primary antibodies against tim 4  (Proteintech)

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: A , B A549 cells overexpressing TIM-4 and control cells were analyzed by RNA-Seq, and cluster analysis and differentially expressed genes (false discovery rate [FDR] < 0.05, fold change ≥2) were visualized in a heatmap and volcano figure. C RNA-Seq analysis in A549 cells. GSEA was used to analyze the OXPHOS-related genes with differential mRNA levels. Normalized enrichment score (NES), P -value <0.05 is considered significant. D , E Mitochondrial oxygen consumption rate (OCR), mitochondrial basal respiration and maximal respiration of A549 cells and H23 cells. F ATP production in A549 and H23 cells overexpressing TIM-4 and the control cells. G , H A549 and H23 cells overexpressing TIM-4 and control cells were treated with rotenone (Rot) (0.5 μM) and oligomycin (Oligo) (2 μM) for 48 h, and PCNA, Cyclin A2, and Cyclin B1 expression were analyzed by western blotting, while Edu staining was measured by flow cytometry assay. Three independent experiments were conducted for each result and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed unpaired t test) for ( D – F ) and ( H ). ns means non-significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Staining, Flow Cytometry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: A , B Mitochondrial membrane potential (JC-1 aggregates) in A549 and H23 cells were accessed by the JC-1 assay kit according to the manufacture’s instruction. C , D Flow cytometry dot plots showed the percentage of depolarized mitochondria in A549 and H23 cell lines transfected with LV-CON or LV-TIM-4. E , F Mitochondria fitness was tested with Mito-tracker Deep Red. G , H Mitochondrial ROS levels of LV-A549 cells and LV-H23 cells were accessed by assay kit. I , J OCR, mitochondrial basal respiration and maximal respiration of LV-A549 cells and LV-H23 cells after fasted (Glucose:1 mM). Three independent experiments were conducted for each result and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed unpaired t test). ns means non-significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Flow Cytometry, Transfection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: A Representative confocal micrographs of mitochondrial morphology in A549 and H23 cells. Mitochondria were visualized by anti-TOM20 immunostaining (gray), Hoechst 33342 labels nuclei (blue). The scale bar represents 10 μm. B Levels of proteins involved in mitochondrial dynamics (MFN2, OPA1, p-DRP1, DRP1, and MFF) were detected by western blotting in A549 and H23 cells. C A549 and H23 cells transfected with LV-CON and LV-TIM-4 were treated with MYLS22 (50 μM) for 24 h, then cells were subjected to anti-TOM20 (gray), DAPI Hoechst 33342 staining (blue), scale bar = 10 μm. D , E OCR, mitochondrial basal respiration and maximal respiration of A549 and H23 cells treated with inhibitor of OPA1 (MYLS22: 50 μM) for 24 h. F The expression levels of cell proliferation marker PCNA and cell cycle-related proteins including Cyclin A2, Cyclin B1 were determined by western blotting in A549 and H23 cells treated with MYLS22 for 24 h. G Flow cytometry histograms showing the level of Ki67 in lung cancer cell lines treated with MYLS22 for 24 h. Three independent experiments were conducted for each result and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed unpaired t test) for ( D ), ( E ), and ( G ). ns means non-significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Immunostaining, Western Blot, Transfection, Staining, Expressing, Marker, Flow Cytometry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: A Venn diagram depicting the comparison of proteins identified in A549-LV-CON, A549-LV-TIM-4, and HEK-293 cells. The shared proteins were excluded from the differential lists. B , C Co-immunoprecipitation (Co-IP) assays were performed in A549 and H23 cells overexpressing TIM-4 or control. D , E OCR, mitochondrial basal respiration and maximal respiration of A549 cells and H23 cells transfected with siRNA of ANXA2 or control. F Mitochondrial membrane potential (JC-1 aggregates) in A549 cells transfected with siRNA targeting ANXA2 were accessed by the JC-1 assay kit according to the manufacture’s instruction. G Mitochondria fitness was tested with Mito-tracker Deep Red. H Flow cytometry dot plots showed the percentage of depolarized mitochondria in A549 cell lines transfected with siRNA targeting ANXA2. I Western blotting analysis of OPA1, p-AKT, PCNA, and ANXA2 expression in siANXA2-transfected lung cancer cell lines. J Flow cytometry histograms showing the level of Ki67 in lung cancer cell lines transfected with siRNA targeting ANXA2. Three independent experiments were conducted for each result and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed unpaired t test) for ( D ), ( E ), ( F – H ), and ( J ). ns means non-significance; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: A Expression value of TIM-4 in tumor samples of 10 patients with NSCLC (TIM-4 High , n = 6; TIM-4 Low , n = 4) (Gene Expression Omnibus accession no. GSE168466). B Uniform Manifold Approximation and Projection (UMAP) showed significant differences between the two groups. C Normalization of the data analyzed by GEO2R. D Volcano plot of differentially expressed genes in tumor samples of 10 patients with NSCLC. E Heatmap of oxidative phosphorylation related genes expression in two groups. F Expression values of OXPHOS-related genes in TIM-4 High and TIM-4 Low patients.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: TIM-4 orchestrates mitochondrial homeostasis to promote lung cancer progression via ANXA2/PI3K/AKT/OPA1 axis

doi: 10.1038/s41419-023-05678-3

Figure Lengend Snippet: TIM-4 interacts with ANXA2 to activate PI3K/AKT signaling and promotes L-OPA1 expression. When TIM-4 expression is elevated, AKT activation is enhanced to maintain L-OPA1 levels and enhance mitochondrial fitness. The maintenance of L-OPA1 leads to a more fusion state of mitochondria, which leads to increased OXPHOS levels, thus promoting lung cancer cell proliferation.

Article Snippet: The membranes were incubated in blocking buffer 5% (w/v) Albumin Bovine V (A8850-5g, Solarbio) in Tris-buffered saline, 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibodies against TIM-4 (1:1000, HPA015625, Sigma), PCNA (1:1000, ab92552, Abcam), Cyclin A2 (1:5000, 18202-1-AP, Proteintech), Cyclin B1 (1:2000, 4138T, CST), β-ACTIN (1:5000, 66009-1-Ig, Proteintech), OPA1 (1:1000, ab42364, Abcam), MFN2 (1:1000, A12771, Abclonal), MFF (1:1000, A4874, Abclonal), DRP1 (1:1000, AP0812, Abclonal), Phospho-Akt (Ser473) (1:1000, 4058s, CST), AKT (1:1000, ab18785, CST), FLAG (1:5000, M185-3, MBL), ANXA2 (1:1000, ab41803, Abcam) and GAPDH (1:5000, 60004-1-Ig, Proteintech) for 16 h at 4 °C.

Techniques: Expressing, Activation Assay