bira r118g bira entry vector  (Addgene inc)

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Metabolomic and transcriptomic analyses reveal that quinacrine downregulates the arginine transporter SLC3A2 and causes arginine deficiency. A) Schematic representation of the metabolic and transcript profiling approaches for H9 cells ( n = 3). DMSO, dimethyl sulfoxide. B) Volcano plot visualization of the log2 fold change ( x ‐axis) and −log10 ( p value; y ‐axis) of intracellular metabolites measured by liquid chromatography–mass spectrometry (LC‐MS/MS). Significantly increased (red) or decreased (blue) metabolites related to arginine metabolism are labeled. C) Metabolic pathway analysis plot showing the most strongly impacted metabolic pathways in treated cells, as shown in (A). Pathway impact value computed from MetaboAnalyst topological analysis ( x ‐axis) versus −In p ‐value obtained from pathway enrichment analysis ( y ‐axis). D) Fold changes in the intracellular metabolite abundance of arginine metabolism detected by LC‐MS/MS in quinacrine‐ and DMSO‐treated cells. The DMSO‐treated levels were set to 1. Significantly increased, decreased, unchanged, and undetected metabolites are color‐labeled as indicated. E) Intracellular arginine concentrations in indicated cell lines treated with quinacrine (2 × 10 −6 m ) for 48 h ( n = 3). F) Uniform manifold approximation and projection (UMAP) plots for quinacrine‐ (light red) and DMSO‐treated (light blue) H9 cells. G) Volcano plot visualization of differentially expressed genes (DEGs) in H9 cells treated with quinacrine or DMSO for 48 h. Significantly downregulated arginine transporters are labeled. H) Table plot showing hallmark analysis results for significantly enriched pathways in quinacrine‐treated cells vs. control cells. NES, normalized enrichment score. I) Gene Ontology (GO) enrichment analysis plots showing the enriched amino acid transmembrane transporter pathways in quinacrine‐ and DMSO‐treated H9 cells. J) Immunoblots of the indicated proteins in PTCL cell lines treated with different quinacrine concentrations for 48 h ( n = 3). β‐actin serves as a loading control. K) Representative SLC3A2 immunofluorescence staining of H9 cells treated with quinacrine or DMSO (2 × 10 −6 m ) for 48 h ( n = 3). Scale bar, 25 × 10 −6 m . L) Arginine levels in H9 cells treated with quinacrine (2 × 10 −6 m ) or DMSO and cultured in H 2 O or arginine (4 × 10 −3 m ) for 48 h ( n = 3). M) Relative cell viability of H9 cells treated with different concentrations of quinacrine and cultured in H 2 O or arginine (4 × 10 −3 m ) for 48 h ( n = 3). For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For B, D, E, and M, p values were generated using Student's two‐tailed unpaired t ‐test. For L, p values were generated using a two‐way ANOVA with multiple comparisons. For I, p values were generated using a permutation test.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Western Blot, Immunofluorescence, Staining, Cell Culture, Generated, Two Tailed Test

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Characterization of arginine metabolism in patients with peripheral T‐cell lymphoma (PTCL). A) Dot plot of arginine transporter expression in the indicated cell types from Group1. pDCs, plasmacytoid dendritic cells; MPs, mononuclear phagocytes; NK cells, natural killer cells; Tfh, follicular helper T cells. B) Density plot of SLC3A2 expression shown in the uniform manifold approximation and projection (UMAP) visualization of T cells. Tfh tumor cells are indicated by dashed circles. C) Violin plot showing SLC3A2 expression in Tfh tumor cells from the newly diagnosed (ND) and relapsed/refractory (RR) groups. D) Box plot of the expression of arginine transporters SLC3A2 , SLC7A4 , SLC7A6 , and SLC7A9 in control and PTCL patients from GSE160119 . E) Table plot showing hallmark analysis results of significantly enriched pathways in SLC3A2 + Tfh tumor cells vs. SLC3A2 − Tfh tumor cells. NES, normalized enrichment score. F) Schematic representation of arginine metabolism. Changes in the mRNA levels of enzymes and transporters in Tfh tumor cells from the RR group compared to those in the ND group are shown. Color‐coding represents the level of log2‐fold change as indicated. G) Violin plot showing the arginine and proline metabolism scores based on KEGG pathway enrichment (has00330) in Tfh tumor cells from the ND and RR groups. H) Schematic of the scRNA‐seq design for Group2. I) UMAP of 11 cell subtypes (left) and two groups (PBMC and LN) (right) identified in 12 specimens. J) Violin plot showing arginine and proline metabolism scores based on KEGG pathway enrichment (has00330) in T cells from the lymph nodes and peripheral blood of patients with angioimmunoblastic T‐cell lymphoma (AITL). K) Arginine levels in the plasma of 15 normal controls (NCs) and 44 PTCL patients. The 44 patients included those with AITL ( n = 12), extranodal NK/T‐cell lymphoma (ENKL) ( n = 12), PTCL‐NOS ( n = 10), ALK‐ ALCL ( n = 4), cutaneous T‐cell lymphoma (CTCL) ( n = 3), and T‐cell large granular lymphocyte leukemia (T‐LGLL) ( n = 3). L) Violin plot showing SLC3A2 expression in Tfh tumor cells from the ND and RR groups. M) Scatter plot showing the correlation between SLC3A2 expression and arginine and proline metabolism scores in Tfh tumor cell clusters. N) Representative SLC3A2 (green) and ASS1 (red) immunofluorescence staining in a PTCL tissue microarray containing 35 PTCL and eight reactive lymphoid hyperplasia (RLH) samples. Scale bar, 50 × 10 −6 m . O,P) Mean fluorescence intensity (MFI) of SLC3A2 (O) and ASS1 (P) in the PTCL tissue microarray. Q) Pie chart showing the percentage of three phenotypes (SLC3A2+/ASS1+, SLC3A2+/ASS1−, and SLC3A2−/ASS1−) in the PTCL samples. R) Patterns of different levels of SLC3A2 expression in normal lymph nodes and AITL tissues, detected using immunohistochemistry (IHC) analysis. Scale bar, 50 × 10 −6 m . S) Distribution of SLC3A2 expression in tissues from 36 patients with AITL and 6 NCs. T) Overall survival (OS) of patients with AITL based on SLC3A2 expression. For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For C, D, G, J, and L, p values were generated using the Wilcoxon rank‐sum test with default parameters. For E, p values were generated using a permutation test. For K, O, and P, p values were generated using one‐way ANOVA with multiple comparisons. For M, p value was generated using Pearson's test. For S, p value was generated using Fisher's exact test. For T, p value was generated using the log‐rank test.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: Expressing, Control, Clinical Proteomics, Immunofluorescence, Staining, Microarray, Fluorescence, Immunohistochemistry, Generated

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Peripheral T‐cell lymphoma (PTCL) relies on SLC3A2‐mediated arginine uptake to proliferate. A) Growth curves of PTCL cell lines under control and arginine‐depleted (‐Arg) conditions ( n = 3). B) Immunoblots of SLC3A2 protein in PTCL cell lines under control and arginine‐restricted conditions ( n = 3). β‐actin serves as a loading control. C) Scatter plot showing the correlation between SLC3A2 expression and arginine levels in PTCL cell lines ( n = 3). D) Immunoblots of Cas9+ H9 and SU‐DHL‐1 cells expressing nontargeting control or independent sgRNAs against SLC3A2 . Whole‐cell lysates were obtained on day 7 after sgRNA expression ( n = 3). β‐actin serves as a loading control. E) Growth curves of Cas9+ H9 (left) and SU‐DHL‐1 (right) cells expressing nontargeting control or independent sgRNAs targeting SLC3A2 . Analysis was performed 96 h after sgRNA expression and 48 h after puromycin selection ( n = 3). F,G) Arginine levels in the cell lysates (F) and supernatants (G) of the indicated cells. FM, fresh medium ( n = 3). H) Growth curves of H9 (left) and SU‐DHL‐1 (right) cells after the indicated treatments ( n = 3). I) Immunoblots of whole‐cell lysates from Karpas 299 and MT‐4 cells with control green fluorescent protein (GFP) or SLC3A2‐FLAG cDNA 7 days after cDNA expression ( n = 3). β‐actin serves as a loading control. J) Fold changes in intracellular amino acid levels detected by liquid chromatography–mass spectrometry (LC‐MS) in Karpas 299 cells expressing control or SLC3A2 sgRNA ( n = 3). K,L) Growth curves of MT‐4 cells expressing a vector (control) or SLC3A2 cDNA after the indicated treatments ( n = 3). M) Half‐maximal inhibitory concentration in PTCL cells expressing a vector (control) or SLC3A2 cDNA treated with different concentrations of quinacrine for 48 h. N) Relative viability of normal T cells isolated from healthy volunteers and tumor cells isolated from patients with angioimmunoblastic T‐cell lymphoma (AITL) treated with different concentrations of quinacrine for 24 h ( n = 3). O,P) Representative staining for SLC3A2, granzyme B, CD8, and CD4 in AITL tissue (O). Scale bar, 100 × 10 −6 m . Correlation between SLC3A2 expression and the percentage of granzyme B + CD8 + cells is shown (P) ( n = 12). Q,R) Flow cytometric analysis of 7‐AAD‐positive CD8 + T cells (Q) and relative mean fluorescence intensity (MFI) of TIM‐3 on CD8 + T cells (R) cocultured with SLC3A2‐WT or SLC3A2‐ knockout SU‐DHL‐1 cells and/or arginine addition ( n = 3). S) Schematic representation of a xenograft mouse model constructed using Cas9+ SU‐DHL‐1 cells expressing a nontargeting control or an sgRNA targeting SLC3A2 . T) Photographs of tumors (left) and tumor growth curves (right) of SLC3A2‐WT and SLC3A2‐knockout groups in the SU‐DHL‐1 xenograft mice ( n = 4). U) Representative immunohistochemistry (IHC) staining showing Ki‐67 and cleaved‐caspase3 expression in the subcutaneous tumors of the SU‐DHL‐1 xenograft mouse model expressing control or SLC3A2 sgRNA (left) and the corresponding quantification (right) ( n = 4). Scale bar, 50 × 10 −6 m . For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For E–H, K, L, Q, and R, p values were generated using one‐way ANOVA with multiple comparisons. For C and P, p values were generated using Pearson's test. For A, J, M, T, and U, p values were generated using Student's two‐tailed unpaired t‐test.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: Control, Western Blot, Expressing, Selection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Concentration Assay, Isolation, Staining, Fluorescence, Knock-Out, Construct, Immunohistochemistry, Generated, Two Tailed Test

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Single‐cell dynamic RNA sequencing reveals that arginine deficiency reduces OXPHOS and induces nascent RNA of SLC3A2 via JUNB. A) Schematic of single‐cell dynamic RNA sequencing. B) Uniform manifold approximation and projection (UMAP) plots of H9 and MT‐4 cells in complete medium (CM) or arginine deprivation (‐Arg) medium. C) Principal component analysis (PCA) showing the four groups (H9 and MT‐4 cells in complete medium or arginine deprivation medium) according to new, old, and total RNAs. D) Heatmap of a subset of metabolic DEGs in the arginine deprivation group compared to those in complete medium group (log2 fold change) according to new, old, and total RNAs. E) The enriched Gene Ontology (GO) pathway –log 10 (adjusted p values) of gene sets significantly changed in the arginine deprivation group versus complete medium group. F) Seahorse analysis of H9 and SU‐DHL‐1 cell oxygen consumption rates (OCRs) after exposure to complete or arginine‐depleted medium for 24 h. Time points of oligomycin (1 × 10 −6 m ), CCCP (1 × 10 −6 m ), and antimycin A (1 × 10 −6 m ) addition are indicated ( n = 3). G) Violin plots of the turnover rate (TOR) of metabolic genes in complete medium and arginine deprivation groups. H) Volcano plot of differentially expressed new RNAs in H9 and MT‐4 cells in the complete medium or the arginine deprivation group. The top 20 of differentially expressed new RNAs are labeled. I) Activity AUC of pySCENIC‐predicted transcription factors (TFs) in new RNAs from the arginine deprivation group. Nine upregulated TFs potentially bind to the SLC3A2 promoter, and the total number of their target genes is labeled. J) Immunoblots of Cas9+ H9 cells expressing nontargeting control or independent sgRNAs targeting JUNB in complete or arginine‐depleted medium ( n = 3). β‐Tubulin serves as a loading control. K) Nuclear extracts of H9 cells incubated in complete or arginine‐depleted medium were immunoprecipitated and analyzed using chromatin immunoprecipitation (ChIP) ( n = 3). IgG serves as a control. L) Schematic representation of control versus arginine‐free diet intervention in a SU‐DHL‐1 xenograft mouse model. M) Photographs of tumors in the control diet and arginine‐free diet‐fed mice ( n = 4). N) Liquid chromatography–mass spectrometry (LC‐MS/MS) detected the arginine levels in the tumors of control and arginine‐free diet‐fed mice ( n = 4). O) Tumor growth curves of the control diet and arginine‐free diet‐fed mice ( n = 4). P) Immunoblots of whole‐cell lysates from tumors of control and arginine‐free diet‐fed mice ( n = 3). GAPDH serves as a loading control. Q) Representative Immunohistochemistry (IHC) staining showing JUNB and SLC3A2 expression in the tumors of the control and arginine‐free diet‐fed mice ( n = 4). Scale bar, 50 × 10 −6 m . For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For E, p values were generated using a permutation test. For H and G, p values were generated using the Wilcoxon‐rank sum test. For K, p values were generated using a two‐way ANOVA with multiple comparisons. For N and O, p values were generated using Student's two‐tailed unpaired t ‐test.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: RNA Sequencing, Labeling, Activity Assay, Western Blot, Expressing, Control, Incubation, Immunoprecipitation, Chromatin Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Generated, Two Tailed Test

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Quinacrine transcriptionally regulates SLC3A2 by targeting SSRP1 in a JUNB‐dependent manner A) ATAC‐seq heatmap comparing the landscapes of accessible chromatin in H9 cells treated with 2 × 10 −6 m quinacrine or dimethyl sulfoxide (DMSO) for 48 h. B) Integrated Genomics Viewer screenshot depicting tracks from ATAC‐seq at the SLC3A2 locus. C) Immunoblot analysis of SLC3A2 expression in whole‐cell lysates from Cas9+ H9 and SU‐DHL‐1 cells expressing control or independent SSRP1 ‐targeting sgRNAs ( n = 3). β‐actin serves as a loading control. D,E) Growth curves of Cas9+ H9 (left) and SU‐DHL‐1 (right) cells expressing nontargeting control or independent sgRNAs against SSRP1 and transfected with flag‐SLC3A2 or a control vector (D). Immunoblots of indicated proteins are shown in E ( n = 3). F) Arginine levels in Cas9+ H9 and SU‐DHL‐1 cells expressing nontargeting control or independent sgRNAs against SSRP1 , transfected with flag‐SLC3A2 or the control vector ( n = 3). β‐actin serves as a loading control. G) A chromatin immunoprecipitation (ChIP) assay was performed in H9 and SU‐DHL‐1 cells using anti‐SSRP1 antibodies, followed by RT‐qPCR. The fold change in the expression of ChIP‐enriched mRNAs relative to the input was calculated ( n = 3). IgG serves as a control. H) A ChIP assay was performed on H9 cells expressing control or JUNB sgRNA using anti‐SSRP1 or anti‐IgG antibodies, followed by RT‐qPCR. The fold change in the expression of ChIP‐enriched mRNAs relative to the input was calculated ( n = 3). IgG serves as a control. I) A ChIP assay was performed on H9 cells expressing control or SSRP1 sgRNA using anti‐JUNB or anti‐IgG antibodies, followed by RT‐qPCR. The fold change in the expression of ChIP‐enriched mRNAs relative to the input was calculated ( n = 3). IgG serves as a control. J) Immunoblots of SSRP1 and JUNB from anti‐JUNB immunoprecipitants (IPs) (left) and anti‐SSRP1 IPs (right) obtained from H9 cells ( n = 3). K) Representative immunofluorescence staining of JUNB and SSRP1 in H9 cells ( n = 3). Scale bar, 25 × 10 −6 m . L) Scatter plot showing the correlation between SSRP1 and SLC3A2 expression in bulk RNA sequencing datasets ( GSE160119 and GSE58445 ). M) Representative images showing the correlation between SSRP1 and SLC3A2 staining in human peripheral T‐cell lymphoma (PTCL) samples. Scale bars, 50 × 10 −6 m . N) Kaplan–Meier curves of overall survival (OS) in PTCL patients with different SSRP1 expression levels in the GSE58445 dataset. For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For D and F, p values were generated using one‐way ANOVA with multiple comparisons. For G, p values were generated using Student's two‐tailed unpaired t‐test. For H and I, p values were generated using two‐way ANOVA with multiple comparisons. For L, p values were generated using Pearson's test. For N, p value was generated using the log‐rank test.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunofluorescence, Staining, RNA Sequencing, Generated, Two Tailed Test

Journal: Advanced Science

Article Title: SSRP1/SLC3A2 Axis in Arginine Transport: A New Target for Overcoming Immune Evasion and Tumor Progression in Peripheral T‐Cell Lymphoma

doi: 10.1002/advs.202415698

Figure Lengend Snippet: Combination epigenetic therapy exerts a synergistic antitumor effect on peripheral T‐cell lymphoma (PTCL). A,B) Matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI) ion images (A) and quantified relative changes (B) for SU‐DHL‐1 xenografts treated with quinacrine or vehicle ( n = 4). Scale bar, 2 mm. C) CCK8 analysis of the relative proliferation activity of the PTCL cell lines treated with quinacrine in combination with chidamide at different concentrations for 48 h ( n = 3). The combination index (CI) is red‐coded. D) Schematic representation of a SU‐DHL‐1 xenograft mouse model treated with vehicle, quinacrine, chidamide, or quinacrine plus chidamide combination therapy in vivo. E) Tumor volume (left) and tumor growth curve (right) in the SU‐DHL‐1 xenograft mouse model treated with vehicle only (black), chidamide (green), quinacrine (blue), and quinacrine in combination with chidamide (red) ( n = 4). F) Representative images (left) of immunohistochemistry (IHC) staining showing Ki‐67 expression in tumor samples from the four groups (vehicle, chidamide, quinacrine, and quinacrine plus chidamide groups) and the corresponding quantification (right) ( n = 4). Scale bar, 50 × 10 −6 m . G) Schematic model. In PTCL, SLC3A2‐mediated excessive arginine uptake by tumor cells produces arginine deficiency in the TME and circulation, thus impairing CD8 + T‐cell survival and function, contributing to the immune escape of PTCL cells. An abundant arginine pool in tumor cells upregulates arginine catabolic activity; induces global metabolic changes, including enhanced oxidative phosphorylation; and inhibits apoptosis, thereby fueling tumor progression. SSRP1 upregulates SLC3A2 as a co‐transcription factor with JUNB. Quinacrine disrupts SLC3A2‐mediated arginine transport by targeting SSRP1 and represents a promising therapeutic strategy for PTCL. For all panels, the data are presented as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant. For B, p values were generated using Student's two‐tailed unpaired t‐test. For E and F, p values were generated using one‐way ANOVA with multiple comparisons.

Article Snippet: Entry vectors bearing SLC3A2 cDNAs were purchased from Nanjing GeneBay Biological Technology Company.

Techniques: Mass Spectrometry, Imaging, Activity Assay, In Vivo, Immunohistochemistry, Expressing, Phospho-proteomics, Generated, Two Tailed Test