Fig 1: miR-328 inhibition in M2 macrophages suppresses the progression of PF by targeting FAM13A. a Masson staining to examine the degree of PF in rats after the inhibition of miR-328 and FAM13A (400 × ). b IHC to analyze the expression of a-SMA and Collagen I in rats after the inhibition of miR-328 and FAM13A (400 × ). c MOD values of a-SMA and Collagen I in rats after the inhibition of miR-328 and FAM13A. miR-328, microRNA-328; PF, pulmonary fibrosis; FAM13A, family with sequence similarity 13, member A; a-SMA, a-smooth muscle actin; IHC, immunohistochemistry; and NC, negative control. * p < 0.05 vs. the control group; # p < 0.05 vs. the antagomir NC + sh-NC group. All results were measurement data and were analyzed using one-way analysis of variance. The results are expressed as the mean ± standard deviation; n = 6
Fig 2: FAM13A and miR-328 may work in tandem to affect PF. a Heat maps of the top 10 DEGs in the PF gene expression dataset GSE44723. The abscissa suggested the sample number, the ordinate indicated the DEGs, and the right upper histogram indicated the color gradation. Each rectangle in the panel corresponded to one sample expression value. b The interaction network of DEGs and disease genes related to PF, where arrows indicate DEGs, and circles indicate disease genes. miR-328 microRNA-328, FAM13A family with sequence similarity 13, member A, PF, pulmonary fibrosis, and DEGs, differentially expressed genes
Fig 3: Silencing of FAM13A decreases lung cancer cell proliferation. Decreased A549 and CORL-105 cell proliferation after FAM13A gene knockdown was assessed using Violet Proliferation Dye (VPD450) and MTS test. After VPD450 staining the cells were cultured for 4–5 days in hypoxia and normal oxygen tension. The fluorescence intensity was measure by flow cytometry. During MTS test the cells were cultured in 96-well plates and cultured for two days in normal and hypoxic atmosphere. After 24/48 h the CellTiter 96® AQueous One Solution Reagent was added and the absorbance was read at 490 nm with a microplate reader. (A) Representative images of A549 cells stained with VPD450. Fluorescence intensity was measured from Day 1 (0 time point) to 5 (96 h time point) by flow cytometry (Flow Sight®, Amnis, Seattle, WA, USA). Each cell is represented by a row of three images acquired simultaneously in flow, from left to right: channel 1—brightfield (gray), channel 2—fluorescence from GFP (transduced cells green) and channel 7—fluorescence from dye VPD450 applied to viable cells (purple). (B) Representative histogram plots displaying changes in VPD450 fluorescence intensity of A549 cells upon FAM13A knockdown (FAM13Ash1, red) and control (CtrNT2, green), in three time points: 0 h, 48 h, 96 h. (C,D) Cell growth kinetics was determined by proliferation index (PI) of FAM13Ash1 (red), FAM13Ash2 (green) and CtrNT2 (black), CtrSCR (grey) control cells in A549 (C) and CORL-105 (D). The difference in PI ratios at 24 h, 48 h, 72 h between FAM13A depleted cells and control cells was evaluated under normoxia (left panel) and hypoxia (right panel). A two-way ANOVA with Bonferroni posttest was used for statistical analysis. * p < 0.05 indicates a significant difference, as marked by an asterisk. The experiments were performed in triplicate and repeated three times. (E,F) Cell proliferation assessed by MTS assay. The results are given for A549 (E) and CORL-105 cells (F). Cell proliferation rate of FAM13Ash1 (red), FAM13Ash2 (green) cells and control cells (mean combined for CtrNT2 and SCR, black) was determined by CellTiter™ AQueous assay (MTS). The difference in cell proliferation rate of FAM13A depleted cells compared to control cells was evaluated under normoxia (bars without filling) or hypoxia (bars with texture) conditions. Data are means ± SEM (n = 4), * p < 0.05, ** p < 0.01, two-way ANOVA.
Fig 4: Stable knockdown of FAM13A promotes F-actin cytoskeletal reorganization. The FAM13A depleted cells (FAM13Ash1, FAM13Ash2) and control (CtrSCR, CtrNT2) cells, were cultured under normoxia and hypoxia for 72 h. After incubation, the cells were fixed, permeabilized and stained with Alexa Fluor® 568 phalloidin for actin filaments visualization and DAPI for nuclei. The cells were observed under a fluorescence microscope. Example images (scale bar 25 µm) of the diverse F-actin organization phenotypes induced by FAM13A gene silencing in normoxia (A) and chronic hypoxia (B). A549 cells with stable FAM13A knockdown (gfp positive) were stained with phalloidin for F-actin (red), with DAPI for nuclei (blue). Representative single and overlaid images of immunofluorescence staining were present (magnification, ×63). The arrowheads indicate F-actin aggregates and accumulations of F-actin punctae.
Fig 5: Generating lung cancer cell lines with FAM13A knockdown. FAM13A shRNAs (FAM13Ash1, FAM13Ash2) and control shRNAs (CtrNT2, CtrSCR) lentiviral particles were used to generate the stable transduction of A549 and CORL-105 cell lines. Transduction efficiency was assessed with the GFP marker. Knockdown of FAM13A mRNA and FAM13A protein was confirmed by real-time quantitative PCR and western-blot analysis. (A) Schematic indicating the binding site for the shRNA in the FAM13A mRNA. Two shRNA transcripts were designed using UCSC Broad Institute GPP Web Portal, against a coding sequence common to the most FAM13A isoforms with the RhoGAP functional domain. (B) RT-qPCR analysis of FAM13A expression in A549 and CORL-105 cell lines cultured under normoxia and hypoxia conditions for 72 h. Relative expression of FAM13A mRNA was determined as the mean normalized expression of FAM13A mRNA/GUSß mRNA. The experiments were performed in triplicate and repeated three times. The data are presented as the mean ± SD (* p < 0.05, ** p < 0.01, unpaired one-tailed t-test). (C) Western blot (WB) analysis of FAM13A expression in A549 and CORL-105 cell lines cultured under normoxia and hypoxia for 72 h. Relative FAM13A protein (117 kDA) amount was significantly decreased in FAM13A sh1/sh2RNA cells. Strong FAM13A expression was induced in controls (CtrNT2, CtrSCR) A549 or CORL105 cells after 72 h of hypoxia (+) compared to normoxia (-). Cropped images are displayed, full-length blots are presented in Supplementary Materials Figure S4. Total protein was used for normalization. Representative stain-free total protein blots are presented. The normalized protein factors are given at bar charts (N-normoxia, H-hypoxia, bars with texture). The data are presented as the mean ± SD (* p < 0.05, ** p < 0.01, unpaired one-tailed t-test).
Supplier Page from Abcam for Anti-FAM13A antibody