Journal: International Journal of Molecular Sciences

Article Title: THSD1 Suppresses Autophagy-Mediated Focal Adhesion Turnover by Modulating the FAK-Beclin 1 Pathway

doi: 10.3390/ijms25042139

Figure Lengend Snippet: THSD1 interacts with talin and modulates focal adhesion stability. ( A ) Western blot detection of co-immunoprecipitated THSD1 in the talin protein complex from HBMECs treated with siRNAs against control (Ctrl, 5 nM) and talin (talin (-), 5 nM) for 48 h. To mitigate the potential off-target effects, we utilized two different siRNAs with non-overlapping sequences. Consequently, data from both clones were pooled for statistical analyses, and representative knockdown results were shown. This method will be used for all other genes to be silenced throughout the study unless indicated otherwise. n = 3 independent experiments. ( B ) Induction of FLAG-THSD1 in HBMECs with doxycycline (100 ng/mL) for 48 h followed by pull-down using anti-FLAG antibody and Western blot analysis against talin or THSD1. n = 3 independent experiments. ( C , D ) Representative images of focal adhesions (FAs) immunostained with paxillin (green) and phalloidin (red) in HBMECs treated with siRNAs against control (Ctrl, 10 nM) and THSD1 (THSD1 (-), 10 nM) for 48 h. Areas highlighted by white dashed boxes (C1 and C2) were selected and shown as enlarged pictures at the bottom (C3 and C4). The number of FAs was quantified from at least 12 different fields (20× objective) for each experiment ( n = 3) and presented in ( D ). Enlarged images were shown in white dashed boxes. ( E , F ) Representative images of cell morphologies revealed by phalloidin staining for control or THSD1-deficient HBMECs at 20 min after re-seeding onto chamber slides. Quantitative analysis was performed by counting the number of cells with surface areas exceeding 1200 nm 2 in each well. At least 9 different fields (10× objective) were randomly recorded for each experiment ( n = 3). ( G ) Measurement of relative fluorescence units of CyQuant Dye at 480/520 nm from control or THSD1-deficient HBMECs after reseeding onto collagen IV pre-coated 48-well plates. n = 3 independent experiments. ( H ) Total mRNA was extracted from both control and THSD1-deficient HBMECs, followed by reverse transcriptase reactions. The mRNA expression levels of paxillin and zyxin were determined through quantitative RT-PCR and subsequently normalized to GAPDH. The fold change was calculated by comparing the mRNA levels in each sample to those in control cells. The statistical comparison was conducted through two-way ANOVA, followed by Bonferroni correction. ns: not significant. * p < 0.05; ** p < 0.01. ns: not significant. Scale bar: 10 µm.

Article Snippet: All stealth small interfering RNAs (siRNAs) were purchased from ThermoFisher Scientific (Waltham, MA, USA) and included siRNAs targeting talin (HSS110804 and HSS186350), THSD1 (HSS148179 and HSS148180), ATG5 (HSS114103 and HSS114104), and PSMB2 (HSS108676 and HSS108677).

Techniques: Western Blot, Immunoprecipitation, Control, Clone Assay, Knockdown, Staining, Fluorescence, CyQUANT Assay, Reverse Transcription, Expressing, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: THSD1 Suppresses Autophagy-Mediated Focal Adhesion Turnover by Modulating the FAK-Beclin 1 Pathway

doi: 10.3390/ijms25042139

Figure Lengend Snippet: Autophagy inhibition rescues focal adhesion stability. ( A ) Representative images of FAs immunostained with paxillin (green) and actin (red) in control or THSD1-deficient HBMECs treated with siRNAs against control (5 nM) or ATG5 (5 nM). Specifically, FAs were examined in control cells (A1), THSD1-deficient cells (A2), ATG5-deficient cells (A3) and cells that deficient in both THSD1 and ATG5 (A4). Representative areas were highlighted by white dashed boxes and accordingly selected as enlarged images at the bottom (A5–A8, respectively). ( B ) Quantification of the number of paxillin puncta per cellular profile from at least 12 different fields (20× objective) for each experiment ( n = 3). ( C , D ) Evaluation of cell spreading ( C ) and attachment ( D ) in control or THSD1-deficient HBMECs in the absence and presence of ATG5 siRNA (5 nM) treatment for 48 h. Data were analyzed by two-way ANOVA followed by Bonferroni correction. * p < 0.05; ** p < 0.01. Scale bar: 10 µm.

Article Snippet: All stealth small interfering RNAs (siRNAs) were purchased from ThermoFisher Scientific (Waltham, MA, USA) and included siRNAs targeting talin (HSS110804 and HSS186350), THSD1 (HSS148179 and HSS148180), ATG5 (HSS114103 and HSS114104), and PSMB2 (HSS108676 and HSS108677).

Techniques: Inhibition, Control

Journal: International Journal of Molecular Sciences

Article Title: THSD1 Suppresses Autophagy-Mediated Focal Adhesion Turnover by Modulating the FAK-Beclin 1 Pathway

doi: 10.3390/ijms25042139

Figure Lengend Snippet: Schematic model of THSD1-mediated autophagy in focal adhesion stability. Focal adhesions (FAs) are composed of integrin α/β transmembrane proteins that link extracellular matrix components like collagens to intracellular adaptors such as talin and paxillin (PXN). The actin cytoskeleton, represented by aligned round grey dots, is tethered to integrins through PXN. Focal adhesion kinase (FAK) plays a central role in focal adhesion regulation, binding to both PXN and THSD1. Under normal conditions, THSD1 interacts with FAK, enhancing its kinase activity, resulting in the phosphorylation of Beclin 1 at tyrosine 233. This phosphorylation event inhibits the binding of ATG14 to Beclin 1, a critical step in autophagy activation. When THSD1 is inactivated, FAK kinase activity is reduced, alleviating its negative control over the formation of the Beclin–ATG14 complex. This, in turn, promotes Beclin 1-dependent autophagosome formation, where LC3 proteins are incorporated on both sides of the phagophore. These autophagosomes can sequester paxillin and other focal adhesion proteins, leading to autophagy-mediated focal adhesion turnover. P: phosphorylation.

Article Snippet: All stealth small interfering RNAs (siRNAs) were purchased from ThermoFisher Scientific (Waltham, MA, USA) and included siRNAs targeting talin (HSS110804 and HSS186350), THSD1 (HSS148179 and HSS148180), ATG5 (HSS114103 and HSS114104), and PSMB2 (HSS108676 and HSS108677).

Techniques: Binding Assay, Activity Assay, Phospho-proteomics, Activation Assay, Negative Control