Journal: The Journal of biological chemistry

Article Title: DNA repair protein RAD52 is required for protecting G-quadruplexes in mammalian cells.

doi: 10.1016/j.jbc.2022.102770

Figure Lengend Snippet: Figure 4. Mitotic recombination at G4s induced by G4-stabilizing drugs or FANCJ deficiency is RAD52-dependent. A, U2OS [HR-EGFP (TPG4)] cells were depleted for XPF or RAD52 by shRNAs with vector (Vec) as control. Mitotic recombination frequency was determined by FACS analysis before (No) and after PDS (50 μM, 72 h) treatment (top). RAD52 and XPF depletion is shown by Western blot with KU70 as a loading control (bottom). B, U2OS [HR-EGFP (TPG4)] cells were depleted for RAD52 by shRNAs using vector (Vec) as the control, followed by depleting FANCJ with shRNAs or expressing shRNA vector (Vec). Mitotic recombination frequency was determined by FACS analysis 4 days after infection of FANCJ shRNA lentiviruses (top). RAD52 and FANCJ depletion is shown by Western blot with KU70 as a loading control (bottom). C, U2OS [HR-EGFP (TPG4)] cells were depleted for MUS81 by shRNAs with vector (Vec) as the control. Mitotic recombination frequency was determined by FACS analysis before (No) and after PDS (50 μM, 72 h) treatment (left). MUS81 depletion is shown by Western blot with KU70 as a loading control (right). D, U2OS and MUS81KO U2OS cells were treated with or without PDS (50 μM, 48 h), followed by γ-H2AX Western blot analysis using GAPDH as the loading control (left). MUS81 Western blot with KU70 as a loading control is present to show MUS81KO (right). In all experiments, error bars represent the SD of at least three independent experiments. EGFP, enhanced GFP; FACS, fluorescence activated cell sorting; G4, G-quadruplexes; HR, homologous recombination; PDS, pyridostatin.

Article Snippet: Commercial antibodies used are as follows: XPF (ABclonal Science, Inc, A8119), MUS81 (Santa Cruz Biotechnology Inc, sc376661), H2AX-S139p (Cell Signaling Technology, #2577), RAD52 (ABclonal Science, Inc, A5186), FANCJ (ABclonal Science, Inc, A6804), KU70 (Santa Cruz Biotechnology, Inc, sc17789), and GAPDH (ABclonal Science, Inc, AC002).

Techniques: Plasmid Preparation, Control, Western Blot, Expressing, shRNA, Infection, FACS, Homologous Recombination

Journal: The Journal of biological chemistry

Article Title: DNA repair protein RAD52 is required for protecting G-quadruplexes in mammalian cells.

doi: 10.1016/j.jbc.2022.102770

Figure Lengend Snippet: Figure 5. XPF recruitment to G4 is dependent on RAD52. A, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-RAD52 before and after PDS (50 μM, 48 h) or CX-5461 (1 μM, 48 h) treatment. Enrichment of RAD52 at G4 was calculated using the ChIP value in PDS- untreated cells as 1 for normalization. B, U2OS [HR-EGFP (TPG4)] cells expressing Flag-RAD52 were depleted for MUS81 by shRNAs with vector (Vec) as the control. Anti-Flag ChIP analysis at G4 locus was performed before and after CX-5461 (1 μM, 48 h) treatment. Enrichment of XPF at G4 locus was calculated using the ChIP value in CX-5461–untreated cells with vector control as 1 for normalization (left). MUS81 depletion is shown by Western blot with KU70 as a loading control (right). C, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-XPF before and after PDS (50 μM, 48 h) or CX-5461 (1 μM, 48 h) treatment. Enrichment of XPF at G4 was calculated using the ChIP value in untreated cells as 1 for normalization. D, anti-Flag ChIP analysis at G4 locus was performed in U2OS [HR-EGFP (TPG4)] cells expressing Flag-XPF and with or without depletion of RAD52 by shRNAs upon PDS (50 μM, 48 h) treatment. Enrichment of XPF at G4 was calculated using the ChIP value in the vector control as 1 for normalization (left). RAD52 depletion is shown by Western blot with KU70 as a loading control (right). In all experiments, error bars represent the SD of at least three independent experiments. ChIP, chromatin immunoprecipitation; EGFP, enhanced GFP; G4, G-quadruplexes; HR, homologous recombination; PDS, pyridostatin.

Article Snippet: Commercial antibodies used are as follows: XPF (ABclonal Science, Inc, A8119), MUS81 (Santa Cruz Biotechnology Inc, sc376661), H2AX-S139p (Cell Signaling Technology, #2577), RAD52 (ABclonal Science, Inc, A5186), FANCJ (ABclonal Science, Inc, A6804), KU70 (Santa Cruz Biotechnology, Inc, sc17789), and GAPDH (ABclonal Science, Inc, AC002).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Chromatin Immunoprecipitation, Homologous Recombination

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: Effects of Arg and Leu on the location of mTOR on LAMP2-positive lysosomal clusters, activation of mTORC1 and expression of SLC38A9 and SLC36A1 in C2C12 cells. ( A ) C2C12 cells were pretreated without serum for 15 h and without amino acids for 3 h then cultured for 10 min in a special medium including the no amino acids group (−AA), the only Arg group (+Arg), and the only Leu group (+Leu). Cells were stained with anti-mTOR (A2445, Abclonal Technology, Wuhan, China) or anti-LAMP2. Scale bar: 10 µM. ( B ) Ratio of mTOR to LAMP2 (%). To assess mTOR translocation, the numbers of mTOR and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. ( C ) Immunoblotting analysis of protein samples from ( A ) with anti-mTOR, anti-phospho-mTOR, anti-S6K, anti-phospho-S6K, anti-SLC38A9, anti-SLC36A1, or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of mTOR relative to β-Actin loading control. ( E ) Densitometric analysis of the immunodetection of phospho-mTOR relative to β-Actin loading control. ( F ) Densitometric analysis of the immunodetection of p70S6K relative to β-Actin loading control. ( G ) Densitometric analysis of the immunodetection of phospho-p70S6K relative to β-Actin loading control. ( H ) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. ( I ) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. ( J ) The mRNA levels of SLC38A9 and SLC36A1 were analyzed by qRT-PCR. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Cell Culture, Staining, Translocation Assay, Software, Western Blot, Immunodetection, Control, Quantitative RT-PCR

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: The effects of SLC38A9 or SLC36A1 overexpression and inhibition on mTOR phosphorylation in C2C12 cells. ( A ) The mRNA expression of SLC38A9 after overexpression of SLC38A9. ( B ) The mRNA expression of SLC38A9 after inhibition of SLC38A9. ( C ) The mRNA expression of SLC36A1 after overexpression of SLC36A1. ( D ) The mRNA expression of SLC36A1 after inhibition of SLC36A1. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct or siRNA to overexpress or knockdown SLC38A9. Immunoblotting analysis of protein samples with anti-SLC38A9, anti-mTOR, anti-phospho-mTOR, or anti-β-Actin antibody. ( F ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct or siRNA to overexpress or knockdown SLC36A1. Immunoblotting analysis of protein samples with anti-SLC36A1, anti-mTOR, anti-phospho-mTOR, or anti-β-Actin antibody. ( G ) Densitometric analysis of the immunodetection of SLC38A9, mTOR or phospho-mTOR relative to β-Actin loading control from ( E ). ( H ) Densitometric analysis of the immunodetection of SLC36A1, mTOR or phospho-mTOR relative to β-Actin loading control from ( F ). Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Inhibition, Phospho-proteomics, Expressing, Transfection, Construct, Knockdown, Western Blot, Immunodetection, Control

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC38A9 increases SLC36A1 expression and promotes the translocation of SLC36A1 to LAMP2-positive lysosomal clusters. ( A ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct to overexpress SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. ( B ) C2C12 cells were transfected with siRNA to knockdown SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. ( C ) Immunoblotting analysis of protein samples from ( A , B ) with anti-SLC36A1 or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct or siRNA to overexpress or knockdown SLC38A9 and stained with anti-SLC36A1 or anti-LAMP2. Scale bar: 10µM. ( F ) Ratio of SLC36A1 to LAMP2 (%). To assess SLC36A1 translocation, the numbers of SLC36A1 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Transfection, Construct, Quantitative RT-PCR, Knockdown, Western Blot, Immunodetection, Control, Staining, Software

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC36A1 increases SLC38A9 expression and promotes the translocation of SLC38A9 to LAMP2-positive lysosomal clusters. ( A ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct to overexpress SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. ( B ) C2C12 cells were transfected with siRNA to knockdown SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. ( C ) Immunoblotting analysis of protein samples from ( A , B ) with anti-SLC38A9 or anti-β-Actin antibody. ( D ) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. ( E ) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct or siRNA to overexpress or knockdown SLC36A1 and stained with anti-SLC38A9 or anti-LAMP2. Scale bar: 10µM. ( F ) Ratio of SLC38A9 to LAMP2 (%). To assess SLC38A9 translocation, the numbers of SLC38A9 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Transfection, Construct, Quantitative RT-PCR, Knockdown, Western Blot, Immunodetection, Control, Staining, Software

Journal: Biomolecules

Article Title: Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

doi: 10.3390/biom11091314

Figure Lengend Snippet: SLC38A9 and SLC36A1 interact. ( A ) SLC38A9 was associated with SLC36A1 at the endogenous level as detected by Co-IP: anti-SLC36A1 (sc-161150, Santa Cruz) was used for pull-down, and anti-SLC38A9 (ab81687, Abcam) and anti-SLC36A1 (sc-368553, Santa Cruz) were used for detection. ( B ) Interaction of SLC38A9 and SLC36A1: C2C12 cells were transfected with SLC38A9 or SLC36A1 in the pCMV-HA vector, and the lysates were prepared and subjected to HA immunoprecipitation followed by immunoblotting for the indicated proteins. ( C ) C2C12 cells were pretreated without serum for 15 h, and without amino acids for 3 h, then cultured for 10 min in a special medium including the no amino acids group (−AA), the only Arg group (+Arg), the only Leu group (+Leu). The interaction of SLC38A9 and SLC36A1 was detected by Co-IP as ( A ). ( D ) Densitometric analysis of the immunodetection of SLC38A9 relative to input control or SLC36A1. Values are the mean ± SEM; n = 3; * p < 0.05.

Article Snippet: The primary antibodies were against SLC38A9 (1:200, ab130398, Abcam, Cambridge, MA, USA), SLC36A1 (1:100, sc-368553, Santa Cruz Biotechnology, Dallas, TX, USA), mTOR (1:100, A2445, Abclonal Technology, Wuhan, China; 1:200, 2983, Cell Signaling Technology, Danvers, MA, USA), and LAMP2 (1:100, sc-20004, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, Immunodetection, Control