rabbit anti atp5a  (Cell Signaling Technology Inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 4. MTA1 interacts with ATP5A. A) Interaction network of 24 mitochondria-associated proteins that bind MTA1 identified by LC–MS/MS. The shade of color indicates the number of proteins interacting with that protein. B) Schematic of the distribution of MTA1-binding proteins, which are highlighted with different colors on ATP synthase. C) Simulation of the interaction between amino acids 670-695 of MTA1 and ATP synthase in state 2. Binding sites are indicated by black boxes and magnified. MTA1 peptides are shown in orange (top) and pink (bottom). The ATP synthase 𝛼subunit is shown in green. The ATP synthase 𝛾subunit is shown in purple. D) Average binding energy of the interaction between MTA1 peptides and ATP synthase

Article Snippet: The coverslips with cells were subsequently incubated overnight with a mouse monoclonal MTA1 primary antibody (ab51266, Abcam) and rabbit antibody against ATP5A (14676-1- AP, Proteintech); and then, incubated with corresponding fluorescenceconjugated secondary antibodies diluted in blocking buffer for 1 h. The coverslips with cells were finally mounted with mounting medium containing DAPI.

Techniques: Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 5. MTA1 promotes the malignant phenotype of CRC via binding with ATP5A. A) The proliferation of MTA1-OE and control HCT116 cells treated with 2.5 mm oligomycin. The values are the mean ± SD (****p < 0.0001), two-way ANOVA, Tukey’s multiple comparisons test, n = 2 replicate per group. B,C) The colony formation ability of MTA1-OE and control HCT116 cells treated with 2 μm oligomycin for 10 days. The values are the mean ± SD (***p < 0.001; ns, not significant), two-way ANOVA, Sidak’s multiple comparisons test, n = 3 replicate/group. D,E) The invasion of MTA1-OE and control HCT116 cells treated with 2 μm oligomycin for 48 h. The values are the mean ± SD (**p < 0.01; ns, not significant), two-way ANOVA, Sidak’s multiple comparisons test, n = 3 replicate/group. Scale bar: 100 μm. F) Western blotting was performed to verify that ATP5A was knocked down in MTA1-OE and control HCT116 cells, and the expression of metastasis related markers in MTA1-OE/ATP5A-KD HCT116 cells. G–I) The proliferation (G) and invasion (H,I) of MTA1-OE/ATP5A-KD and control HCT116 cells. The cells were incubated in transwell plates for 48 h. The values are the mean ± SD (****p < 0.0001, ***p < 0.001; ns, not significant), two-way ANOVA, Tukey’s multiple comparisons test, n = 3 replicate/group. Scale bar: 200 μm. J,K) The ECAR (J) and OCR (K) of MTA1-OE/ATP5A-KD and control HCT116 cells. The values are the mean ± SD (****p < 0.0001, ***p<0.001, and *p<0.05), two-way ANOVA, Tukey’s multiple comparisons test. L) Representative image of the liver metastatic burden in BALB/c-nu/nu mice injected with MTA1-OE/ATP5A-KD and control cells via the spleen tail. The group names are shown in (M); n = 3 mice/group. M) The liver weight of mice with liver metastasis shown in (L). The values are the mean ± SD (*p < 0.05; ns, not significant), one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: The coverslips with cells were subsequently incubated overnight with a mouse monoclonal MTA1 primary antibody (ab51266, Abcam) and rabbit antibody against ATP5A (14676-1- AP, Proteintech); and then, incubated with corresponding fluorescenceconjugated secondary antibodies diluted in blocking buffer for 1 h. The coverslips with cells were finally mounted with mounting medium containing DAPI.

Techniques: Binding Assay, Control, Western Blot, Expressing, Incubation, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 6. MTA1 increases the efficacy of mTOR inhibitors both in vitro and in vivo. A) Schematic of the experiment. MTA1-KO and control HCT116 cells were seeded in 384-well plates (800 cells per well) 16 h before the experiment, and the cells were treated with 237 metabolism-related anticancer drugs for 72 h (five dilutions per drug, two replicates). Cell viability was assessed by the CCK8 assay. B) Fold change in the IC50 of metabolism-related anticancer drugs in MTA1-KO cells versus control cells. The red dots are mTOR inhibitors, and the green dots are mTOR activators. C) Western blot analysis of the levels of MTA1, mTOR, p-mTOR, AMPK𝛼, and p-AMPK𝛼in MTA1-KO cells, MTA1-OE cells, and control HCT116 cells. D) IC50 curves of the mTOR inhibitors AZD8055, temsirolimus, zotarolimus, and Torin-1 in MTA1-KO and control HCT116 cells for individual pharmacological validation. The values are the mean ± SD. E) The viability of MTA1-KO and control HCT116 cells treated with 5 μm temsirolimus. The values are the mean ± SD (****p < 0.0001), two-way ANOVA, and Tukey’s multiple comparisons test. F) Image of liver metastatic burden in the eight groups of BALB/c-nu/nu mice injected with MTA1-OE/ATP5A-KD via the spleen tail. The mice were injected intraperitoneally with 100 μL of 8.8 mg mL−1 rapamycin every other day 2 weeks after spleen-tail injection; n = 5 mice per group. G) The liver weight of mice with liver metastasis shown in (F). The values are the mean ± SD (****p < 0.0001, **p < 0.01; ns, not significant), one-way ANOVA, and Tukey’s multiple comparisons test. H) Representative HE staining images of the liver tumors of the mice in (F). Scale bar: 3 mm.

Article Snippet: The coverslips with cells were subsequently incubated overnight with a mouse monoclonal MTA1 primary antibody (ab51266, Abcam) and rabbit antibody against ATP5A (14676-1- AP, Proteintech); and then, incubated with corresponding fluorescenceconjugated secondary antibodies diluted in blocking buffer for 1 h. The coverslips with cells were finally mounted with mounting medium containing DAPI.

Techniques: In Vitro, In Vivo, Control, CCK-8 Assay, Western Blot, Biomarker Discovery, Injection, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 7. MTA1 and ATP5A levels are correlated with sensitivity to sirolimus in CRC patients. A) Representative images of IHC staining for ATP5A in human tumors and adjacent tissues on 180 CRC tissue arrays. The sample dots (left) were magnified (right) three times. Scale bar: 600 μm. B–D) ATP5A (B), mTOR (C), and p-mTOR (D) IHC staining scores in 180 paired CRC tissues (****p < 0.0001), Wilcoxon matched-pairs signed rank test. E) Correlation plot showing the Spearman correlation between MTA1 levels and ATP5A levels, mTOR staining, and p-mTOR staining in 180 paired CRC tissues (R = 0.23, p = 0.002), Spearman’s rank order correlation coefficient. F) Histogram of cytoplasmic MTA1 protein levels in 58 sirolimus- or everolimus-treated responder and nonresponder breast cancer patients. The Chi-square test was used to compare the IHC staining score between the above two groups. p < 0.05 was considered significant. G) Representative images of IHC staining for MTA1, ATP5A, mTOR, and p-mTOR proteins in 58 sirolimus- or everolimus-treated responder and nonresponder breast cancer patients. Scale bar: 50 μm.

Article Snippet: The coverslips with cells were subsequently incubated overnight with a mouse monoclonal MTA1 primary antibody (ab51266, Abcam) and rabbit antibody against ATP5A (14676-1- AP, Proteintech); and then, incubated with corresponding fluorescenceconjugated secondary antibodies diluted in blocking buffer for 1 h. The coverslips with cells were finally mounted with mounting medium containing DAPI.

Techniques: Immunohistochemistry, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 4. MTA1 interacts with ATP5A. A) Interaction network of 24 mitochondria-associated proteins that bind MTA1 identified by LC–MS/MS. The shade of color indicates the number of proteins interacting with that protein. B) Schematic of the distribution of MTA1-binding proteins, which are highlighted with different colors on ATP synthase. C) Simulation of the interaction between amino acids 670-695 of MTA1 and ATP synthase in state 2. Binding sites are indicated by black boxes and magnified. MTA1 peptides are shown in orange (top) and pink (bottom). The ATP synthase 𝛼subunit is shown in green. The ATP synthase 𝛾subunit is shown in purple. D) Average binding energy of the interaction between MTA1 peptides and ATP synthase

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense with an endogenous peroxidase blocker (PV-9000, Zsgb-Bio) for 10 min to block endogenous peroxidase activity, and incubated with normal goat serum (ZLI-9022, Zsgb-Bio) for 10 min. Next, the slides were incubated overnight with rabbit monoclonal primary antibodies against ATP5A (14676-1-AP, Proteintech), mTOR (#2972, Cell Signaling Technology), and phosphorylated mTOR (#2971, Cell Signaling Technology) at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 5. MTA1 promotes the malignant phenotype of CRC via binding with ATP5A. A) The proliferation of MTA1-OE and control HCT116 cells treated with 2.5 mm oligomycin. The values are the mean ± SD (****p < 0.0001), two-way ANOVA, Tukey’s multiple comparisons test, n = 2 replicate per group. B,C) The colony formation ability of MTA1-OE and control HCT116 cells treated with 2 μm oligomycin for 10 days. The values are the mean ± SD (***p < 0.001; ns, not significant), two-way ANOVA, Sidak’s multiple comparisons test, n = 3 replicate/group. D,E) The invasion of MTA1-OE and control HCT116 cells treated with 2 μm oligomycin for 48 h. The values are the mean ± SD (**p < 0.01; ns, not significant), two-way ANOVA, Sidak’s multiple comparisons test, n = 3 replicate/group. Scale bar: 100 μm. F) Western blotting was performed to verify that ATP5A was knocked down in MTA1-OE and control HCT116 cells, and the expression of metastasis related markers in MTA1-OE/ATP5A-KD HCT116 cells. G–I) The proliferation (G) and invasion (H,I) of MTA1-OE/ATP5A-KD and control HCT116 cells. The cells were incubated in transwell plates for 48 h. The values are the mean ± SD (****p < 0.0001, ***p < 0.001; ns, not significant), two-way ANOVA, Tukey’s multiple comparisons test, n = 3 replicate/group. Scale bar: 200 μm. J,K) The ECAR (J) and OCR (K) of MTA1-OE/ATP5A-KD and control HCT116 cells. The values are the mean ± SD (****p < 0.0001, ***p<0.001, and *p<0.05), two-way ANOVA, Tukey’s multiple comparisons test. L) Representative image of the liver metastatic burden in BALB/c-nu/nu mice injected with MTA1-OE/ATP5A-KD and control cells via the spleen tail. The group names are shown in (M); n = 3 mice/group. M) The liver weight of mice with liver metastasis shown in (L). The values are the mean ± SD (*p < 0.05; ns, not significant), one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense with an endogenous peroxidase blocker (PV-9000, Zsgb-Bio) for 10 min to block endogenous peroxidase activity, and incubated with normal goat serum (ZLI-9022, Zsgb-Bio) for 10 min. Next, the slides were incubated overnight with rabbit monoclonal primary antibodies against ATP5A (14676-1-AP, Proteintech), mTOR (#2972, Cell Signaling Technology), and phosphorylated mTOR (#2971, Cell Signaling Technology) at 4 °C.

Techniques: Binding Assay, Control, Western Blot, Expressing, Incubation, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 6. MTA1 increases the efficacy of mTOR inhibitors both in vitro and in vivo. A) Schematic of the experiment. MTA1-KO and control HCT116 cells were seeded in 384-well plates (800 cells per well) 16 h before the experiment, and the cells were treated with 237 metabolism-related anticancer drugs for 72 h (five dilutions per drug, two replicates). Cell viability was assessed by the CCK8 assay. B) Fold change in the IC50 of metabolism-related anticancer drugs in MTA1-KO cells versus control cells. The red dots are mTOR inhibitors, and the green dots are mTOR activators. C) Western blot analysis of the levels of MTA1, mTOR, p-mTOR, AMPK𝛼, and p-AMPK𝛼in MTA1-KO cells, MTA1-OE cells, and control HCT116 cells. D) IC50 curves of the mTOR inhibitors AZD8055, temsirolimus, zotarolimus, and Torin-1 in MTA1-KO and control HCT116 cells for individual pharmacological validation. The values are the mean ± SD. E) The viability of MTA1-KO and control HCT116 cells treated with 5 μm temsirolimus. The values are the mean ± SD (****p < 0.0001), two-way ANOVA, and Tukey’s multiple comparisons test. F) Image of liver metastatic burden in the eight groups of BALB/c-nu/nu mice injected with MTA1-OE/ATP5A-KD via the spleen tail. The mice were injected intraperitoneally with 100 μL of 8.8 mg mL−1 rapamycin every other day 2 weeks after spleen-tail injection; n = 5 mice per group. G) The liver weight of mice with liver metastasis shown in (F). The values are the mean ± SD (****p < 0.0001, **p < 0.01; ns, not significant), one-way ANOVA, and Tukey’s multiple comparisons test. H) Representative HE staining images of the liver tumors of the mice in (F). Scale bar: 3 mm.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense with an endogenous peroxidase blocker (PV-9000, Zsgb-Bio) for 10 min to block endogenous peroxidase activity, and incubated with normal goat serum (ZLI-9022, Zsgb-Bio) for 10 min. Next, the slides were incubated overnight with rabbit monoclonal primary antibodies against ATP5A (14676-1-AP, Proteintech), mTOR (#2972, Cell Signaling Technology), and phosphorylated mTOR (#2971, Cell Signaling Technology) at 4 °C.

Techniques: In Vitro, In Vivo, Control, CCK-8 Assay, Western Blot, Biomarker Discovery, Injection, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

doi: 10.1002/advs.202300756

Figure Lengend Snippet: Figure 7. MTA1 and ATP5A levels are correlated with sensitivity to sirolimus in CRC patients. A) Representative images of IHC staining for ATP5A in human tumors and adjacent tissues on 180 CRC tissue arrays. The sample dots (left) were magnified (right) three times. Scale bar: 600 μm. B–D) ATP5A (B), mTOR (C), and p-mTOR (D) IHC staining scores in 180 paired CRC tissues (****p < 0.0001), Wilcoxon matched-pairs signed rank test. E) Correlation plot showing the Spearman correlation between MTA1 levels and ATP5A levels, mTOR staining, and p-mTOR staining in 180 paired CRC tissues (R = 0.23, p = 0.002), Spearman’s rank order correlation coefficient. F) Histogram of cytoplasmic MTA1 protein levels in 58 sirolimus- or everolimus-treated responder and nonresponder breast cancer patients. The Chi-square test was used to compare the IHC staining score between the above two groups. p < 0.05 was considered significant. G) Representative images of IHC staining for MTA1, ATP5A, mTOR, and p-mTOR proteins in 58 sirolimus- or everolimus-treated responder and nonresponder breast cancer patients. Scale bar: 50 μm.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense with an endogenous peroxidase blocker (PV-9000, Zsgb-Bio) for 10 min to block endogenous peroxidase activity, and incubated with normal goat serum (ZLI-9022, Zsgb-Bio) for 10 min. Next, the slides were incubated overnight with rabbit monoclonal primary antibodies against ATP5A (14676-1-AP, Proteintech), mTOR (#2972, Cell Signaling Technology), and phosphorylated mTOR (#2971, Cell Signaling Technology) at 4 °C.

Techniques: Immunohistochemistry, Staining