells from representative experiments. (H) U2OS cells transfected with non-targeting siRNA (siNT) or siRNA targeting LPIN1 (siLPN1) were treated with or without oleate plus DGATi for 24 h. Lysates were immunoblotted with antibodies against Lipin1, CCTα, and actin. (I and J) siNT and siLPN1 transfected cells treated with oleate plus DGATi were immunostained for PML along with BODIPY 493/503 and the percentage cells with PML patches (I) and PML patches per cell (J) was quantified. Results are from two representative experiments (22 fields of cells).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Expressing, Imaging, Transfection

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: The CCTα activators oleoyl alcohol and farnesol induce PML patches. (A) U2OS cells treated with oleate and DGATi, 100 µM farnesol or 100 µM oleyl alcohol (Oleyl-OH) for 4 h were immunostained for PML and CCTα along with BODIPY 493/503 (bar, 10 µm). The percentage of cells with PML patches and (B) CCTα NE enrichment (C) was quantified. Results are the mean and SD of five fields of cells ( n = 303) from a representative experiment. (D) U2OS cells treated with no addition (NA) or increasing concentrations of oleyl alcohol for 16 h were immunoblotted for CCTα and OSBP. (E) U2OS cells treated with NA or increasing concentrations of farnesol for 16 h were immunoblotted for CCTα and OSBP. (F) U2OS cells were treated with 100 µM oleyl alcohol for the indicated times and lysates were immunoblotted for PML, CCTα, and OSBP. (G) U2OS cells treated with NA or 100 µM farnesol for the indicated times and immunoblotted antibodies for PML, CCTα, and OSBP. Results for panels D–G were repeated three times with similar results. Statistical significance was determined using Student's t test. * p <0.05, ** p <0.01, **** p < 0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Knockout of the terminal enzymes in PC synthesis increases PML patch formation. (A) U2OS, CEPT1 KO, and CHPT1 KO cells were immunostained for PML. DNA was stained with Hoechst 33342 (bar, 5 µm). (B) The percentage of cells with PML patches was quantified from images in panel A and is the mean and SD from 12 fields of cells ( n = 911) from three experiments. (C) U2OS, CEPT1 KO, and CHPT1 KO cells transiently expressing nGFP-DAG were immunostained for CCTα and LMNA/C (bar, 10 µm). (D and E) The NE enrichment of the nGFP-DAG (D) and CCTα (E; in GFP-positive nuclei) was quantified from two independent experiments. (F) control SH-SY5Y and SH-SY5Y CEPT1 KO cells were immunostained with antibodies against PML and CCTα (bar, 10 µm). (G and H) the percentage of cells with PML patches (G) and NE enrichment of CCTα (H) were quantified from eight fields of cells ( n = 411) from a representative experiment. (I) Lysates from SH-SY5Y and SH-SY5Y CEPT1 KO cells were immunoblotted for CCTα and OSBP or CEPT1 and actin (asterisk indicates a non-specific band). (J) CCTα expression relative to OSBP was quantified from two experiments. Statistical significance determined by Student's t test; *** p <0.001, **** p <0.0001 (ns, not significant).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Knock-Out, Staining, Expressing, Control