internal reflection fluorescence tirf microscopy  (Nikon)

Journal: The Journal of Pathology

Article Title: C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter‐mediated doxorubicin resistance

doi: 10.1002/path.6384

Figure Lengend Snippet: Targeting C1GALT1 promotes doxorubicin‐induced apoptosis while impairing doxorubicin efflux. (A) Immunoblotting analysis for C1GALT1 expression in various OS cell lines, using GAPDH as an internal control. (B) RT‐qPCR analysis showing mRNA expression levels of C1GALT1 in SaOS‐2 and HOS cells stimulated with 50 n m doxorubicin for 48 h, using ACTB as an internal control. Immunoblotting analysis for C1GALT1 and GAPDH protein expression levels in the same treated cells. (C) Immunoblotting analysis of C1GALT1 expression in SaOS‐2 and HOS OS cell lines transfected with C1GALT1 shRNA or treated with itraconazole (2.5 μ m ), using GAPDH as an internal control. (D) Representative flow cytometry plots showing percentages of apoptotic cells in doxorubicin‐treated cells transfected with C1GALT1 shRNA or scramble control. Overlay histogram plots are shown on the right‐hand side of the panel. (E) Representative fluorescence images taken by TIRF/confocal microscope display residual intensity of doxorubicin (red) in cells over 1 h. Scale bar, 50 μm. * p < 0.05, **** p < 0.0001. Bars represent mean fluorescence intensity changes of randomly selected cells ( n = 30).

Article Snippet: Doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Transfection, shRNA, Flow Cytometry, Fluorescence, Microscopy

Journal: The Journal of Pathology

Article Title: C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter‐mediated doxorubicin resistance

doi: 10.1002/path.6384

Figure Lengend Snippet: ITZ mimics the effects of boosting doxorubicin‐induced apoptosis and reducing doxorubicin efflux. (A) Representative flow cytometry plots showing percentages of apoptotic cells in doxorubicin (50 n m ) or ITZ) (2.5 μ m )‐treated OS cells. (B and C) Representative fluorescence images taken by TIRF/confocal microscope display residual intensity of doxorubicin (red) in cells over 1 h. Scale bar, 50 μm. **** p < 0.0001. Bars represent the mean fluorescence intensity changes of randomly selected cells ( n = 30).

Article Snippet: Doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine).

Techniques: Flow Cytometry, Fluorescence, Microscopy

Journal: The Journal of Pathology

Article Title: C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter‐mediated doxorubicin resistance

doi: 10.1002/path.6384

Figure Lengend Snippet: ABCC1 identification in doxorubicin resistance and efflux and clinical correlation to C1GALT1 in OS. (A) Bar charts depict ABC transporter gene expression using the TaqMan™ Human ABC Transporter Array in doxorubicin‐selected HOS and SaOS‐2 cell lines, covering 50 genes across seven ABC transporter families. Arrows indicate the relatively high expression of ABC transporters, with GAPDH used as an internal control. (B) Representative flow cytometry plots show percentages of apoptotic cells in doxorubicin‐treated cells transfected with corresponding ABC shRNA or scramble control. (C and D) Representative fluorescence images captured by TIRF/confocal microscopy demonstrate residual intensity of doxorubicin (red) in cells over 1 h. Scale bar, 50 μm. Bars represent mean fluorescence intensity changes of randomly selected cells ( n = 30). **** p < 0.0001. (E) Representative IHC images display C1GALT1 and ABCC1 protein staining in OS tumors with and without recurrence. Brown color indicates positive staining. Scale bars, 100 μm; high‐magnification images of marked area are shown in lower right corner. (F) Kaplan–Meier curves depict overall survival and PFS according to ABCC1 expression in 29 OS patients ( p = 0.0135 and 0.0086, respectively, log‐rank test). (G) Pearson correlation analysis of C1GALT1 and ABCC1 protein expression in the same patient cohort ( r = 0.45, p < 0.05).

Article Snippet: Doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine).

Techniques: Gene Expression, Expressing, Control, Flow Cytometry, Transfection, shRNA, Fluorescence, Confocal Microscopy, Staining

Journal: The Journal of Pathology

Article Title: C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter‐mediated doxorubicin resistance

doi: 10.1002/path.6384

Figure Lengend Snippet: ABCC1 is involved in the phenotypic changes mediated by C1GALT1 in OS cells both in vitro and in vivo . (A) Fluorescence images captured using TIRF/confocal microscopy display the residual intensity of doxorubicin (red) in mock, C1GALT1‐overexpressing, and C1GALT1‐overexpressing with ABCC1‐silenced cells over 1 h. Scale bar, 50 μm. Bars represent mean fluorescence intensity changes of randomly selected cells ( n = 30). **** p < 0.0001. (B) Representative flow cytometry plots display percentages of apoptotic cells in doxorubicin‐treated samples. (C) IVIS images of representative mice taken at 2‐week intervals, highlighting the regions of interest (ROIs, marked by ovals) around tibial tumors in mice injected with either G292 mock‐transfected cells (black bar and circles), C1GALT1‐overexpressing cells (ovC1GALT1, green bar and squares), or C1GALT1 ‐overexpressing cells with ABCC1 knockdown (ovC1GALT1/shABCC1, purple bar and triangles). H&E‐stained sections of tibial tumors are shown on the right. (D) Bars represent mean bioluminescent signal intensity within tumor ROIs ( n = 6). ** p < 0.01. Tumor growth curves show tumor volume (mm 3 ). (E) Tumor volumes were measured by calipers at 0, 14, and 28 days after injection. p < 0.01 at 28 days, indicating significant differences between ovC1GALT1 group and both the mock and ovC1GALT1/shABCC1 groups.

Article Snippet: Doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine).

Techniques: In Vitro, In Vivo, Fluorescence, Confocal Microscopy, Flow Cytometry, Injection, Transfection, Knockdown, Staining

Journal: The Journal of Pathology

Article Title: C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter‐mediated doxorubicin resistance

doi: 10.1002/path.6384

Figure Lengend Snippet: O‐glycosylation changes induced by silencing C1GALT1 hinder ABCC1 cell‐surface targeting and promote its lysosomal degradation. (A) Histograms showing VVA lectin binding fluorescence intensity in mock, C1GALT1‐silenced, and itraconazole‐treated cells. HOS cells were stained with FITC‐conjugated VVA lectin or without lectin (n.c.). (B) Western blot showing proteins pulled down (PD) by VVA lectin in mock, C1GALT1‐silenced, and itraconazole‐treated cells, then immunoblotted (IB) with anti‐ABCC1. GAPDH was the internal control. (C) Western blot demonstrating ABCC1 expression in HOS cells treated with CQ and MG132 inhibitors (10 μ m each). (D) Immunofluorescence staining of mock, C1GALT1‐silenced, and itraconazole‐treated cells for ABCC1 (green) and GM130 (red). Colocalization at the Golgi apparatus is shown in yellow in the merged image of C1GALT1‐silenced samples. (E) Representative image showing tumors from paratibial injection of HOS cells in mice. Blue arrowheads indicate mock group; red arrowheads indicate the C1GALT1 knockdown group. Tumor volume, calculated as (length × width 2 )/2, is plotted over time ( n = 6). * p < 0.05, ** p < 0.01. (F) IHC images showing ABCC1 staining in tibia tumors. Brown indicates positive staining. Scale bars, 100 μm. High‐magnification images of marked areas are in lower right corner.

Article Snippet: Doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine).

Techniques: Glycoproteomics, Binding Assay, Fluorescence, Staining, Western Blot, Control, Expressing, Immunofluorescence, Injection, Knockdown