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 ) Immunofluorescence analysis of hSPAR using an anti-hSPAR antibody in MDA-MB-231 cells ( n = 3 independent biological samples). Scale bar, 5 µm. ( B ) Immunoblotting against hSPAR, FIBRILLARIN (nuclear marker), β-Tubulin (cytoplasmic marker), and ATP1V1A (membrane marker) in nuclear, cytoplasmic (membrane components removed), and membrane fractions prepared from MDA-MB-231 cells (n = 3 independent biological samples). ( C ) Diagram of GFP-tagged hSPAR. ( D ) Living-cell images of GFP-hSPAR and lysosomes (Red, labeled with Lyso-Tracker) in MDA-MB-231 cells. The graphs display the fluorescence intensity (arbitrary units) of GFP-hSPAR and Lyso-Tracker over the distance from adjacent image (depicted by the arrow). The value of Pearson’s correlation Rr of SPAR and Lyso-Tracker is 0.907408. Scale bar, 5 µm, ( n = 3 independent biological samples). ( E ) Co-immunofluorescence staining of Flag (green) and the lysosomal marker LAMP1 (red) in MDA-MB-231 cells transfected with Vector Ctrl, ΔATG1 + 2, or Flag-hSPAR. Nuclei were stained with Hoechst (blue). The Vector Ctrl and ΔATG1 + 2 are negative control groups, and Flag proteins are not expressed (The white “X” in Vector Ctrl and ΔATG1 + 2 groups indicates no green fluorescent signals). The graphs display the fluorescence intensity (arbitrary units) of Flag and the lysosomal marker LAMP1 over the distance from adjacent image (depicted by the arrows). The value of Pearson’s correlation Rr of Flag-hSPAR and LAMP1 is 0.934597. Scale bar, 25 µm ( n = 3 independent biological samples). ( F ) Immunoblotting against p-mTOR, mTOR, p-S6K, S6K, p-S6, S6, Flag and GAPDH in extracts from MDA-MB-231 cells transfected with Vector Ctrl, ΔATG1 + 2, or Flag-hSPAR ( 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. ( H ) Immunoblotting against p-mTOR, mTOR, p-S6K, S6K, p-S6, S6, hSPAR, and GAPDH in extracts from the breast cancer tumoral tissues and their adjacent non-tumoral tissues ( n = 18 independent biological samples). ( I ) Quantified relative levels of p-mTOR/mTOR, p-S6K/S6K, p-S6/S6, and hSPAR/GAPDH from panel ( H ) ( n = 18 independent biological samples). Data are presented as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction. ( J ) Immunoblotting against p-mTOR, mTOR, p-S6K, S6K, p-S6, S6, Flag, and GAPDH in extracts from MDA-MB-231 cells transfected with Vector Ctrl, ΔATG1 + 2, or Flag-hSPAR, with or without Rapamycin treatment (an mTOR inhibitor; 10 μM for 12 h) ( n = 3 independent biological samples). ( K ) Representative images of EdU assay in the presence of Vector Ctrl, ΔATG1 + 2 or Flag-hSPAR, with or without Rapamycin treatment (10 μM for 12 h) ( n = 3 independent biological samples). Scale bar, 50 µm. ( L ) Quantification of cell proliferation rate from panel ( K ) ( 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: Immunofluorescence, Western Blot, Marker, Membrane, Labeling, Fluorescence, Staining, Transfection, Plasmid Preparation, Negative Control, Two Tailed Test, EdU 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: 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