Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) Western blot of protein extracts from MM231 SEC16A-iKD cells with or without DOX treatment, blotted with anti-SEC16A and anti-Vinculin used as loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to loading control levels for each experimental condition (B) Confocal images of MM231 SEC16A-iKD cells untreated or treated with DOX, stained with DAPI (blue) and anti-SEC16A (grey). Scale bars indicate 15 µm. (C) (Left panels) Phase contrast (upper panels) or confocal images (lower panels) of MM231 SEC16A-iKD #1 spheroids in Matrigel untreated or treated with DOX. (Lower panels) cells were stained with DAPI (blue) and Phalloidin (magenta). Scale bars, 50 μm. (Right panel) Quantification of the invasiveness of MM231 SEC16A-iKD #1 spheroids untreated or treated with DOX. Statistical significance was calculated using unpaired t-test ****p<0.0001. (D) Western blot on protein extracts from TNBC PDXOs SEC16A-iKD untreated or treated with DOX, cultured in invasive conditions and blotted with anti-SEC16A and anti-HSC70 used as a loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to HSC70 levels for each experimental condition. (E) (Left panels) DIC images of TNBC PDXOs SEC16A-iKD cultured in invasive conditions, untreated or treated without DOX. (Right panel) Quantification of the invasiveness of TNBC PDXOs SEC16A-iKD cultured in invasive condition, untreated or treated without DOX. Data are from three biological replicates and include more than 30 PDXOs per condition and per replicate. Statistical significance was calculated using unpaired t-test. ∗∗∗∗p<0.0001. (F) (Left panels) Confocal images of MM231 SEC16A-iKD cells untreated or treated with DOX, stained with DAPI (blue), Phalloidin (magenta), and Paxillin (grey). Scale bars indicate 15 µm. (Right panel) Quantification of the number of FAs per MM231 SEC16A-iKD cells treated with DOX (n=71) or without DOX (n=64). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. Statistical significance was calculated using unpaired t-test ****p<0.0001. (G) (Left panels) Confocal images of MM231 SEC16A-iKD cells untreated or treated without DOX, stained with DAPI (blue), Phalloidin (magenta) and anti-Rab4 (grey). Scale bars indicate 15 µm. (Right panel) Quantification of the number of Rab4 vesicles in MM231 SEC16A-iKD cells treated with DOX (n=336) or without DOX (n=496). Data are from three biological replicates, with at least three fields analysed per condition and per replicate. Each dot represents one field. Scale bars indicate 15 µm. Error bars indicate standard deviation. Statistical significance was calculated using unpaired t-test. ****p<0.0001.

Article Snippet: MDA-MB-231 cells (MM231) were provided by Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control, Staining, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) MS/MS spectrum of the SEC16A phosphopeptide FTGS[pho]FDDDPDPHRDPYGEEVDR (4+), showing phosphorylation at Ser4, corresponding to Ser1327 in the full-length protein. Labelled peaks correspond to the main fragment ions observed in the spectrum. Identified b- and y-series ions, together with the precursor ion, confirm the peptide sequence. The b- ions identified support the presence and location of the phosphate group. The site was identified with a localization probability of 0.92 and a PEP score of 0.0085. (B) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (cyan blue), or GFP-SEC16A-S1327A (cyan blue) or GFP-SEC16A-S1327D (cyan blue), treated with DOX and stained with DAPI (blue) and anti-SEC16A (magenta). Scale bars represent 15μm. (C) (Left panels) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT, or GFP-SEC16A-S1327A or GFP-SEC16A-S1327D, treated with DOX and stained with DAPI (blue), anti-SEC16A (magenta) and anti-Rab4 (grey). Scale bars represent 15μm. (Right panel) Quantification of the number of Rab4 vesicles in MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (n=76) or GFP-SEC16A-S1327A (n=93) or GFP-SEC16A-S1327D (n=85). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. ****p<0.0001 between cells expressing WT SEC16A and S1327A SEC16A. ***p=0.0004 between cells expressing S1327A SEC16A and S1327D SEC16A. ns indicates non-significant between cells reconstituted with WT SEC16A and S1327D SEC16A (p=0.9291). Statistical significance was calculated using one-way ANOVA. (D) (Left panels) Confocal images of MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (cyan blue), or GFP-SEC16A-S1327A (cyan blue) or GFP-SEC16A-S1327D (cyan blue), treated with DOX and stained with DAPI (blue) and anti-Paxillin (grey). Scale bars represent 15 μm. (Right panel) Quantification of the number of FAs in MM231 SEC16A iKD cells expressing GFP-SEC16A-WT (n=65) or GFP-SEC16A-S1327A (n=78) or GFP-SEC16A-S1327D (n=59). Data are from three biological replicates with at least three fields analysed per condition and per replicate. Each dot represents one cell. ****p<0.0001 between cells expressing WT SEC16A and S1327A SEC16A and between cells expressing S1327A SEC16A and S1327D SEC16A. ns indicates non-significant (p=0.4673) between cells expressing WT SEC16A and S1327D SEC16A. Statistical significance was calculated using one-way ANOVA. (E) (Left panel) Aminoacid sequence of the region comprising amino-acids 1307-1378. Phosphorylatable residues are highlighted: serine (magenta), threonine (green), or tyrosine (blue). Purple arrow indicates Ser1327. (Right panel) Ratios of serine (magenta), threonine (green), or tyrosine (blue) residues in the region comprising amino-acids 1307-1378.

Article Snippet: MDA-MB-231 cells (MM231) were provided by Cell Lines Service (Eppelheim, Germany).

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Sequencing, Expressing, Staining

Journal: bioRxiv

Article Title: Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as key event associated with FER-mediated invasion

doi: 10.64898/2025.12.18.695070

Figure Lengend Snippet: (A) Western blot on protein extracts from MM231 FER-iKD cells untreated or treated with DOX, blotted with anti-SEC16A or anti-HSC70 used as a loading control. Numbers on the bottom indicate the fold-changes in SEC16A levels normalised to HSC70 levels for each experimental condition. (B) (Left panels) Confocal images of MM231 FER-iKD cells untreated or treated with DOX, stained with DAPI (blue) and anti-SEC16A (grey). Scale bars represent 15 µm. (Right panel) Quantification of the shortest distance between SEC16A signals and the nucleus in MM231 FER-iKD cells treated with DOX (n=113) or without DOX (n=188). Data are from three biological replicates and include at least three fields analysed per condition and per replicate. Each dot represents one field. Error bars indicate standard deviation. Statistical significance was calculated using unpaired t-test. ****p<0.0001. (C and D) Multispectral immunofluorescence imaging of a TNBC PDXO #1 tumour xenograft invading on CAM. Scale bars represent 30 µm. Tissue samples were stained with anti-SEC16A (magenta), anti-FER (yellow), anti-Rab4 (orange), anti-Integrin β1 (cyan blue), anti-E-cad (red) and DAPI (blue). (D) Inset images highlight the levels of FER and SEC16A in leader invading cells. White arrows indicate leader cells. Scale bars represent 10 µm. (E) IHC images of TNBC clinical specimens, stained for SEC16A and FER. Scale bars: 50 µm. (F) Correlation between SEC16A and FER expression in Luminal A, Luminal B, HER2 and TNBC/basal breast cancer. Statistical significance estimated by χ 2-test. P-values are indicated. Statistically significant p-values are indicated in bold. (F) Model by which phosphorylated SEC16A acts, downstream of FER, to coordinate the formation of Rab4-positive endosomes and focal adhesions and sustain invasion in TNBC. In TNBC, high levels of FER correlate with low SEC16A levels. Still, a certain amount of SEC16A protein is necessary to sustain FER-dependent endosomal recycling and invasion of integrins in TNBC.

Article Snippet: MDA-MB-231 cells (MM231) were provided by Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control, Staining, Standard Deviation, Immunofluorescence, Imaging, Expressing