Figure 3.
ORP9 is recruited to damage sites during PM repair. (A) Schematic of the photodamage assay. (B) Representative confocal images of GFP accumulation at the site of UV damage in HeLa cells transfected with GFP empty vector (EV), GFP-ORP9L, or GFP-ORP9S for 24 h. Cells were imaged in phenol red-free culture medium supplemented with FBS and PI (Propidium Iodide, 50 μg/ml). UV damage occurred 30 s after imaging as indicated by the red arrow with an area of 5 × 5 pixels. Scale bars = 10 µm for all images. (C) Relative fold change of GFP accumulation at the site of photodamage as in B. Data shown are the mean ± 95% CI, n = 10–15 cells. (D) Representative confocal images of cells transfected with GFP-ORP9L HH/AA, GFP-ORP9L L393D, GFP-ORP9S HH/AA, or GFP-ORP9S L228D for 24 h, Images are shown before and after UV damage, targeting an area of 2 × 2 pixels. Scale bars = 10 µm for all images. (E and F) Relative fold change of GFP accumulation at the site of photodamage (2 × 2 pixels) for ORP9L (E) and ORP9S (F) and their mutants as in D. Data shown are the mean ± SEM, n = 7–21 cells. (G) Representative confocal images of time-lapse observation of mNeonGreen accumulation in mNeonGreen empty vector (EV) transfected or mNeonGreen-ORP9 KI cells. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (H) Relative fold change of mNeonGreen accumulation at the damage site (5 × 5 pixels) with photodamage as in G. Data shown are the mean ± SEM, n = 21–35 cells. (I) Representative time-lapse confocal images of GFP accumulation in HeLa cells transfected with GFP empty vector (EV) or GFP-OSBP-PH for 24 h. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (J) Relative fold change of GFP accumulation at the damage site as in I. Data shown are the mean ± 95% CI, n = 21 cells. (K) Representative time-lapse confocal images of CFP accumulation in HeLa cells transfected with CFP empty vector (EV) or CFP-PI4K2a or CFP-PI4K2b for 24 h. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (L) Relative fold change of CFP accumulation at the damage site (5 × 5 pixels) with photodamage as in K. Data shown are the mean ± 95% CI, n = 32–35 cells.

ORP9 is recruited to damage sites during PM repair. (A) Schematic of the photodamage assay. (B) Representative confocal images of GFP accumulation at the site of UV damage in HeLa cells transfected with GFP empty vector (EV), GFP-ORP9L, or GFP-ORP9S for 24 h. Cells were imaged in phenol red-free culture medium supplemented with FBS and PI (Propidium Iodide, 50 μg/ml). UV damage occurred 30 s after imaging as indicated by the red arrow with an area of 5 × 5 pixels. Scale bars = 10 µm for all images. (C) Relative fold change of GFP accumulation at the site of photodamage as in B. Data shown are the mean ± 95% CI, n = 10–15 cells. (D) Representative confocal images of cells transfected with GFP-ORP9L HH/AA, GFP-ORP9L L393D, GFP-ORP9S HH/AA, or GFP-ORP9S L228D for 24 h, Images are shown before and after UV damage, targeting an area of 2 × 2 pixels. Scale bars = 10 µm for all images. (E and F) Relative fold change of GFP accumulation at the site of photodamage (2 × 2 pixels) for ORP9L (E) and ORP9S (F) and their mutants as in D. Data shown are the mean ± SEM, n = 7–21 cells. (G) Representative confocal images of time-lapse observation of mNeonGreen accumulation in mNeonGreen empty vector (EV) transfected or mNeonGreen-ORP9 KI cells. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (H) Relative fold change of mNeonGreen accumulation at the damage site (5 × 5 pixels) with photodamage as in G. Data shown are the mean ± SEM, n = 21–35 cells. (I) Representative time-lapse confocal images of GFP accumulation in HeLa cells transfected with GFP empty vector (EV) or GFP-OSBP-PH for 24 h. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (J) Relative fold change of GFP accumulation at the damage site as in I. Data shown are the mean ± 95% CI, n = 21 cells. (K) Representative time-lapse confocal images of CFP accumulation in HeLa cells transfected with CFP empty vector (EV) or CFP-PI4K2a or CFP-PI4K2b for 24 h. UV damage occurred 30 s after imaging as indicated by the red arrow with a damage area of 5 × 5 pixels. Scale bars = 10 µm for all images. (L) Relative fold change of CFP accumulation at the damage site (5 × 5 pixels) with photodamage as in K. Data shown are the mean ± 95% CI, n = 32–35 cells.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal