Characterization of CRISPR/Cas9 knock-in HeLa cells and endogenous localization of MOSPD2. (A) Live confocal images of HeLa cells expressing mClover3-MOSPD2 (green) at the endogenous level and transfected with control siRNAs (siCtrl) and siRNAs targeting MOSPD2 (siMOSPD2) to confirm the specificity of mClover3 signal. Scale bars: 10 µm. (B) Western blot (WB) analysis of MOSPD2 expression in WT (HeLa WT) and mClover3-MOSPD2 knock-in (HeLa KI) HeLa cells transfected with control siRNAs (siCtrl) and siRNAs targeting MOSPD2 (siMOSPD2). mClover3 was detected using anti-GFP antibodies. (C) Western blot analysis of MOSPD2 expression in HeLa, Huh-7, and 501-MEL cells. (D and E) Colocalization of endogenous MOSPD2 (labeled with anti-MOSPD2, in green) and LDs (labeled with LipidTOX, in magenta) in Huh-7 cells (D) or 501-MEL (E) after OA treatment. Panels on the right show the background signal of anti-MOSPD2 antibodies in cells transfected with siRNAs targeting MOSPD2 (siMOSPD2). Subpanels show the LD staining. Left: Subpanels on the bottom are higher magnification images of the area outlined. The overlay panel shows merged channels. Scale bars: 10 µm (insets, 2 µm). (A, D, and E) Images were acquired on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). Source data are available for this figure: SourceData FS3.