Fig 1: Lap2α is degraded in a lamin A/C-dependent manner under HS. (A) Proximity ligation assay (PLA) with antibodies against lamin A/C and Lap2α was carried out on control and heat-shocked cells. Maximum projections of confocal images are shown. Scale bars: 10 μm. Lamin A/C KO1 cells were used as a negative control. (B) Quantification of PLA signals per nucleus (n=200, three biological replicates). Data show individual observations and mean values of each replicate. ***P<0.001 (two-tailed unpaired Student's t-test). (C) Western blot analysis showing lamin A/C and Lap2α levels in HeLa, NT HeLa and LAC KO1 cells at different time points under HS and at the recovery. The average numerical values of signal intensities relative to the loading control (GAPDH) are shown below the blot. Each cell line has been normalized to its own control sample to highlight the change of Lap2α protein levels between the time points (n=5). (D) Confocal microscopy images showing Lap2α aggregation upon 4 h heat shock in HeLa, NT HeLa and LAC KO1 cells. Scale bars: 5 μm. (E) Western blot analysis of LAC KO1 cells transfected with different GFP-tagged lamin A vectors and detected with antibodies against pSer22 lamin A, HSF1 and Lap2α. The average numerical values of signal intensities relative to the loading control (actin) are shown below the blot (n=3). (F) Western blot analysis of Lap2α protein levels in heat-shocked parental HeLa cells treated with either 10 μM cycloheximide (CHX), 10 μM epoxymycin (EPO), 10 μM chloroquine (CQ) or both EPO and CQ. The average numerical values of signal intensities relative to the loading control (GAPDH) and percentage of intensity change between control and heat-shocked cells are shown below the blot (n=2).
Fig 2: Cx30 controls the recruitment and activation of Cdc42 in migrating astrocytes. (A) Primary astrocyte cultures were transfected with Cx30 and GFP-Cdc42. 8 h after wounding, cells were immunostained for Cx30 and the intensity of the GFP-Cdc42 signal at the leading edge was measured. Scale bar: 20 µm. (B) Quantitative analysis of the linear intensity profile of GFP-Cdc42 at the leading edge. Cx30 reduced significantly the recruitment of GFP-Cdc42 at the leading edge (Ct, n=19 cells; Cx30, n=15 cells). (C) Cdc42 pull-down activation assay in astrocytes transfected with Cx30 or GFP 30 min after wounding. Immunoblots showing Cdc42 protein levels in the total (Input) and GTP-bound fractions (GST-PAK-PBD). GAPDH (total) and Ponceau staining (GTP) were used as loading controls. (D) Quantitative analysis of Cdc42 activation (n=3 cultures). Cdc42-GTP values were normalized to Cdc42 levels in the total fractions. Cx30 inhibited Cdc42 activation. Asterisk indicates statistical significance (*P<0.05, paired t-test).
Fig 3: Cx30 regulates the secretion of laminin in migrating astrocytes. (A) Immunoblot detection of laminin expression in control (Ct) or Cx30-expressing (Cx30) astrocytes before (no treatment, NT) or after wounding (wound, 6 h). GAPDH was used as a loading control. (B) Quantitative analysis of laminin expression (n=6 cultures). Laminin expression values after wounding were normalized to levels before wounding (NT) in Ct and Cx30-expressing astrocytes. Cx30 prevented the increase in relative laminin levels after wounding. (C) Primary astrocytes were transfected with GFP (control) or Cx30-venus (Cx30). 24 h after wounding, cells were immunolabeled for laminin. Scale bar: 20 μm. (D) Quantitative analysis of laminin expression (area occupied by laminin puncta normalized to total cell area) and axial distribution (distance of laminin puncta to leading edge normalized to cell length). Cx30 impaired the proper recruitment of laminin toward the leading edge (Ct, n=14 cells; Cx30, n=15 cells). Asterisks indicate statistical significance (*P<0.05, **P<0.01).
Fig 4: TRPS1 regulates BC cell migration and EMT in vitro. (A) The protein levels of TRPS1 in human BC cell lines determined by western blot. (B) Western blot were used to confirm TRPS1 expression in transfected human BC cell lines with different metastatic characteristics. T47D and MCF-7 were transfected with TRPS1-specific shRNA. MDA-MB-468 and MDA-MB-231 were transfected with TRPS1 cloning vector. (C-D) Migration capacity of TRPS1 in transfected BC cell lines T47D, MCF-7, MDA-MB-468 and MDA-MB-231 were examined by transwell migration assay. (E) Cell proliferation was examined by MTS assay. (F) GSEA identified significant association between TRPS1 and EMT. Genes defining epithelial-mesenchymal transition were enriched for shTRPS1. (G) The expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and Vimentin) in T47D and MDA-MB-468 cells were determined by western blot analysis. GAPDH was used as a loading control. (H) Analysis showing correlation of TRPS1 and CDH1 expression in human breast cancer samples in publicly accessible breast cancer datasets. GSE2990, n = 189,P<0.0001.*P < .05, **P < .01, based on Student’s t test.
Fig 5: circARF3 Acts as an Endogenous miR-103 Sponge to Relieve the Inhibitory Effect on TRAF3(A) Divergent primers amplify circRNAs in cDNA, but not in genomic DNA (gDNA). (B) Relative miR-103-related circRNA expression in ND and HFD mouse adipose tissue. (C) circARF3 contains a site complementary to miR-103, as analyzed by the bioinformatic program RNAhybrid. (D) Double luciferase assay was performed in 293T cells 48 hr after miR-103 mimic transfection with pGL3-TRAF3-WT 3′ UTR and pc-circARF3. (E) qPCR was used to detect circARF3 and GAPDH levels after streptavidin capture. (F) The expression of TRAF3 in adipocytes after circARF3 lentivirus, circARF3 lentivirus + miR-103 adenovirus, or control empty virus treatment. (G) The expression of TRAF3 in adipose tissue of HFD mice after injection with LV-circARF3 or Ad-miR103. Error bar, SE. Data are represented as the mean ± SEM (*p < 0.05 and **p < 0.01; n ≥ 3).
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