human osteogenic sarcoma cell line u 2 os u2os  (ATCC)

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: UFL1 and C53 interact with γTuRC proteins. ( A ) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1 301–389 , C53, γ-tubulin (γ-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ). ( B ) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, γ-tubulin (γ-Tb), GCP2, or GCP4. Load ( lane 1 ), immobilized Abs not incubated with cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and carriers without Abs incubated with cell extracts ( lane 4 ). ( C ) The size distribution of UFL1, C53, γ-tubulin (γ-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. ( D ) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel ( C ) were precipitated with Ab to γ-tubulin. The blots were probed with Abs to UFL1, C53, and γ-tubulin (γ-Tb). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carriers incubated with cell extracts ( lane 4 ).

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Immunoprecipitation, Negative Control, Incubation, Fractionation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Subcellular localization of UFL1 and C53. ( A ) U2OS cells expressing TagRFP-tagged proteins were fixed and stained with Ab to γ-tubulin. Localization of C53-TagRFP ( a , d ) and γ-tubulin ( b , e ). Superposition of images ( c , f ) C53-TagRFP, red; γ-tubulin, green; DAPI, blue). Localization of UFL1-TagRFP ( g ) and γ-tubulin ( h ). Superposition of images ( i , UFL1-TagRFP, red; γ-tubulin, green; DAPI, blue). Arrows indicate the same positions. Fixation D/F/M. Scale bar, 20 μm ( a – c , g – i ), 5 μm ( d – f ). ( B ) Association of proteins with purified centrosomes. Centrosomes enriched by centrifugation onto a Ficoll cushion were further purified by sucrose gradient centrifugation. The gradient was fractionated from the bottom. Individual fractions are indicated on the top, sucrose density in the fractions is shown at the bottom. Blots were probed with Abs to pericentrin, CDK5RAP2, ODF2, γ-tubulin (γ-Tb), C53, UFL1, PKCα, histone H1.4, and calnexin.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Staining, Purification, Centrifugation, Gradient Centrifugation

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Deletion of UFL1 or C53 induces UPR and expansion of ER. ( A ) Immunoblot analysis of calnexin and PDI in whole-cell lysates of U2OS cells lacking UFL1 or C53. GAPDH served as a loading control. Densitometric quantification of immunoblots is shown on the right. Relative intensities of corresponding proteins normalized to control cells and the amount of GAPDH in individual samples. Values indicate mean ± SD ( n = 4 for calnexin; n = 3 for PDI). ( B ) Immunofluorescence microscopy. ( a – d ) Control U2OS cells, ( e – h ) UFL1-deficient cells (UFL1_KO) and ( i – l ) C53-deficient cells (C53_KO). Cells were fixed and double-labeled for calnexin ( a , e , i ) and β-tubulin ( b , f , j ; Microtubules). Higher magnification views of the regions delimited by rectangles are shown on the right of images from control ( c , d ), UFL1_KO ( g , h ), and C53_KO ( k , l ) cells. The images ( a , e , i ) and ( c , g , k ) were collected and processed in the same manner. Fixation F/Tx. Scale bars, 20 μm ( j ), and 5 µm ( l ). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. ER area coefficients (area occupied by ER/area free of ER) were calculated from fluorescence intensities for calnexin. The distributions of ER area coefficients (arbitrary units [AU]) are shown as box plots (four independent experiments, ≥16 cells counted for each experimental condition). Box plot of area coefficients in UFL1_KO ( n = 111) and C53_KO cells ( n = 127) relative to control cells ( n = 149). The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( A , C ) One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine statistical significance. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Labeling, Staining, Fluorescence

Journal: Cells

Article Title: C53 Interacting with UFM1-Protein Ligase 1 Regulates Microtubule Nucleation in Response to ER Stress

doi: 10.3390/cells11030555

Figure Lengend Snippet: Generation of ER stress by tunicamycin increases centrosomal microtubule nucleation. U2OS cells were treated with 1 µg/mL tunicamycin (+Tunicam.) or DMSO carrier (Control) for 24 h. ( A ). Effect of tunicamycin on ER expansion and subcellular distribution of calnexin, PDI, and ER stress-induced transcription factor DDIT3. ( a , b ) Visualization of ER in live cells by ER-Tracker. ( c – h ) Fixed cells stained for calnexin ( c , d ), PDI ( e , f ), and DDIT3 ( g , h ). Insets represent an enlargement of the boxed area. Fixation F/Tx. Pairs of images ( a , b ), ( c , d ), ( e , f ), and ( g , h ) were collected and processed in the same manner. Scale bars, 20 μm ( h ) and 5 µm (inset in f ). ( B ) ER area quantification in live cells stained with ER-Tracker. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥23 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 81) relative to control cells ( n = 108). ( C ) ER area quantification in fixed cells stained with Ab to calnexin. The distributions of ER area coefficients (area occupied by ER/area free of ER; arbitrary units [AU]) are shown as box plots (three independent experiments, ≥19 cells counted for each experimental condition). Box plot of ER area coefficients in tunicamycin-treated cells ( n = 80) relative to control cells ( n = 65). ( D , E ) The distributions of α-tubulin or γ-tubulin fluorescence intensities (arbitrary units [AU]) in 2-μm ROIs at 3.0 min of microtubule regrowth in control and tunicamycin-treated cells are shown as box plots (four independent experiments, >27 cells counted for each experimental condition). Box plot of α-tubulin ( D ) and γ-tubulin ( E ) fluorescence intensities in tunicamycin-treated cells ( n = 234) relative to control cells ( n = 181). ( F ) Time-lapse imaging of control and tunicamycin-treated cells expressing EB3-mNeonGreen. Still images of EB3 (Single frame) and tracks of EB3 comets over 10 s (10 frames project.). Scale bar, 5 µm. ( G ) Microtubule nucleation rate (EB3 comets/min) in tunicamycin-treated cells relative to control cells. Three independent experiments (at least 9 cells counted in each experiment). Control ( n = 31), tunicamycin-treated cells ( n = 31). The bold and thin lines within the dot plot represent mean ± SD. ( B – E ) The bold and thin lines within the box represent mean and median (the 50th percentile), respectively. The bottom and top of the box represent the 25th and 75th percentiles. Whiskers below and above the box indicate the 10th and 90th percentiles. ( B – E , G ) Two-tailed, unpaired Student’s t -test was performed to determine statistical significance. **** p < 0.0001.

Article Snippet: Human osteogenic sarcoma cell line U-2 OS (U2OS) (Catalog No. ATCC, HTB-96), human glioblastoma cell line T98G (Catalog No. ATCC, CRL-1690), and human cervix adenocarcinoma HeLa S3 (Catalog. No. ATCC, CCL-2.2) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Staining, Fluorescence, Imaging, Expressing, Two Tailed Test