Journal: The Journal of Biological Chemistry
Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner
doi: 10.1016/j.jbc.2024.107761
Figure Lengend Snippet: LAT1 promotes Golgi unlinking along with Aurora A recruitment to the centrosomes . A , HeLa S3 cells were fixed with formaldehyde and stained for LAT1 ( green ) and TGN46 ( red ) or calnexin ( red ). Representative images are shown. Scale bar, 10 μm. B–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 19 h and washed with PBS(−). B and C , the cells were cultured for a further 9 h or 10 h in siControl or siLAT1 cells, respectively, to analyze the Golgi structure in prophase. The cells were fixed with MeOH and stained for TGN46 ( gray or green ) and DNA ( red ). B , representative z-stack images are shown, and Golgi objects based on TGN46 staining are numbered (see “ ”). Scale bar, 10 μm. C , the number of Golgi object within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 40). Statistical analysis was performed using Welch’s ANOVA ( F = 319, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , The cells were cultured for a further 9.5 h or 10.5 h in siControl or siLAT1 cells, respectively, to analyze Aurora A recruitment in prometaphase cells. Then, the cells were fixed with MeOH and stained for Aurora A ( green ), phospho-Aurora A (pT288) ( red ), and DNA ( cyan ). D , representative z-stack images are shown. Scale bar, 10 μm. E , the fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured (see “ ”), and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Statistical analysis was performed using Welch’s ANOVA ( F = 99.8, p = 0.000 in the left panel ; F = 88.6, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). F and G , HeLa S3 cells were treated with DMSO or 30 μM SP600125 for 2 h. F , the cells were fixed with MeOH and stained for TGN46 and DNA. The number of Golgi objects within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). G , the cells were fixed with MeOH and stained for Aurora A, phospho-Aurora A (pT288), and DNA. The fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured, and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Asterisks indicate significant differences [Student’s t test in panel ( F and G ), ∗∗ p < 0.01; ∗∗∗ p < 0.001].
Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.
Techniques: Staining, Transfection, Control, Cell Culture, Fluorescence
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![LAT1 promotes Golgi unlinking along with Aurora A recruitment to the centrosomes . A , HeLa S3 cells were fixed with formaldehyde and stained for LAT1 ( green ) and TGN46 ( red ) or calnexin ( red ). Representative images are shown. Scale bar, 10 μm. B–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 19 h and washed with PBS(−). B and C , the cells were cultured for a further 9 h or 10 h in siControl or siLAT1 cells, respectively, to analyze the Golgi structure in prophase. The cells were fixed with MeOH and stained for TGN46 ( gray or green ) and DNA ( red ). B , representative z-stack images are shown, and Golgi objects based on TGN46 staining are numbered (see “ ”). Scale bar, 10 μm. C , the number of Golgi object within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 40). Statistical analysis was performed using Welch’s ANOVA ( F = 319, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , The cells were cultured for a further 9.5 h or 10.5 h in siControl or siLAT1 cells, respectively, to analyze Aurora A recruitment in prometaphase cells. Then, the cells were fixed with MeOH and stained for Aurora A ( green ), phospho-Aurora A <t>(pT288)</t> ( red ), and DNA ( cyan ). D , representative z-stack images are shown. Scale bar, 10 μm. E , the fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured (see “ ”), and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Statistical analysis was performed using Welch’s ANOVA ( F = 99.8, p = 0.000 in the left panel ; F = 88.6, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). F and G , HeLa S3 cells were treated with DMSO or 30 μM SP600125 for 2 h. F , the cells were fixed with MeOH and stained for TGN46 and DNA. The number of Golgi objects within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). G , the cells were fixed with MeOH and stained for Aurora A, phospho-Aurora A (pT288), and DNA. The fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured, and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Asterisks indicate significant differences [Student’s t test in panel ( F and G ), ∗∗ p < 0.01; ∗∗∗ p < 0.001].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0712/pmc11490712/pmc11490712__gr6.jpg)
