Fig 1: SEC23A expression is negatively associated with miR-375 levels in the thyroidA. Nthy-ori 3-1 were transfected with pre-miR-375 or pre-miR-CTL for 48h. TT cells were transfected with antagomiR-375 (anti-miR-375) or antagomiR-CTL (anti-miR-CTL) for 48h. SEC23A protein levels were quantified by immunoblotting. Tubulin (TUBA) and actin B (ACTB) protein levels were used as loading controls. Light exposure (upper band): unsaturated signal for all samples. Dark exposure (lower band): unsaturated signals for TT cells only. B. Immunohistochemistry with anti-SEC23A. (a) MTC: weak expression in tumor cells; (b) Papillary thyroid carcinoma: intense cytoplasmic expression in tumor cells; (c) Normal thyroid tissue: intense cytoplasmic expression in normal follicular cells; (d) C-cells: weak expression (arrows). (a-d: immunoperoxdiase, original magnification × 400).
Fig 2: Additional FA phenotype data and transport experiments. (A) Venn diagram representing the intersection between the six interactors and the adhesome proteins. (B) Confocal images of FAs assessed by vinculin staining after 48 h of the indicated treatment. Arrows show the FAs in cells plated in control (plastic) plates. Arrowheads show the enlarged FAs in cells plated in ECM- or Matrigel-treated plates. (C) SEC23A mRNA levels were assessed by RT-qPCR in cells treated with the indicated siRNAs together with the FAK inhibitor PND-1186 at the indicated time and concentration. The data were normalized to cells treated with the indicated siRNA but no FAK inhibitor. The dots represent the values obtained for independent experiments, and the bar, the mean value. (D) VSVG assay after 48 h of treatment with the indicated siRNAs. Cells were divided according to the level of VSVG expression. Bars represent the average transport ratio per condition of one representative experiment in which =100 cells were quantified. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control, Student’s t test.
Fig 3: The transport defect phenotype is caused by the downregulation of SEC23A. (A) Differential expression analysis of a subset of 595 manually curated secretory pathway genes for the indicated knockdowns (48 h) compared with control siRNA. The zoomed-in region highlights the COPII genes. (B) RT-qPCR quantification of SEC23A levels after 48 h of knockdown with the indicated siRNAs. Bars represent averages of three independent experiments, and dots, the individual values. The dark gray bar represents SEC23A levels after 48 h of MACF1 siRNA treatment in human lung fibroblasts. (C) Western blot analysis of SEC23A protein levels after 48 h of depletion with the indicated siRNAs. a-Tubulin was used as a loading control. (D) SEC23A rescue experiment. Cells were cotransfected with the indicated siRNAs and a cDNA encoding SEC23A-YFP. After 60 h of transfection, the VSVG assay was performed. Based on the intensity of the YFP channel, cells were divided into non-expressing, low-expressing, and high-expressing. Bars represent averages of three independent experiments, and dots, the individual values. Statistical significance: *, P < 0.05 and **, P < 0.01 compared with control, Student’s t test. Source data are available for this figure: SourceData F3.
Fig 4: Differential expression analysis. (A) Cells were treated with the indicated siRNAs, and after 48 h, total RNA was extracted, and mRNA was sequenced using an Illumina workflow. The differentially expressed (DE) genes were obtained by comparing expression to control-treated cells with a minimum of three biological replicas per condition. The false discovery rate was set at 0.1, and the log2 fold-change at 0.58. (B) The differential expression analysis was hierarchically clustered and represented as a heatmap using Morpheus (https://software.broadinstitute.org/morpheus). (C) Scheme representing the transport phenotypes obtained during the screen and their relationship to SEC23A and SEC23B levels.
Fig 5: A functional interaction screen between SEC23 and cytoskeleton-associated proteins uncovers new proteins connected to secretion. (A) Schematic representation of the high-throughput screen workflow. HeLa cells were cotransfected with siRNAs targeting 378 cytoskeleton-associated proteins and siRNAs targeting the COPII subunits SEC23A or SEC23B. Using an automated microscopy acquisition and analysis pipeline, VSVG transport to the cell surface was quantified after 60 h of depletion in single- and double-knockdown (k.d.) conditions. For each condition, a transport score was calculated. (B) Hit selection. The conditions in which the double-knockdown score was significantly different from the expected additive effect of the single knockdowns were selected. Gray circles denote single knockdowns. Red circles denote the positive controls. Interactors in double knockdown are shown in orange (SEC23A interactors) or blue (SEC23B interactors). (C) Annotation of the subcellular location of the interactors using the Human Protein Atlas and Compartments as the main sources. For proteins with multiple locations, only the two main locations were annotated (plasma membrane [PM]). Others include mitochondria, centrioles, FAs, etc. (D) STRING network (Szklarczyk et al., 2019) using medium confidence and removing the text mining. Proteins in gray are not interactors but were included to fill up the gaps in the network. (E) VSVG transport assay. Wide-field images of the VSVG-YFP after 1 h of temperature shift from 40° to 32°C. Transport was assessed after 60 h of knockdown with the indicated siRNAs. Arrowhead indicates plasma membrane. Arrows indicate ER membranes. Asterisk indicates Golgi or post-Golgi membranes.
Supplier Page from Abcam for Anti-SEC23A antibody