Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 expression is negatively correlated with TNBC prognosis. A) The UMAP plot in GSE176078 and t‐SNE plot in GSE75688 were performed to visualize cell types and SAT1 expression of each cell from breast cancer patients. B) The representative images of SAT1 expressions in different subtypes (n = 5) of breast cancer from SYSUCC using IHC. C) The bar plot of IHC scores in different subtypes of breast cancer from SYSUCC. D) Comparison of the SAT1 expression across normal tissues and TNBC tissues in GSE38959, GSE45827 and GSE65194. E–G) The SAT1 expressions were detected in paired tumor‐adjacent normal tissues and TNBC tissues (n = 4) through RT‐qPCR (E), western blots (F) and IHC (G). H,I) The representative IHC images (H) of various SAT1 expressions in TNBC and the Kaplan–Meier survival analysis (I) for OS performed in 100 TNBC patients based on different SAT1 levels.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Expressing, Comparison, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 knockdown inhibits TNBC progression in vivo. A) The in vivo bioluminescence imaging and tumor images of xenograft models generated by SAT1 WT and SAT1 KD cells (n = 5). B) The tumor growth curves of mice xenograft models generated by SAT1 WT and SAT1 KD cells. C) IHC staining of EMT‐related markers in tumor tissues derived from mice xenograft models in both SAT1 WT and SAT1 KD groups. D) The in vivo bioluminescence imaging (left) and luciferase activity analysis (right) of mice liver metastases models in both SAT1 WT and SAT1 KD groups (n = 3). E,F) The bioluminescence images and HE staining of mice liver metastases (E), and statistics on the number of liver metastatic nodules (F) in both SAT1 WT and SAT1 KD groups.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Knockdown, In Vivo, Imaging, Generated, Immunohistochemistry, Derivative Assay, Luciferase, Activity Assay, Staining

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: JUN boosts SAT1 transcription by binding to its promoter sequence. A) The top 10 transcription factors (TFs) binding to SAT1 promoter were obtained from the JASPAR database. B) The relationship between SAT1 expression and the top three transcription factors was determined with Spearman's correlation analyses in TNBC samples from TCGA‐BRCA cohort. C) The binding motif of JUN on SAT1 promoter. D) Four potential sites where JUN binds to SAT1 promoter were obtained from the JASPAR database. E,F) The alterations of SAT1 in response to JUN inhibition were detected by RT‐qPCR (E) and western blots (F), respectively. G) The dual‐luciferase reporter assay was used to confirm the binding of JUN to SAT1 promoter. H) The ChIP assay with JUN antibody was employed to verify the direct binding of JUN to SAT1 promoter. I) The dual‐luciferase reporter assay reaffirmed the key binding site of JUN to SAT1 promoter sequence.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Binding Assay, Sequencing, Expressing, Inhibition, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 stabilizes YBX1 protein through deubiquitylation. A) Identification of 79 interacted proteins of SAT1 through CoIP‐MS analyses in both BT549 and SUM159PT cells. B) The silver staining after immunoblotting of SAT1 in BT549 cells. C) The interaction between SAT1 and YBX1 was verified using CoIP followed by immunoblotting. D,E) Co‐localizations of SAT1 and YBX1 in both TNBC cells (D) and tissues (E) were visualized with immunofluorescence. F) The RT‐qPCR for YBX1 mRNA expression in SAT1 WT and SAT1 KD cells. G) Western blots for YBX1 protein expression in SAT1 WT and SAT1 KD cells. H) Immunofluorescence images for YBX1 protein in SAT1 WT and SAT1 KD cells. I) Western blots for SAT1 in SAT1 KD cells with or without YBX1 overexpression. J) Western blots for YBX1 at different times (left) and quantization of half‐life time (right) of YBX1 in SAT1 WT and SAT1 KD cells in response to CHX (50 µ m ) treatment. K) Western blots for YBX1 in SAT1 WT and SAT1 KD cells after MG132 (50 µ m ) treatment for 12 h. L) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells transfected with the indicated plasmids to detect the alteration of HA‐YBX1 ubiquitylation level.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Silver Staining, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Over Expression, Transfection

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: HERC5 mediates YBX1 ubiquitination induced by SAT1 knockdown. A) The protein mass spectrometry data of YBX1 immunoprecipitants from BT549 cells. B) Co‐immunoprecipitation for indicated protein followed by immunoblots for HERC5 in BT549 cells. C) Co‐localization of HERC5 and YBX1 in BT549 cells was visualized with immunofluorescence. D) Western blots for YBX1 in BT549 cells with HERC5 inhibition or overexpression. E) Western blots for YBX1 in TNBC cells with overexpressed HERC5 in response to CHX (50 µM) treatment for different periods. F,G) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with HERC5 inhibition (F) or overexpression (G). H) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with inhibition of both SAT1 and HERC5.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Knockdown, Mass Spectrometry, Immunoprecipitation, Western Blot, Immunofluorescence, Inhibition, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 deficiency activates autophagy in TNBC. A) The Venn diagram showed pathway enrichment analyses of SAT1 in three TNBC cohorts (GSE38959, GSE45827 and GSE65194). B) The GSEA results of autophagy in the above three datasets. C,D) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells after EBSS treatment for indicated hours (C); The fold change of LC3 II was calculated by bar plot (D). E) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells with or without BafA1 (100 n m ) treatment for 12 h. F) Western blots for LC3 and p62 in indicated cells with or without chloroquine treatment for 12 h. G,H) SAT1 WT and SAT1 KD BT549 cells transfected with an mRFP‐GFP‐LC3 reporter were treated with or without BafA1 (100 n m ) for 12 h. Representative confocal images were presented G), and the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) per cell was quantified H). I) Western blots for LC3 and p62 in SAT1 KD cells with or without Baf‐A1 (100 nM) treatment in response to YBX1 overexpression. J) The representative confocal images of mRFP‐GFP‐LC3 assay in SAT1 WT and SAT1 KD BT549 cells which were transfected with or without YBX1 overexpression and treated with or without Baf‐A1 (100 nM) for 12 h.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Western Blot, Transfection, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 suppresses autophagy through YBX1‐mediated m5C modification of mTOR mRNA. A) The GSEA results of mTOR signaling pathway in a TNBC cohort‐GSE38959. B) Western blots for mTOR and its downstream effectors in SAT1 WT and SAT1 KD cells. C) Western blots for mTOR and its downstream effectors in SAT1 KD cells with or without overexpressed YBX1. D) qPCR for the mTOR mRNA expression in indicated cells with knockdown of SAT1 or YBX1. E) mRNA quantification of mTOR in BT549 cells with YBX1 knockdown or overexpression in response to actinomycin D treatment for indicated hours. F) mRNA quantification of mTOR in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression in response to actinomycin D treatment. G) The RIP‐qPCR assay to detect the binding between YBX1 and mTOR mRNA in BT549 cells. H) Lysates of BT549 cells were pulled down with biotinylated probe recognizing mTOR mRNA, and then YBX1 in the precipitates was detected by western blots. I) Dot blot assay for m5C levels in BT549 cells with YBX1 knockdown or overexpression. The intensity of dot immunoblotting (above) represented the m5C levels while methylene blue staining (below) indicated the amount of loaded RNA. J) Dot blot assay for m5C levels in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. K) The RIP‐qPCR assay to verify the binding between m5C and mTOR mRNA in BT549 cells. L) The RIP‐qPCR assay to qualify m5C enrichment levels of mTOR mRNA in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. (M) A schematic of the conclusion in this current study.

Article Snippet: The antibodies used were: anti‐SAT1 (NB110‐41622, Novus Biologicals); anti‐vimentin (10366‐1‐AP, Proteintech); anti‐slug (12129‐1‐AP, Proteintech); anti‐N‐cadherin (13116, CST); anti‐LC3A/B (12741, CST); anti‐p62 (18420‐1‐AP, Proteintech).

Techniques: Modification, Western Blot, Expressing, Knockdown, Over Expression, Binding Assay, Dot Blot, Staining

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-SAT1 , Novus Biological , Cat#110-41622.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing, Control, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 expression is negatively correlated with TNBC prognosis. A) The UMAP plot in GSE176078 and t‐SNE plot in GSE75688 were performed to visualize cell types and SAT1 expression of each cell from breast cancer patients. B) The representative images of SAT1 expressions in different subtypes (n = 5) of breast cancer from SYSUCC using IHC. C) The bar plot of IHC scores in different subtypes of breast cancer from SYSUCC. D) Comparison of the SAT1 expression across normal tissues and TNBC tissues in GSE38959, GSE45827 and GSE65194. E–G) The SAT1 expressions were detected in paired tumor‐adjacent normal tissues and TNBC tissues (n = 4) through RT‐qPCR (E), western blots (F) and IHC (G). H,I) The representative IHC images (H) of various SAT1 expressions in TNBC and the Kaplan–Meier survival analysis (I) for OS performed in 100 TNBC patients based on different SAT1 levels.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Expressing, Comparison, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 knockdown inhibits TNBC progression in vivo. A) The in vivo bioluminescence imaging and tumor images of xenograft models generated by SAT1 WT and SAT1 KD cells (n = 5). B) The tumor growth curves of mice xenograft models generated by SAT1 WT and SAT1 KD cells. C) IHC staining of EMT‐related markers in tumor tissues derived from mice xenograft models in both SAT1 WT and SAT1 KD groups. D) The in vivo bioluminescence imaging (left) and luciferase activity analysis (right) of mice liver metastases models in both SAT1 WT and SAT1 KD groups (n = 3). E,F) The bioluminescence images and HE staining of mice liver metastases (E), and statistics on the number of liver metastatic nodules (F) in both SAT1 WT and SAT1 KD groups.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Knockdown, In Vivo, Imaging, Generated, Immunohistochemistry, Derivative Assay, Luciferase, Activity Assay, Staining

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: JUN boosts SAT1 transcription by binding to its promoter sequence. A) The top 10 transcription factors (TFs) binding to SAT1 promoter were obtained from the JASPAR database. B) The relationship between SAT1 expression and the top three transcription factors was determined with Spearman's correlation analyses in TNBC samples from TCGA‐BRCA cohort. C) The binding motif of JUN on SAT1 promoter. D) Four potential sites where JUN binds to SAT1 promoter were obtained from the JASPAR database. E,F) The alterations of SAT1 in response to JUN inhibition were detected by RT‐qPCR (E) and western blots (F), respectively. G) The dual‐luciferase reporter assay was used to confirm the binding of JUN to SAT1 promoter. H) The ChIP assay with JUN antibody was employed to verify the direct binding of JUN to SAT1 promoter. I) The dual‐luciferase reporter assay reaffirmed the key binding site of JUN to SAT1 promoter sequence.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Binding Assay, Sequencing, Expressing, Inhibition, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 stabilizes YBX1 protein through deubiquitylation. A) Identification of 79 interacted proteins of SAT1 through CoIP‐MS analyses in both BT549 and SUM159PT cells. B) The silver staining after immunoblotting of SAT1 in BT549 cells. C) The interaction between SAT1 and YBX1 was verified using CoIP followed by immunoblotting. D,E) Co‐localizations of SAT1 and YBX1 in both TNBC cells (D) and tissues (E) were visualized with immunofluorescence. F) The RT‐qPCR for YBX1 mRNA expression in SAT1 WT and SAT1 KD cells. G) Western blots for YBX1 protein expression in SAT1 WT and SAT1 KD cells. H) Immunofluorescence images for YBX1 protein in SAT1 WT and SAT1 KD cells. I) Western blots for SAT1 in SAT1 KD cells with or without YBX1 overexpression. J) Western blots for YBX1 at different times (left) and quantization of half‐life time (right) of YBX1 in SAT1 WT and SAT1 KD cells in response to CHX (50 µ m ) treatment. K) Western blots for YBX1 in SAT1 WT and SAT1 KD cells after MG132 (50 µ m ) treatment for 12 h. L) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells transfected with the indicated plasmids to detect the alteration of HA‐YBX1 ubiquitylation level.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Silver Staining, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Over Expression, Transfection

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: HERC5 mediates YBX1 ubiquitination induced by SAT1 knockdown. A) The protein mass spectrometry data of YBX1 immunoprecipitants from BT549 cells. B) Co‐immunoprecipitation for indicated protein followed by immunoblots for HERC5 in BT549 cells. C) Co‐localization of HERC5 and YBX1 in BT549 cells was visualized with immunofluorescence. D) Western blots for YBX1 in BT549 cells with HERC5 inhibition or overexpression. E) Western blots for YBX1 in TNBC cells with overexpressed HERC5 in response to CHX (50 µM) treatment for different periods. F,G) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with HERC5 inhibition (F) or overexpression (G). H) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with inhibition of both SAT1 and HERC5.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Mass Spectrometry, Immunoprecipitation, Western Blot, Immunofluorescence, Inhibition, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 deficiency activates autophagy in TNBC. A) The Venn diagram showed pathway enrichment analyses of SAT1 in three TNBC cohorts (GSE38959, GSE45827 and GSE65194). B) The GSEA results of autophagy in the above three datasets. C,D) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells after EBSS treatment for indicated hours (C); The fold change of LC3 II was calculated by bar plot (D). E) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells with or without BafA1 (100 n m ) treatment for 12 h. F) Western blots for LC3 and p62 in indicated cells with or without chloroquine treatment for 12 h. G,H) SAT1 WT and SAT1 KD BT549 cells transfected with an mRFP‐GFP‐LC3 reporter were treated with or without BafA1 (100 n m ) for 12 h. Representative confocal images were presented G), and the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) per cell was quantified H). I) Western blots for LC3 and p62 in SAT1 KD cells with or without Baf‐A1 (100 nM) treatment in response to YBX1 overexpression. J) The representative confocal images of mRFP‐GFP‐LC3 assay in SAT1 WT and SAT1 KD BT549 cells which were transfected with or without YBX1 overexpression and treated with or without Baf‐A1 (100 nM) for 12 h.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Western Blot, Transfection, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 suppresses autophagy through YBX1‐mediated m5C modification of mTOR mRNA. A) The GSEA results of mTOR signaling pathway in a TNBC cohort‐GSE38959. B) Western blots for mTOR and its downstream effectors in SAT1 WT and SAT1 KD cells. C) Western blots for mTOR and its downstream effectors in SAT1 KD cells with or without overexpressed YBX1. D) qPCR for the mTOR mRNA expression in indicated cells with knockdown of SAT1 or YBX1. E) mRNA quantification of mTOR in BT549 cells with YBX1 knockdown or overexpression in response to actinomycin D treatment for indicated hours. F) mRNA quantification of mTOR in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression in response to actinomycin D treatment. G) The RIP‐qPCR assay to detect the binding between YBX1 and mTOR mRNA in BT549 cells. H) Lysates of BT549 cells were pulled down with biotinylated probe recognizing mTOR mRNA, and then YBX1 in the precipitates was detected by western blots. I) Dot blot assay for m5C levels in BT549 cells with YBX1 knockdown or overexpression. The intensity of dot immunoblotting (above) represented the m5C levels while methylene blue staining (below) indicated the amount of loaded RNA. J) Dot blot assay for m5C levels in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. K) The RIP‐qPCR assay to verify the binding between m5C and mTOR mRNA in BT549 cells. L) The RIP‐qPCR assay to qualify m5C enrichment levels of mTOR mRNA in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. (M) A schematic of the conclusion in this current study.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Modification, Western Blot, Expressing, Knockdown, Over Expression, Binding Assay, Dot Blot, Staining