Journal: iScience
Article Title: The microcephaly-associated protein YIPF5 differentially regulates ER export
doi: 10.1016/j.isci.2026.114791
Figure Lengend Snippet: YIPF5 regulates the secretome composition (A) Schematic representation of the experimental workflow for analyzing the glycoprotein secretome. MCF10A cells were metabolically labeled with the clickable sugar analog ManNAz, followed by a biotin-azide click reaction to tag glycoproteins ( n = 4). Secreted glycoproteins were concentrated from the culture medium, selectively purified, and identified using mass spectrometry. The image was created with BioRender.com . (B) Quantitative comparison of protein abundance in the secretome versus the total proteome of MCF10A cells. Proteins with secretion changes reflecting a similar change in total expression (“explained by proteome”) (q value ≤ 0.05) are shown in green. Proteins with increased or decreased secretion independent of total proteome changes are highlighted in red and blue, respectively. Gray (not affected) represents unchanged proteins. (C) Analysis of protein abundance changes in the secretome of YIPF5 KO versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 KO secretome are shown. Proteins with secretion changes explained by total proteome abundance are shown in green, while unaffected proteins are in gray. The vertical axis represents –log10(q value), and the horizontal axis represents log2(fold change). (D and E) Heatmap depicting proteins showing increased secretion in the YIPF5 KO secretome with a gene ontology (GO) annotation for the ER (GO: 0007029) and the Golgi apparatus (GO: 0005794) (D) or that contain a KDEL-ER retrieval sequence (E). (F) MCF10A WT and YIPF5 KO cells stably expressing the inducible ER-Ca 2+ sensor GCampER were analyzed using live fluorescence microscopy at identical settings. (G) Mean ± SD fluorescence intensity of GCampER was quantified in MCF10A WT and YIPF5 KO cells in 72 wells per cell line from n = 3 independent experiments. Unpaired t test, two-tailed p < 0.0001. (H) HeLa cells stably expressing shScramble or shRNAs against YIPF5 were transiently transfected with CNPY3-mCherry, supernatants were collected and cell lysates prepared after 24 h followed by western blot analysis using a CNPY3 antibody. A representative blot of n = 3 independent experiments is shown. For full size blot see C. (I) Quantification of CNPY3-secretion from (H). Displayed are arbitrary units normalized to the values of shScramble-expressing cells from n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test, with ∗ indicating p < 0.05. (J) Analysis of protein abundance changes in the secretome of YIPF5 KO re-expressing YIPF5 I98S mutant versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 I98S secretome are shown ( n = 4). Proteins with secretion changes explained by total proteome abundance are denoted in green, while unaffected proteins are in gray. (K) Venn Diagram of protein abundance changes in YIPF5-KO cells and YIPF5-KO re-expressing YIPF5 I98S cells. Proteins with decreased (blue) or increased (red) secretome abundance are shown. See also and , , , , and .
Article Snippet: The GCamPer coding sequence was amplified from pMOS003-lenti-CMV-GCaMPer (Addgene #65227, ) using primers GCamPer-F and GCamPer-R containing attB sites (sequences see ), gel-purified, and recombined with pDONR221 by BP ClonaseTM II (Thermo Fisher Scientific).
Techniques: Metabolic Labelling, Labeling, Purification, Mass Spectrometry, Comparison, Quantitative Proteomics, Expressing, Control, Sequencing, Stable Transfection, Fluorescence, Microscopy, Two Tailed Test, Transfection, Western Blot, Mutagenesis
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