Fig 2: Enrichment of CD14 in golgin-97-positive TGN in flotillin-depleted cells. A Raw264 cells transfected with control shRNA, variant NC1. B Cells transfected with shRNA specific against Flot2, variant No. 5. Localization of CD14 (A1, B1), golgin-97 (A2, B2) and merged images of CD14 and golgin-97 localization (A3, B3). z-Stack images of three optical sections taken in the middle of a cell are shown. Colocalized CD14 and golgin-97 appear as white spots. A4, B4 Enlarged images of golgin-97-positive TGN indicated by arrowheads in A3, B3. A5, B5 Reconstructed 3D images of golgin-97-positive TGN delineated in green with CD14 visualized in pink. Contours of the nucleus detected by Hoechst 33342 staining are shown in grey. ROI as these were used for quantitative analysis of CD14 localization in the compartment—see Table 4. C Ratio of the fluorescence intensity of CD14 in golgin-97-positive TGN calculated as the sum of CD14 voxel intensities to the total CD14 fluorescence detected in z-projections of the respective cell. n, number of cells analyzed. Results of two independent experiments are shown. Significantly different values as indicated by Student's t-test are marked

Fig 3: Internalization of exogenous fibulin-4 and its function in LOX activation, and a model for the role of fibulin-4 in LOX activation.(A) Western blot analysis of LOX secreted from WT and Fbln4−/− MEFs, with or without addition of recombinant fibulin-4 (rFBLN4) in the medium. (B to E) Internalization of FLAG-rFBLN4 by Fbln4−/− MEFs. Cell surface–bound FLAG-rFBLN4 after 4°C incubation (B) could be removed by acid wash (D). The cells were warmed at 37°C followed by acid wash, and internalized FLAG-rFBLN4 was visualized (E). (F) Quantification of internalized FLAG-rFBLN4 with or without Dynasore (100 μM) and Filipin (2.5 μg/ml) treatment. Bar graphs show mean ± SEM (n = 6 to 8). **P < 0.01 and ***P < 0.001 compared with dimethyl sulfoxide (DMSO). (G) Inhibition of rFBLN4-induced LTQ formation in LOX secreted from Fbln4−/− MEFs by Dynasore (100 μM). The ratio of the quantified intensity of lower and upper bands of LOX is shown. Bar graphs show mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with DMSO. (H) Immunofluorescent staining showing colocalization of internalized FLAG-rFBLN4 and Golgin-97, a TGN marker. Scale bars, 10 μm. (I) A proposed model for the role of fibulin-4 in LOX activation. ER, endoplasmic reticulum.

Fig 4: Intracellular localization of GnT-IVa and GnT-IVb. DKO cells transfected with a plasmid for expression of GnT-IVa or GnT-IVb were stained with anti-myc (green), antibodies for marker proteins (Golgin-97 for the Golgi, calnexin for the ER, or N-cadherin for the plasma membrane) (red), and DAPI (blue). The scale bar represents 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; DKO, double KO; ER, endoplasmic reticulum; GnT, N-acetylglucosaminyltransferase.

Fig 5: Targeting ERK/MAPK kinase attenuates golgin-97 KO-induced breast cancer malignancy and enhances paclitaxel treatment efficacy. A Wound healing assay of MDA-MB-231 and 2–9 KO cells treated with vehicle (veh.) control, SB203580 (20 μM), U0126 (20 μM), or SB203580 (20 μM) + U0126 (20 μM) for 17 h (left panel) and quantification of cell migration (right panel). The quantitative data were analyzed via one-way ANOVA. qRT‒PCR analysis of IL-1β, IL-6, and MMP1 mRNA levels in 2–9 KO cells treated with inhibitors (B) or gene-specific knockdown (C) as indicated. The data were analyzed via one-way ANOVA. Wound healing assays with gene-specific knockdown of p38 MAPK and ERK1/2 as indicated were performed on 2–9 KO cells (D) and MCF-7 cells (E). The data were analyzed via one-way ANOVA. F MDA-MB-231 and 2–9 KO cells were treated with vehicle control, SB203580, U0126, or a combination (SB203580 + U0126) for the indicated times, followed by a cell viability assay. G 2–9 KO cells were treated with vehicle control, SB203580 + U0126, PTX, or PTX + SB203580 + U0126 for the indicated times, followed by a cell viability assay. The data were analyzed via two-way ANOVA. The data are presented as the means ± SDs. *p < 0.05; **p < 0.01; ***, P < 0.001; ****, P < 0.0001. Images from each group in all the wound healing assays were acquired in three random fields of view via microscopy