dermal fibroblasts hdf α  (Cell Applications Inc)

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: Characterization of autophagy machinery required for STING-dependent LC3B lipidation. (a) WT, STING KO, and STING KO HeLa cells stably expressing GFP-STING were treated with 15 µg/ml or the indicated concentrations of cGAMP for 8 h in normal DMEM. (b) WT and FIP200 KO HeLa cells were treated with 15 µg/ml cGAMP for 8 h in normal DMEM (all the following experiments were treated the same unless specified). (c) WT HeLa cells were treated with cGAMP alone or with cGAMP and wortmannin (200 nM) or VPS34-IN1 (300 nM) for 8 h. (d) Primary HDFs were treated with 60 µg/ml cGAMP and wortmannin or VPS34-IN1 at the same concentrations as in c for 4 h. (e) WT, WIPI2 KO, and WIPI 4KO HeLa cells stably expressing GFP-STING were treated with 60 µg/ml cGAMP. (f) Representative Airyscan-processed confocal imaging of WT HeLa cells stably expressing GFP-LC3B (green) and mCh-STING (red) were treated with 60 µg/ml cGAMP or starvation media (HBSS with Ca2+ and Mg2+) for 8 h before immunofluorescent labeling of endogenous WIPI2 (magenta). Scale bar, 10 µm, 2 µm (inset). (g and h) WT, ATG16L1 KO, and NDP52 (N), optineurin (O), and TAX1BP1 (Tx) triple KO (N/O/Tx TKO) HeLa cells stably expressing GFP-STING were treated with 60 µg/ml cGAMP. Each Western blotting experiment was independently replicated three times. Starv, starvation; Wort, wortmannin.

Article Snippet: Cell culture, reagents, and antibodies HeLa cells (CCL-2) and primary neonatal HDFs (PCS-201-010) were purchased from American Type Culture Collection.

Techniques: Stable Transfection, Expressing, Imaging, Labeling, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: Experiments supplementary to Figs. 1 and ​and22. (a and b) Primary HDFs (a) and primary MEFs (b) were treated with 60 μg/ml cGAMP, and cell lysates were collected at the indicated time points. (c) WT HeLa cells were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 and with or without wortmannin (200 nM) or VPS34-IN1 (300 nM) for 4 h. All the following experiments were treated the same unless specified. (d) MEFs were treated with 60 μg/ml cGAMP alone and with wortmannin for 2 h. All the following experiments using MEFs use the same conditions. (e and f) WT, WIPI2 KO (e), and WIPI 4KO (f) HeLa cells stably expressing GFP-STING were incubated in starvation media alone and with BafA1 for 4 h. (g) MEFs were incubated in starvation media alone and with BafA1 for 4 h. (h and i) MEFs were incubated with cGAMP alone and with BafA1 (h) or with BafA1 and wortmannin (i). (j) WT and FIP200 KO HeLa cells were incubated with 15 µg/ml cGAMP alone and with 20 μM chloroquine (CQ) for 8 h. (k) Representative Airyscan-processed confocal imaging of FIP200 KO HeLa cells with stable expression of RFP-LC3B and GFP-STING treated with cGAMP (60 µg/ml) for 8 h, fixed, and immunostained for endogenous LAMP2. Scale bar, 10 µm. Western blotting experiments were independently replicated two (b, c, e, and g) or three (a, d, f, h, i, and j) times. Starv, starvation; Wort, wortmannin.

Article Snippet: Cell culture, reagents, and antibodies HeLa cells (CCL-2) and primary neonatal HDFs (PCS-201-010) were purchased from American Type Culture Collection.

Techniques: Incubation, Stable Transfection, Expressing, Imaging, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: cGAMP induces LC3B lipidation and redistribution of the V1 complex in the perinuclear region that is sensitive to pharmacological inhibition of the V-ATPase. (a and b) WT HeLa cells and primary HDFs were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 for 8 h (a) and 4 h (b). (c) WT HeLa cells and WT HeLa cells with stable expression of GFP-STING were incubated with 15 µg/ml (WT) or 60 µg/ml (WT GFP-STING) cGAMP alone and with 100 nM BafA1 for 8 h. All the following experiments were treated the same unless specified. (d) HDFs were incubated with 60 µg/ml cGAMP alone and with BafA1 for 4 h. (e) FIP200 KO HeLa cells were incubated with cGAMP alone and with BafA1. (f) HDFs were incubated with 60 µg/ml cGAMP alone, with BafA1 and 200 nM wortmannin (Wort) alone, and with cGAMP, BafA1, and Wort for 4 h. (g) Representative Airyscan-processed confocal imaging of FIP200 KO HeLa cells with stable expression of RFP-LC3B and GFP-STING treated with cGAMP (60 µg/ml) alone and with BafA1, fixed, and immunostained for endogenous ATP6V1D. Scale bar, 10 µm, 2 µm (inset). Each Western blotting experiment was independently replicated three times. Starv, starvation.

Article Snippet: Cell culture, reagents, and antibodies HeLa cells (CCL-2) and primary neonatal HDFs (PCS-201-010) were purchased from American Type Culture Collection.

Techniques: Inhibition, Incubation, Expressing, Imaging, Western Blot

Journal: The Journal of Cell Biology

Article Title: STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain

doi: 10.1083/jcb.202009128

Figure Lengend Snippet: Experiments supplementary to Figs. 3 and ​and55. (a) WT and ATG16L KO HeLa cells with stable expression of GFP-STING and BFP-ATG16L-α, BFP-ATG16L-β, or ATG16L-ΔWD40 were incubated in starvation media (HBSS with Ca2+ and Mg2+) alone and with 100 nM BafA1 for 8 h. All the following experiments use the same conditions unless specified. (b and c) WT HeLa cells and WT HeLa cells with stable expression of BFP-SopF were incubated in starvation media alone and with BafA1 for 4 h (b) and treated with 15 µg/ml cGAMP for 8 h (c). Western blotting experiments were independently replicated three times. (d and e) Representative Airyscan confocal imaging of primary HDFs and HDFs with stable expression of BFP-SopF were treated with 60 μg/ml cGAMP for 4 h, fixed, and immunostained for endogenous ATP6V1D (d) or LC3 (e). Scale bar, 10 µm.

Article Snippet: Cell culture, reagents, and antibodies HeLa cells (CCL-2) and primary neonatal HDFs (PCS-201-010) were purchased from American Type Culture Collection.

Techniques: Expressing, Incubation, Western Blot, Imaging

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Induction of Apoptosis in A375 Malignant Melanoma Cells by Sutherlandia frutescens

doi: 10.1155/2016/4921067

Figure Lengend Snippet: The effect of S. frutescens extract on the viability of (a) A375 melanoma, (b) Colo-800 melanoma, (c) HDF α fibroblast, and (d) Hek 293 cells after 24, 48, and 72 h of treatment. The viability was assessed using the alamarBlue assay and expressed as percentage relative to the control-treated cells (0 mg/mL). Error bars represent the SEM ( n ≥ 3) and ∗ indicates a significant difference from 0 mg/mL ( P < 0.05).

Article Snippet: The human primary dermal fibroblast cell line (HDF α ) was purchased from ScienCell, CA, USA.

Techniques: Alamar Blue Assay, Control