Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: AIMP3 expression in clinical non‐small cell lung tumour and adjacent tissues. A, Representative IHC staining of AIMP3 on NSCLC and adjacent tissues. B, AIMP3 expression plotted based on immunohistochemical score. C, Seven random selected human NSCLC tumours and their paired adjacent normal tissues were tested using AIMP3 specific antibody. A, adjacent normal tissues; T, tumours. D, Density analysis of Western blot results. * P < .05 compared with adjacent normal tissues

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: Overexpression of AIMP3 suppresses the proliferation of NSCLC cells. A, The expression of AIMP3 protein in six NSCLC cell lines indicated and HBE cells. Relative fold changes were determined by densitometry and normalized to GAPDH. B, H1299 and A549 cells were transiently transfected with different amounts of HA‐AIMP3 for 24 h. Cell lysates were extracted and used to detect the protein levels of AIMP3 and p53. C, Saturation density assay. Cells transfected with indicated amount of AIMP3 were cultured in full medium for 6 days, trypsinized and counted. Data are presented as mean ± SEM (n = 3). D, Low serum assay. Cells transiently transfected with AIMP3 were cultured in 1% FBS RPMI1640 medium. At the indicated times, cells were trypsinized and counted. Data are presented as mean ± SEM (n = 3). E, Colony formation assay. 500 transiently transfected cells were seeded in 6‐well plates in 5% FBS RPMI1640 medium, respectively. After 14 days, cells were fixed and stained with 2% crystal violet. F, The number of colonies was counted and data are presented as mean ± SEM (n = 3). * P < .05, ** P < .01, *** P < .001 by Student's t test

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Over Expression, Expressing, Transfection, Cell Culture, Serum Assay, Colony Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: AIMP3 suppresses the migration of NSCLC cells. A, Transwell migration assay. 10 4 transiently transfected H1299 and A549 cells were cultured in 1% FBS RPMI1640 medium and seeded in transwell chambers. 10 h later, cells were fixed, stained and photographed. B, The crystal violet was dissolved in DMSO, and the absorbance was tested. Data are presented as mean ± SEM (n = 3). C, Cells were transiently transfected with AIMP3 or control vector. The expression of N‐cadherin, E‐cadherin, Vimentin, Snail and Slug was analysed by Western blot. D, Relative fold changes were determined by densitometry and normalized to β‐actin

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Migration, Transwell Migration Assay, Transfection, Cell Culture, Staining, Control, Plasmid Preparation, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: AIMP3 induces cell cycle arrest at G2/M phase and apoptosis depends on p53. A, Cell cycle analysis of A549 cells transfected with different amounts of AIMP3 for 48 h. Cells were collected and analysed by flow cytometry after propidium iodide staining. The inserts showed the proportion of cells for each phase and were marked with different colours (violet: G0/G1 phase, green: S phase and gold: G2/M phase). B, The quantification of cell number in each phase of cell cycle. C, Examination of apoptotic cells in A549 cells transfected with indicated amount of AIMP3. D, Quantitative analysis of the percentage of apoptotic cells was shown in Figure C. Data are presented as mean ± SEM (n = 3); * P < .05, ** P < .01. E, The effect of AIMP3 on p53‐dependent transcription of NOXA , PUMA and p21 . A549 cells were transiently transfected with indicated amount of AIMP3 and incubated for 24 h. Total RNAs were extracted and used for RT‐PCR detection. F, A549 cells were co‐transfected with p21 promoter‐reporter vector and different amounts of AIMP3 expression plasmid (0, 50, 100 ng). Relative luciferase activity values are presented as mean ± SEM (n = 3); ** P < .01 by Student's t test. G, A549 cells were transiently transfected with HA‐AIMP3 for 24 h, and whole cell lysates were immunoprecipitated with HA antibody (mouse) or mouse IgG as control. The precipitates were immunoblotted with ATM antibody. H, Cells were transiently transfected with AIMP3 or control vector. The expression of indicated proteins was analysed by Western blot

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Cell Cycle Assay, Transfection, Flow Cytometry, Staining, Incubation, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Immunoprecipitation, Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: AIMP3 suppresses tumour growth in mouse xenograft model. A, A549 cells stably expressing empty vector or AIMP3 were subcutaneously injected into nude mice , and the expression of AIMP3 was confirmed by western blot. Tumour volume was measured every 3 days and is shown as the mean ± SEM (empty vector, Mock: n = 8, AIMP3: n = 8). Significant differences were determined by one‐way ANOVA, followed by Dunnett's test; * P < .05 (versus empty). A representative image of tumour xenografts harvested at day 30. B, Tumour weights from the two groups are represented. C, BALB/c nude mice (n = 8 per group) received a tail vein injection of A549 cells stably expressing empty vector or AIMP3. The number of metastatic lung nodules in individual mice was counted under a dissection microscope. Data represent the mean ± SEM * P < .05 vs empty vector

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Injection, Western Blot, Dissection, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: AIMP3 is a direct target of miR‐96‐5p. A, Prediction of miR‐96‐5p binding sites in 3’UTR of AIMP3 using Target Scan Human 7.2 software. B, A549 cells were co‐transfected with AIMP3 3’‐UTR plasmid and miR‐96‐5p mimic (left panel) or miR‐96‐5p inhibitor (right panel). Relative luciferase activity values are presented as mean ± SEM (n = 3); * P < .05, ** P < .01. C, The expression of miR‐96‐5p in six NSCLC cell lines and HBE cells. U6 was used as an internal control. * P < .05, ** P < .01, *** P < .001. D, miR‐96‐5p expression in 36 pairs of NSCLC and matched adjacent normal tissues. *** P < .001. E, Correlation between AIMP3 and miR‐96‐5p expression in 36 NSCLC tissues. F, Up‐regulation of miR‐96‐5p suppressed the expression of AIMP3 protein and mRNA in A549 cells. G, Inhibition of miR‐96‐5p increased both the mRNA and protein levels of AIMP3

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Binding Assay, Software, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Control, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR‐96‐5p‐AIMP3‐p53 axis

doi: 10.1111/jcmm.16344

Figure Lengend Snippet: The effect of miR‐96‐5p on the growth of NSLSC cells. A, Saturation density assay. H1299 and A549 cells transiently transfected with miRNA control, miR‐96‐5p mimic or inhibitor were cultured in full medium. 6 days later, cells were trypsinized and counted. Data are presented as mean ± SEM (n = 3); * P < .05, ** P < .01 by Student's t test. B, Low serum assay. H1299 and A549 cells transiently transfected with miRNA control, miR‐96‐5p mimic were cultured in 1% FBS RPMI1640 medium. Cells were trypsinized and counted at indicated times. Data are presented as mean ± SEM (n = 3); ** P < .01 by Student's t test. C, Colony formation assay. Five hundred transiently transfected H1299 and A549 cells were seeded in 6‐well plates in 5% FBS RPMI1640 medium, respectively. After 14 days, cells were fixed and stained. The number of colonies was counted and data are presented as mean ± SEM (n = 3); ** P < .01 by Student's t test. D, Transwell migration assay. 10 4 transiently transfected H1299 and A549 cells were cultured in 1% FBS RPMI1640 medium and seeded in transwell chambers. 10 h later, cells were fixed, stained and photographed. Then, the crystal violet was dissolved in DMSO and the absorbance was tested. Data are presented as mean ± SEM (n = 3); ** P < .01 by Student's t test. E, qPCR was used to examine the miR‐96‐5p level in transfected A549 cells. F, Western blot was performed to detect the expression of AIMP3 and p53 in these cells. G, Tumour weights of tumours from control or miR‐96‐5p mimic transfected A549 xenografts at sacrifice. n = 8 for each group. H, The number of metastatic lung nodules in individual mice received a tail vein injection of A549 cells transfected control or miR‐96‐5p mimic was counted. Data represent the mean ± SEM* P < .05 vs control

Article Snippet: The proteins were detected by the following primary antibodies: AIMP3 (Proteintech, 10805‐1‐AP), p53 (Proteintech, 10442‐1‐AP), N‐cadherin(Cell Signaling, 13116), E‐cadherin (Cell Signaling, 3195), Vimentin (Cell Signaling, 5741), Snail (Cell Signaling, 3879), Slug (Cell Signaling, 9585), ATM (Proteintech, 27156‐1‐AP), p‐ATM (Abcam, ab81292), β‐actin (Cell Signaling, 3700) and GAPDH (Proteintech, 60004‐1‐Ig) and followed by incubation with a secondary antibody.

Techniques: Transfection, Control, Cell Culture, Serum Assay, Colony Assay, Staining, Transwell Migration Assay, Western Blot, Expressing, Injection

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 1. Loss of AIMP3 is a feature in bladder cancer and AIMP3 promoter methylation is cancer-specific. (a) AIMP3 TMA-IHC. Top: Normal urothelium demonstrating high levels of cytoplasmic AIMP3 expression (L 320; R 3100). Bottom: AIMP3 is not expressed in an invasive T2G3 tumour (MIBC) (L 320; R 3100). In A, the dashed square indicates the area at a higher magnification (3100) in the photomicro- graphs on the right (R). (b) Distribution of AIMP3 expression levels across tumours of different stages and grades and normal tissues. There is a reduction in AIMP3 expression in cancer as compared to normal bladder tissue. A downward-shift in the percentage of moderate/high expressing tumours exists with stage/grade progression. (c) Promoter methylation of AIMP3 was detected in about 10% of bladder tumours (both MIBC and NMIBC) but was undetected in normal tissues. (d) Relative AIMP3 mRNA expression stratified by tumour stage/grade and methylation status. Methylated tumours demonstrated a significant loss in expression, while a marked difference was also observed between Ta/T1 and T2/higher tumours (p < 0.001). Bars, SE. [Color figure can be viewed in the online issue, which is available at wileyon- linelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Methylation, Expressing

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 2. Association between AIMP3 expression, Tp53 transactivity and genomic stability. (a) Relative TP53TG1 mRNA expression stratified by AIMP3 expression levels and TP53 mutation status. Low expressing AIMP3 wt TP53 tumours presented with a loss of TP53TG1 expres- sion comparable to that of mutant Tp53 tumours. Bars, SE. wt, wild-type. (b) Relative AIMP3 expression stratified by FGA. FGA of all tumours were defined as a quantitative expression of genomic instability, assessed by 1-Mb aCGH. Tumours with high levels of AIMP3 exhibited a lower fraction of genome altered (FGA) (p < 0.001). Bars, SE. (c) Dose–response curves of cell lines HeLa, T24, 253J, RT112 and RT4 irradiation (0–5Gy). Bars, SD. (d) AIMP3 protein expression increased with time upon exposure to IR as assessed by western blots and normalised with b actin. Bars, SD (e) Confocal immunofluorescence imaging showing subcellular localisation of AIMP3 in RT4 cells without irradiation (IR2) and with irradiation. (IR1) (3600) (AIMP3-FITC (green-yellow); Actin-TRITC (red-pink); Nuclear DAPI staining (blue); and merged images). Following IR, there is increased nuclear localisation of AIMP3 (Manders coefficient: 0.362 without irradiation com- pared to 0.778 with irradiation). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Expressing, Mutagenesis, Irradiation, Western Blot, Imaging, Staining

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 3. Reduction of AIMP3 in vitro is associated with increased radioresistance. (a) Western Blot demonstrating significant siRNA- mediated knockdown of AIMP3 between 24 and 96 hrs post-transfection. (b) AIMP3-knockdown cells demonstrate a significant increase in clonogenic survival following irradiation (p < 0.05*). Bars, SD. Relative to cells exposed to IR at IC50 (vertical-banded bars) and those with just transfection-medium exposure with IR at IC50 (horizontal-banded bars), those with AIMP3-knockdown and exposed to IR at IC50 (grey, unbanded bars) have a higher clonogenic survival. Untreated cells were used as reference (100%) for relative changes in clonogenic sur- vival (bars not shown). (c) Clonogenic assay images demonstrating stained and counted colonies in the respective cell lines. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: In Vitro, Western Blot, Knockdown, Transfection, Irradiation, Clonogenic Assay, Staining

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 4. Reduced expression of AIMP3 is associated with reduced survival following radiotherapy and higher risk of recurrence. (a) AIMP3 expression in the BCON TMA. Example of AIMP3-negative (top) and AIMP3-positive (bottom) tumours that were treated with either radical radiotherapy alone or radical radiotherapy supplemented with carbogen and nicotinamide. (b) Kaplan-Meier plot illustrates that the adjusted HR for death in AIMP3-positive group was half the HR for death in the AIMP3-negative group in the BCON cohort. (c) Kaplan Meier plot depicts the HR for death in patients with tumour recurrence following radiotherapy was 8.8 relative to patients without recurrence. (d) AIMP3 expression was evaluated on a radical cystectomy TMA set. Kaplan Meier plot demonstrates no significant differences in the HR for death between the AIMP3-positive and negative groups. [Color figure can be viewed in the online issue, which is available at wileyonlineli- brary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Expressing