Fig 1: PDAC cell‐derived sEV‐EZR exacerbates cancer metastasis to the liver. (A) Representative images and the quantified bar chart of soft agar colony formation of PDAC cells co‐cultured with HPaSteC treated with 5 μg·mL−1 of PDAC‐shLacz or shEZR cell‐derived sEVs for 5 days. Scale bar, 500 μm. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (***P < 0.001). (B) Schematic illustration of animal study setup and time course for the in vivo mouse liver metastasis model. Pan18 and mouse fibroblasts were co‐injected into the spleen of 6‐week‐old male C57BL/6J mice. Approximately 14 days postinjection, the mice were found with metastatic liver tumors derived from the injected PDAC cells. The total number of mice examined in each group was six (n = 6). (C) IVIS images showed liver metastasis on day 14. (D) The bioluminescence photon counts in the region of liver metastasis (n = 6) were measured by IVIS software. The bioluminescent signal (pseudocolor) was recorded as photons per second (p/s). Values are mean ± SD. The red horizontal line indicates mean. Level of significance was determined using the Student's t test (**P < 0.01). N = 1.
Fig 2: Inhibition of EZR/STAT3 or EZR/YAP‐1 signaling pathways abrogates the EZR‐induced activation of Fibroblasts. (A) qRT‐PCR analysis of expressions of Col1a1, Col1a2, Col3a1, and Cal12a1 in HPaSteC, whose EZR was overexpressed (transfected with EZR OE or treated with Lip‐EZR) and then transfected with siRNA‐STAT3 or treated with 5 μM Stattic. GAPDH was used as an internal control. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). (B) qRT‐PCR analysis of the expressions of Col1a1, Col1a2, Col3a1, and Cal12a1 in HPaSteC whose EZR was overexpressed (transfected with EZR OE or treated with Lip‐EZR) and then transfected with siRNA‐ YAP‐1 or treated with 100 nm CA3. GAPDH was used as an internal control. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Representative images and the quantified bar chart of soft agar colony formation of PDAC cells co‐cultured with HPaSteC treated with Lip‐EZR and then transfected with siRNA‐STAT3 or treated with 5 μm Stattic for 5 days. Scale bar, 100 μm. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (***P < 0.001). (D) Representative images and the quantified bar chart of soft agar colony formation of PDAC cells were co‐cultured with HPaSteC treated with Lip‐EZR and then transfected with siRNA‐YAP‐1 or treated with 5 μm CA3 for 5 days. Scale bar, 100 μm. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (***P < 0.001).
Fig 3: PDAC cell‐derived sEV‐EZR activates fibroblasts. (A) Schematic illustration of animal study setup and time course. PDAC cell‐derived sEVs (PC080‐shLacz sEVs/shEZR‐sEVs) were administered every day for 1 week before PC080 cells injection in NSG mice, followed by administration on every other day until day 21. Representative photomicrographs and the dot chart of Trichrome staining and IHC staining of α‐SMA, FAP, PDGFRA, and PDGFRB positive fibroblasts in mouse pancreatic cancer tissues. Each dot represents the datum of a single mouse. Scale bar, 200 μm. 40× magnification. Data presented as mean ± SD. Level of significance was determined using the Student's t test (*P < 0.05). N = 1. (B) Results of western blotting showing expressions of EZR, α‐SMA, FAP, PDGFRA, and PDGFRB in HPaSteC treated with 5 μg·mL−1 sEVs derived from BXPC‐3, PC080, or PC084 with shLacz sEVs/shEZR‐sEVs for 48 h. GAPDH was used as a protein loading control. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH. N = 2. (C) Representative images and the quantified bar chart of Transwell migration assays of HPaSteC treated with 5 μg·mL−1 sEV derived from PC080 or PC084 shLacz sEVs/shEZR‐sEVs for 24 h. Scale bar, 200 μm. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (**P < 0.01 and ***P < 0.001). (D) qRT‐PCR analysis of Col1a1, Col1a2, Col3a1, and Cal12a1 in HPaSteC treated with 5 μg·mL−1 sEVs derived from BXPC‐3, PC080 or PC084 with shLacz sEVs/shEZR‐sEVs for 48 h. GAPDH was used as an internal control. Values are mean ± SD (n = 3). N = 2. Level of significance was determined using the Student's t test (***P < 0.001). shEZR, short hairpin RNA ezrin; shLacz, short hairpin RNA β‐galactosidase (negative control hairpin).
Fig 4: PDAC cell‐derived sEVs induces α‐SMA and PDGFRB expression in fibroblasts via EZR/STAT3 and EZR/YAP‐1 signaling pathways. (A) Results of western blotting of EZR, α‐SMA, PDGFRB, PDGFRA, FAP, YAP‐1, active YAP‐1, STAT3, AKT, PI3K, and their phosphorylation in HPaSteC transfected with siRNA‐EZR or NS (nonspecific siRNA) for 48 h. N = 2. (B) Expressions of EZR, α‐SMA, PDGFRB, PDGFRA, FAP, YAP‐1, active YAP‐1, STAT3, AKT, PI3K, and their phosphorylation in HPaSteC transfected with EZR OE (EZR expression vector) or Vec (control vector) for 48 h. The numerical values for protein band intensities were corrected with the values for the loading control GAPDH bands. N = 2. (C) Results of western blotting of EZR, α‐SMA, PDGFRB, STAT3, and phospho‐STAT3 in HPaSteC transfected with EZR OE or Vec and then transfected with siRNA‐STAT3 or NS (left) or treated with 5 μm Stattic (STAT3 inhibitor; right). Values are mean ± SD (n = 3). Level of significance was determined using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). (D) Results of western blotting showing the expressions of EZR, α‐SMA, PDGFRB, STAT3, and phospho‐STAT3 in HPaSteC treated with Lip‐EZR (Liposomal‐EZR) or Lip‐ctrl (Liposomal‐control) and then transfected with siRNA‐STAT3 or NS (left) or treated with 5 μm Stattic (right). Values are mean ± SD (n = 3). Level of significance was determined using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). (E) Results of western blotting showing the expressions of EZR, α‐SMA, PDGFRB, YAP‐1, and active YAP‐1 in HPaSteC transfected with EZR OE or Vec and then transfected with siRNA‐YAP‐1 or NS (left) or treated with 100 nm CA3 (YAP‐1 inhibitor; right). Values are mean ± SD (n = 3). Level of significance was determined using the Student's t test (*P < 0.05 and **P < 0.01). (F) Results of western blotting showing the expressions of EZR, α‐SMA, PDGFRB, YAP‐1, and active YAP‐1 in HPaSteC treated with Lip‐EZR or Lip‐ctrl and then transfected with siRNA‐YAP‐1 or NS (left) or treated with 100 nm CA3 (right). Values are mean ± SD (n = 3). Level of significance was determined using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH was used as control. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH.
Supplier Page from Abcam for Recombinant Human Ezrin protein