Additional file 4 of AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells
Creators: name: Yuan, Stephen Hsien-Chi
name: Wu, Chih-Ching
name: Wang, Yu-Chih
name: Chan, Xiu-Ya
name: Chu, Hao-Wei
name: Yang, Youngsen
name: Liu, Hao-Ping
Publisher: name: figshare
Year: 2024
Subjects: Biochemistry
Molecular Biology
Chemical Sciences not elsewhere classified
Biological Sciences not elsewhere classified
Cancer

description: Supplementary Material 4: Fig. S1. Ectopic expression of AGR2 modulated extracellular milieu of several cancer cell lines to promote cell chemotaxis. The CMT cell line DMGT (A, B) was transfected with pcDNA3.1-myc.His-AGR2 or the mock vector and grown in 1% FBS-containing DMEM for 24 h. Additionally, the human breast adenocarcinoma cell line MDA-MB-231 (C, D) or MCF7 (E, F) was transfected with an expression vector for human AGR2 under similar condition settings. (A, C, E) Whole-cell lysates (WCL) of the transfectants were analyzed by immunoblotting to confirm the expression of Myc-tagged AGR2. Conditioned media (CM) of the transfectants were collected and placed in the bottom well for a transwell migration assay. Cells in the insert were fixed for image acquisition using an epifluorescence microscope with a 10 × objective. (B, D, F) The number of migrated cells was counted and presented as the mean + SD of three independent experiments. Statistical significance was determined by a two-tailed unpaired t-test. *, p &lt; 0.05; **, p &lt; 0.01. Fig. S2. AGR2 modulated the release of 14-3-3ε and α-actinin 4 in several cancer cell lines. (A) MCF7 transfected with pcDNA3.1-myc.His-hAGR2 or the mock control were subsequently cultured in DMEM containing 1% FBS for 24 h. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting. (B) DMGT or MDA-MB-231 transfected with pcDNA3.1-myc.His-AGR2, pcDNA3.1-myc.His-hAGR2, or the mock control were subsequently cultured in DMEM containing 1% FBS for 14 h with or without addition of 50 nM tunicamycin (Tm) following transfection. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting using antibodies specific to indicated proteins. (C) DMGT, MDA-MB-231, or MCF7 were cultured in 1% FBS-containing DMEM and treated with 100 nM rapamycin (Rm) or 40 µM chloroquine (CQ) for 20 h. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting. These results represent data from three independent experiments. Fig. S3. Extracellular vesicles were dispensable for the chemotaxis conferred by AGR2-modulated CM. (A) Schematic diagram for comprehensive isolation of extracellular vesicles (EVs) with varied sizes by differential ultracentrifugation. (B) Nanoparticle tracking analysis (NTA) was performed on the isolated large (l) EVs and small (s) EVs derived from control Ctrl-S3 cells. (C) Transmission electron microscopy (TEM) images depict the isolated lEV and sEV (indicated by arrowheads). (D) The CM samples before and after EV depletion, and the WCL of CMT-U27e, Ctrl-S3, KO-S10, and KO-S4, respectively, were analyzed by immunoblotting with antibodies specific to the indicated proteins. CD9 and CD63 are considered markers for exosomes with smaller sizes, and Annexin A1 is considered a marker for microvesicles with larger sizes. (E) Isolated lEVs and sEVs originating from individual cells were analyzed by immunoblotting with specific antibodies as indicated. (F) CM samples with and without EV depletion (denoted EV-present and EV-depleted CM, respectively) were applied to a transwell migration assay. Data are presented as the mean + SD of three independent experiments. Fig. S4. AGR2 knockout led to a reduction in the EV-delivered 14-3-3ε and α-actinin 4 and the EV-mediated chemotaxis. (A) The schematic diagram for isolation of EV-enriched fractions by the size exclusion chromatography (SEC) using the qEV Isolation Columns. (B) CM from CMT-U27e cells was first concentrated with VivaSpin 20 (&gt; 100 kD) and applied to the SEC. Individual fractions collected throughout the SEC, along with the concentrated CM (&gt; 100 kD) and the flowthrough (&lt; 100 kD) subsequently concentrated for the second time, were analyzed by immunoblotting with antibodies specific to 14-3-3ε, α-actinin 4, CD9, or Albumin. Albumin was used as an indicator for the EV-free protein fractions. (C) Nanoparticle tracking analysis (NTA) was conducted on the isolated EVs derived from CMT-U27e cells in qEV Fraction 1. (D) Transmission electron microscopy (TEM) images illustrate the isolated EVs (indicated by arrowheads) in qEV Fraction 1. (E) The CM fractions of CMT-U27e, Ctrl-S3, KO-S10, or KO-S4 collected during the SEC, together with the corresponding input CM (&gt; 100 kD) and the flowthrough (&lt; 100 kD), were analyzed by immunoblotting. (F) The EV-enriched fractions (Fraction 1) derived from individual cells were applied to a transwell migration assay. Data are presented as the mean + SD of three independent experiments, and the statistical significance was determined by a two-tailed unpaired t-test. *, p &lt; 0.05; **, p &lt; 0.01. Fig. S5. Experimental replicates of the immunoblotting analysis which are shown in Fig. 6 (A to D). (A, B) Experimental replicates of the immunoblotting data shown in Fig. 6A and Fig. 6B. (C, D) Experimental replicates of the immunoblotting data shown in Fig. 6C and Fig. 6D. Fig. S6. Experimental replicates of the immunoblotting analysis which are shown in Fig. 6 (E to H). (A, B) Experimental replicates of the immunoblotting data shown in Fig. 6E and Fig. 6F. (C, D) Experimental replicates of the immunoblotting data shown in Fig. 6G and Fig. 6H.