Additional file 1 of Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer
Creators: name: Wang, Caiqin
name: Xie, Bo
name: Yin, Shi
name: Cao, Jianghua
name: Huang, Junhao
name: Jin, Longyang
name: Du, Ge
name: Zhai, Xiaohui
name: Zhang, Rongqin
name: Li, Shanshan
name: Cao, Taiyuan
name: Yu, Hongen
name: Fan, Xinjuan
name: Yang, Zuli
name: Peng, Junsheng
name: Xiao, Jian
name: Lian, Lei
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1. The promotion of gastric cancer cell motility by ACTN2 was independent of other genes. Cell migration and invasion were detected when AGS cells were co-transfected with ACTN2 and ADIPOQ (A), and SNU-16 cells were co-transfected with ACTN2 and SYT12/SPINK6 (B, C). The bars indicate the SD. The results are expressed as the mean ± SD of five independent experiments. *p &lt; 0.05, **p &lt; 0.01 using Ordinary one-way ANOVA. ns, no significance. Figure S2. (A) α-Actinin-2 promotes GC cell migration by facilitating the increase and growth of filopodia. SNU-16 cells were transfected with GFP or GFP-α-Actinin-2 for 24 h, then cells were stained for DAPI (Nucleus, blue), GFP/GFP-α-Actinin-2 (green) and F-Actin (red). (B-E) Overexpression of α-actinin-2 had no influence of cell viability and apoptosis. (B) Cell viability were detected when AGS/ SNU-16 cells were transfected with ACTN2 from day 1 to 7. (C) Clone formation assay in AGS and SNU-16 cells with stable ACTN2 overexpression. (D) Cell cycle data were measured by flow cytometry in AGS and SNU-16 cells with stable ACTN2 overexpression. (E) Cell apoptosis were detected by Annexin V/PI assay after transfection with ACTN2. The bars indicate the SD. The results are expressed as the mean ± SD of five independent experiments. Figure S3. (A) The identify of N-terminal and C-terminal amino acid sequence between α-Actinin 1, 2, 3 and 4. (B) GFP-α-Actinin 1, 2, 3 or 4 was overexpressed in AGS cells for 24 h, then cell lysates were harvested and subjected to Western blotting using indicated antibodies. (C) AGS cells were transfected with α-Actinin-2 for 24 h, then the membrane extract (PM), cytoplasmic extract (Cyto) and nuclear extract (Nu) of cells were separated by the Subcellular Protein Fractionation Kit. Glut1, the protein marker of PM; Tubulin, the protein marker of Cyto; Histone H1, the protein marker of Nu. (D) GFP-α-Actinin-2 was co-transfected with or without α-Actinin-2 siRNAs in AGS cells for 24 h, then cells were treated as in (Figure 3B) to separate the supernatant and pellet. The pellet was used for co-Immunoprecipitation (IP) assay by using α-Actinin-2 antibodies. Figure S4. (A) α-Actinin-2 was co-transfected with or without α-Actinin-1 siRNAs in AGS cells for 24 h, then cells were lysed for co-Immunoprecipitation (IP) assay by using α-Actinin-2 antibodies. (B and C) In vitro G-Actin/F-Actin and α-Actinin-1:α-Actinin-4 complex or α-Actinin-2:α-Actinin-4 complex binding studies were performed and analyzed by Western blotting using the indicated antibodies, (B) and (C) show inputs of the experiment. Figure S5. NF-κB pathway was activated in HAGC. (A, B) GO analysis with differentially expressed genes in NAGC vs normal as well as HAGC vs normal. (C) Pathway specifically enriched in HAGC by GO analysis. (D) Heatmap representing differential expressed genes (fold change &gt; 2) which were transcriptional targets of RelA in HAGC and NAGC. Figure S6. (A) AGS cells were transfected with GFP-RelA for 2 days following transfect with negative control (NC) siRNAs or α-Actinin-2 siRNAs for 24 h, then cells were stained for quantification of the number of filopodia per cell or the average length of filopodia. GFP/GFP-α-Actinin-2 (green), F-Actin (red) and Nucleus (DAPI, blue). Scale bar, 10 μm. (B) GFP-RelA was co-transfected with or without α-Actinin-2 siRNAs in AGS cells for 24 h, then the ratio of F-Actin/G-Actin was determined by G-Actin and F-Actin ratio assays. P: pellet; S: supernatant. (C) AGS cells were transfected with RelA, α-Actinin-2 or RelA plus α-Actinin-2 for 2 days following transfect with negative control (NC) siRNAs or α-Actinin-2 siRNAs for 24 h, then the total RNA of cell were extracted for qRT-PCR analysis. The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *P &lt; 0.05 using Ordinary one-way ANOVA for (A and B). *P &lt; 0.05 using Student’s t-test for (C). Table S1. Clinical characteristics of patients with HAGC and NAGC. Table S2. Up-regulated genes in HAGC and/or NAGC. Table S3. Plasmids and qRT-PCR primers. Table S4. Clinical characteristics of patients detected by multiple immunofluorescence staining of α-Actinin-2 and p-RelA.