Journal: The Journal of Cell Biology

Article Title: Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

doi:

Figure Lengend Snippet: Loss of endogenous Tctex-1 protein by ectopic expression of RP3. MDCK cells were transiently transfected with expression vectors encoding FLAG-RP3 (A) or GFP (B) and then allowed to polarize on Transwell filters. After fixation, cells were immunolabeled with either anti-FLAG and Tctex-1 antibodies (A) or Tctex-1 antibody alone (B). Bar, 20 μm.

Article Snippet: For transient transfections, cells were transfected at ∼50% confluence on 10-cm dishes and then plated at high density (∼4 × 10 5 cells/cm 2 ) onto 12-mm Transwell filters (Corning-Costar).

Techniques: Expressing, Transfection, Immunolabeling

Journal: The Journal of Cell Biology

Article Title: Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

doi:

Figure Lengend Snippet: Specific inhibition of rhodopsin's apical polarity in MDCK cells by Tctex-1 downregulation. (A–C) MDCK cells were transiently transfected with FLAG-RP3 (A), GFP (B), or FLAG–Tctex-1 (C) and then allowed to form polarized monolayers. Monolayers were then infected with rhodopsin adenovirus for 24 h. Cells were double-labeled with Tctex-1 rabbit antibody (green) and rhodopsin mAb (red) in A. Cells were singly labeled with rhodopsin mAb (red; GFP is green) in B. In these experiments, FLAG-RP3–transfected cells are identifiable by their loss of endogenous Tctex-1 expression. In C, cells were doubled labeled with antirhodopsin rabbit antibody (red) and anti-FLAG mAb (green). Confocal sections in the x-z plane is shown, with the apical surface toward the top of the frame. (D) T23/FLAG-RP3 cells were left uninduced (top) or were induced to express FLAG-RP3 (bottom) before infection with rhodopsin adenovirus. The distribution of rhodopsin and Tctex-1 expression was determined by double-labeling for rhodopsin (red) and Tctex-1 (green). Tctex-1 was undetectable in almost all induced cells. (E) CD7-Rho39 was overexpressed in uninduced (top) or induced (bottom) T23/FLAG-RP3 monolayers via an adenovirus particle transfection method. The distribution of CD7-Rho39 in relationship to the expression of Tctex-1 was examined by double labeling of CD7 mAb (red) and Tctex-1 rabbit antiserum (green). (F) The steady-state distribution of adenovirally expressed rhodopsin in uninduced (left) or induced (right) T23/FLAG-RP3 cells was determined by domain-selective surface biotinylation followed by immunoprecipitation of rhodopsin. Immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose. Biotinylated rhodopsin was quantitated by chemifluorescence. In three independent trials using triplicate measurements on a single cell line, rhodopsin was highly polarized to the apical surface in uninduced cells. FLAG-RP3 induction resulted in a significant loss of steady-state rhodopsin polarity. Note that rhodopsin is prone to forming higher-order aggregates on SDS-PAGE; signals from multimers were also taken into consideration in quantitation. Rhodopsin expressed in tissue culture is heterogeneously N-glycosylated , accounting for the broadened bands. No signal was detected from cells infected with a control recombinant adenovirus (data not shown), indicating that all of the signal seen on this blot represents rhodopsin. (G) Uninduced or induced polarized T23/FLAG-RP3 cells grown on Transwell filters were infected with rhodopsin-encoding adenovirus before pulse labeling with [ 35 S]cysteine/methionine. At 1 or 2 h of chase, cells underwent domain-selective surface biotinylation. Biotinylated rhodopsin was recovered by rhodopsin immunoprecipitation followed by precipitation with streptavidin-agarose. Samples were then digested with N-glycosidase F to reduce its aggregation on SDS-PAGE. Biotinylated, radiolabeled rhodopsin was separated by SDS-PAGE and quantitated by phosphorimaging. The percentage of total surface rhodopsin present on the apical surface at each chase timepoint is plotted. Asterisk, P < 0.05 between uninduced and induced cells by two-sample t test. Bar, 10 μm.

Article Snippet: For transient transfections, cells were transfected at ∼50% confluence on 10-cm dishes and then plated at high density (∼4 × 10 5 cells/cm 2 ) onto 12-mm Transwell filters (Corning-Costar).

Techniques: Inhibition, Transfection, Infection, Labeling, Expressing, Immunoprecipitation, SDS Page, Quantitation Assay, Control, Recombinant

Journal: Scientific Reports

Article Title: Prognostic factors and Doxorubicin involved in malignant progression of meningioma

doi: 10.1038/s41598-023-28996-0

Figure Lengend Snippet: Doxorubicin inhibits proliferation, migration and invasion of meningioma. ( A ) Correlation of compound with differently expressed mRNA: Heatmap showing compounds (top) from the CMap shares mechanisms of action and the mechanisms of action was sorted by descending number of compounds (right); “ComplexHeatmap” R package (version 3.15) was used to graph the result. ( B ) The molecular structure of Doxorubicin; ( C ) The dose-curve of Doxorubicin in IOMM-Lee cell line; ( D ) CCK-8 assay of the cell proliferation ability in IOMM-Lee cells upon Doxorubicin or DMSO treatment; ( E ) EdU assay of the cell proliferation ability in IOMM-Lee cells upon Doxorubicin or DMSO treatment. Scale bar: 125 μm; ( F ) Statistical analysis of the EdU-positive cell ratio; ( G ) Wound healing assay showed delayed closure of the wound gap upon Doxorubicin treatment. ( H ) Statistical analysis of the cell migration in the scratch wound healing assays. ( I ) Transwell invasion and migration assay of IOMM-Lee cells upon Doxorubicin or DMSO treatment. ( J ) Statistical analysis of the cell numbers passing through the transwell chamber. All data were the means ± SD. Results triplicate and repeated at least three times. Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Migration or invasion assays were performed using 24-well plates inserted by 8-μm pore size transwell filter insert (Costar 3422 [Corning, Corning, New York, USA]) with or without pre-coated diluted Matrigel (1:8) (Becton Dickinson and Co., Franklin Lakes, NJ, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Wound Healing Assay