Fig 1: FAK signaling and cell adhesion to ECM are connected to SEC23A expression. (A) SEC23A and SEC23B mRNA levels were assessed by RT-qPCR after treatment with the FAK inhibitor PND-1186 for the indicated times at the indicated concentrations. (B) SEC23A and SEC23B mRNA levels were assessed by RT-qPCR after 48 h of plating cells in dishes coated with fibroblast-derived ECM or Matrigel (4 µg/ml) and treated with the FAK inhibitor PND-1186 for 24 h at the indicated concentrations. (C) Western blot analysis of SEC23A protein levels after 72 h of plating cells in dishes covered with the indicated concentrations of Matrigel. (D) Quantification of the Western blots. (E) VSVF transport experiment. Cells were plated at the indicated concentration of Matrigel, and the VSVG was performed 72 h later. Some dishes were treated with PND-1186 48 h before the VSVG release. Cells were divided according to the level of expression of VSVG. A minimum of 50 cells per condition were analyzed. (F) Model representing the proposed relationship between adhesion, FA signaling, and SEC23A. (1) Interaction of cells with ECM or substrate through FAs. (2) Activation of FAK and a downstream signaling cascade. (3) Transcriptional regulation of SEC23A. (4) SEC23A-mediated ER-to-Golgi transport. In a condition of low FAs or low FAK signaling (left), SEC23A gene expression is not repressed. SEC23A levels are high (blue circles) and SEC23A-dependent transport proceeds normally (green arrow). In a condition of high FA or high FAK signaling (right) the repression is more prominent, and SEC23A levels decrease, possibly affecting SEC23A-dependent transport. The elements of the signaling cascade and the transcriptional regulation remain to be identified. For the experiments presented in B–D, dots represent values obtained from independent experiments, and the bar, the mean value per condition. For E, dots represent the values per cell, and the lines represent the mean value per condition. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control; ###, P < 0.001 compared with untreated cells, Student’s t test. In D, n.s., non significant, by one-way ANOVA among different Matrigel concentrations. Source data are available for this figure: SourceData F5.
Fig 2: 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 3: 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 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 [EPR13270(B)]