Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-LAMP2 , CST, USA , Cat #49067.

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: See also Fig. . ( A ) Immunoblotting of nuclear, cytoplasmic (membrane components removed), and membrane fractions prepared from MDA-MB-231 cells transfected with the indicated constructs against P27KIP1, TRIM21, FIBRILLARIN (nuclear marker), β-Tubulin (cytoplasmic marker), ATP1V1A (membrane marker), and Flag ( n = 3 independent biological samples). ( B ) Immunoblotting of whole-cell extracts (left panel), cytoplasmic (lysosome components removed) and lysosomal extracts (right panel) prepared from MDA-MB-231 cells transfected with the indicated constructs against TRIM21, P27KIP1, Flag, GAPDH, LAMP2 (lysosomal marker), and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( C ) Heatmap shows the relative expression analysis of the displayed experiment for TRIM21 and P27KIP1 to β-Tubulin (cytoplasmic fractions) or LAMP2 (lysosomal fractions) from panel ( B ) ( n = 3 independent biological samples). ( D ) Immunoblotting of whole-cell extracts (left panel), cytoplasmic (lysosome components removed) and lysosomal extracts (Right panel) prepared from MDA-MB-231 cells transfected with siCtrl or indicated siTRIM21s against TRIM21, P27KIP1, GAPDH, LAMP2 (lysosomal marker), and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( E ) Volcano plot showing differential amino acid metabolite levels between MDA-MB-231 (right panel) or HEK293T cells (left panel) transfected with ΔATG1 + 2 and Flag-hSPAR ( n = 3 independent biological samples). The regulated metabolites between ΔATG1 + 2 and Flag-hSPAR are determined by fold change analysis. The significance is presented as P value and analyzed using hypergeometric test. Red dots, significantly upregulated metabolites. Green dots, significantly downregulated metabolites. Black dots, non-different metabolites. ( F ) Levels of glutamine in MDA-MB-231 or HEK293T cells transfected with Vector Ctrl, ΔATG1 + 2 or Flag-hSPAR ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( G ) Co-immunofluorescence staining of P27KIP1 (green) and the lysosomal marker LAMP1 (red) in MDA-MB-231 cells cultured with or without glutamine. Cells were permeabilized with digitonin to remove the soluble P27KIP1. Nuclei were stained with Hoechst (blue). The graphs display the fluorescence intensity (arbitrary units) of P27KIP1 and LAMP1 over the distance from adjacent image (depicted by the arrows). The graphs display the values of Pearson’s correlation Rr of P27KIP1 and LAMP1 with or without glutamine. Scale bar, 25 µm ( n = 3 independent biological samples). ( H ) Immunoblotting of whole-cell extracts (Left panel), cytoplasmic (lysosome components removed) and lysosomal extracts (right panel) prepared from MDA-MB-231 cells cultured with or without glutamine against P27KIP1, GAPDH, LAMP2 (lysosomal marker) and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( I ) The protein levels of SLC38A2 detected by immunoblotting with the anti-SLC38A2 antibody after transfection with Vector Ctrl, ΔATG1 + 2 or Flag-hSPAR in MDA-MB-231 and HEK293T cells. GAPDH, loading control ( n = 3 independent biological samples). ( J ) Levels of glutamine in MDA-MB-231 cells in the presence of siCtrl, si hSPAR s, siSLC38A2s, or si hSPAR s together with siSLC38A2s ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( K ) Immunoblotting of whole-cell extracts (left panel), cytoplasmic (lysosome components removed) and lysosomal extracts (right panel) prepared from MDA-MB-231 cells transfected with siCtrl or indicated siSLC38A2s against SLC38A2, P27KIP1, GAPDH, LAMP2 (lysosomal marker), and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). The hSPAR-regulated proteins shown by immunoblotting are marked by red text. .

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Western Blot, Membrane, Transfection, Construct, Marker, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Cell Culture, Fluorescence, Control

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: ( A ) Levels of glutamine in MCF10A cells transfected with Vector Ctrl, ΔATG1 + 2 or Flag-hSPAR ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( B ) Co-immunofluorescence staining of P27KIP1 (green) and Lyso-Tracker (red) in MDA-MB-231 cells cultured with or without glutamine. Cells were permeabilized with digitonin to remove the soluble P27KIP1. Nuclei were stained with Hoechst (blue). The graphs display the fluorescence intensity (arbitrary units) of P27KIP1 and Lyso-Tracker over the distance from adjacent image (depicted by the arrows). Scale bar, 5 µm ( n = 3 independent biological samples). ( C ) Immunoblotting against SLC7A1, SLC7A5, Flag and GAPDH in extracts from MDA-MB-231 cells transfected with Vector Ctrl, ΔATG1 + 2, or Flag-hSPAR ( n = 3 independent biological samples). ( D ) Quantified relative levels of SLC7A1/GAPDH and SLC7A5/GAPDH from panel ( C ) ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. ( E ) Immunoblotting of whole-cell extracts (upper panel), cytoplasmic (lysosome components removed) and lysosomal extracts (lower panel) prepared from MDA-MB-231 cells cultured with or without arginine, leucine or glutamine against P27KIP1, GAPDH, LAMP2 (lysosomal marker) and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( F ) Changes of interaction between P27KIP1 and TRIM21, P27KIP1 and LAMTOR1 were detected by Co-IP and immunoblotting in the indicated fractions extracts from MDA-MB-231 cells after transfection with the indicated constructs. Left, immunoblotting of inputs. Right, immunoblotting using antibodies against P27KIP1, TRIM21 and LAMTOR1 following IP of P27KIP1 ( n = 3 independent biological samples).

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Cell Culture, Fluorescence, Western Blot, Marker, Co-Immunoprecipitation Assay, Construct

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: See also Figs. and . ( A ) Immunoblotting of whole-cell extracts (left panel), cytoplasmic (lysosome components removed) and lysosomal extracts (right panel) prepared from MDA-MB-231 cells transfected with the indicated constructs against p-mTOR, mTOR, RagA, LAMTOR1-5, Flag, GAPDH, LAMP2 (lysosomal marker), and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( B ) Heatmap shows the relative expression analysis of the displayed experiment for each protein to β-Tubulin (cytoplasmic fractions) or LAMP2 (lysosomal fractions) from panel ( A ) ( n = 3 independent biological samples). ( C ) Interaction of LAMTOR1 with P27KIP1, LAMTOR2-5, RagA and mTOR, or P27KIP1 with LAMTOR1 detected by Co-IP and immunoblotting from MDA-MB-231 cells transfected with the indicated constructs. Left: immunoblotting of inputs. Upper right: immunoblotting using antibodies against LAMTOR1 and P27KIP1 after IP of P27KIP1. Lower right: immunoblotting using antibodies against P27KIP1, LAMTOR1-5, RagA and mTOR after IP of LAMTOR1 ( n = 3 independent biological samples). ( D ) Immunoblotting of cytoplasmic (lysosome components removed) and lysosomal extracts (right panel) prepared from MDA-MB-231 cells transfected with siCtrl, indicated siTRIM21s, or siTRIM21s together with siSLC38A2s against P27KIP1, TRIM21, SLC38A2, p-mTOR, mTOR, RagA, LAMTOR1-5, LAMP2 (lysosomal marker) and β-Tubulin (cytoplasmic marker) ( n = 3 independent biological samples). ( E ) Heatmap shows the relative expression analysis of the displayed experiment for each protein to β-Tubulin (cytoplasmic fractions) or LAMP2 (lysosomal fractions) from panel ( D ) ( n = 3 independent biological samples). ( F ) Immunoblotting against TRIM21, SLC38A2, p-mTOR, mTOR, p-S6K, S6K, p-S6, S6 and GAPDH for extracts from MDA-MB-231 cells transfected with siCtrl, indicated siTRIM21s or siSLC38A2s, or siTRIM21s together with siSLC38A2s ( n = 3 independent biological samples). ( G ) Quantified relative levels of p-mTOR/mTOR, p-S6K/S6K, and p-S6/S6 from panel ( F ) ( n = 3 independent biological samples). Data are presented as the mean ± SEM and analyzed using one-way ANOVA with Dunnett’ multiple comparisons test. The hSPAR-regulated proteins shown by immunoblotting are marked by red text. .

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Western Blot, Transfection, Construct, Marker, Expressing, Co-Immunoprecipitation Assay

Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: DNAJC6 deficiency causes downregulated expression of clathrin heavy chain, LAMP1, LAMP2 or cathepsin D and upregulated expression of p62/SQSTM1 in dopaminergic neurons. After transfection of shRNAs targeting DNAJC6 for 3 days, the protein level of cytosolic clathrin heavy chain was significantly decreased in dopaminergic neurons. Cytosolic protein levels of lysosomal marker proteins LAMP1 or LAMP2 and lysosomal aspartic protease cathepsin D were downregulated in DNAJC6 shRNA-transfected dopaminergic neurons. The protein level of cytosolic autophagy receptor p62/SQSTM1 was upregulated in dopaminergic neurons transfected with shRNAs targeting DNAJC6. Each bar shows the mean ± S.E. value of 6 experiments. ** p < 0.01 compared with control dopaminergic neurons.

Article Snippet: Then, dopaminergic neurons were incubated at 4 °C overnight with diluted rabbit anti-LAMP2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) and interacted with Alexa Fluor 488-conjugated secondary antibody.

Techniques: Expressing, Transfection, Marker, shRNA, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: Paucity of DNAJC6 decreases LysoTracker staining intensity of lysosomes and immunofluorescence staining intensity of LAMP2-positive lysosomes in dopaminergic neurons. ( A ) Live cell imaging of lysosomes was conducted by treating differentiated SH-SY5Y dopaminergic neurons with 100 nM LysoTracker Yellow HCK-123, which stains acidic compartments and visualizes lysosomes, for 1 h. A three-day transfection of shRNA1 or shRNA2 targeting DNAJC6 significantly decreased the fluorescence intensity of LysoTracker Yellow staining in dopaminergic neurons. SC shRNA failed to affect lysosomal staining of LysoTracker Yellow HCK-123. Scale bar is 50 μm. ( B ) Immunofluorescence staining of lysosomal marker protein LAMP2 was performed to visualize lysosomes of dopaminergic neurons. Following a 3-day transfection of shRNAs targeting DNAJC6, the fluorescence intensity of LAMP2-positive puncta was significantly decreased in dopaminergic neurons. SC shRNA did not affect the immunofluorescence staining intensity of LAMP2. Scale bar is 60 μm. Each bar shows the mean ± S.E. value of 6 experiments. ** p < 0.01 compared with control dopaminergic neurons.

Article Snippet: Then, dopaminergic neurons were incubated at 4 °C overnight with diluted rabbit anti-LAMP2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) and interacted with Alexa Fluor 488-conjugated secondary antibody.

Techniques: Staining, Immunofluorescence, Live Cell Imaging, Transfection, Fluorescence, shRNA, Marker, Control