Journal: Journal of Inflammation Research

Article Title: Plasma Exosomal LncRNA XIST Impairs CD4 + T Cell Autophagy and Promotes Activation via the miR-98/RICTOR/Akt-mTOR Axis in Systemic Lupus Erythematosus

doi: 10.2147/JIR.S574715

Figure Lengend Snippet: SLE plasma exosomes (Sexo) inhibit autophagy and promote activation of CD4 + T cells from healthy controls (HCs). (A) Western blot analysis of autophagy markers LC3B-II and SQSTM1/p62 in HC CD4 + T cells following co-culture with Sexo (n = 16). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls presented as bar graphs. (B) Flow cytometric assessment of autophagy in HC CD4 + T cells (n = 6). (C) Surface activation markers CD69, CD70, and CD40L detected by flow cytometry (n = 14). (D) ELISA measurement of cytokines IL-6, IL-10, and TNF-α in cell culture supernatants (n = 17). (E) qRT-PCR quantification of lncRNA XIST, miR-98, and RICTOR expression in HC CD4 + T cells (n = 54). (F) Western blot analysis of Rictor, Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K (n = 21). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls. The co-cultured Sexo were derived from six independent SLE donors, where n represents the number of independent HC CD4 + T cell donors. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: CD63 (ab193349, Abcam), Alix (ab186429, Abcam), LC3B (#2775, CST), SQSTM1/p62 (#5114, CST), Rictor (#2140, CST), p-AKT (#4058, CST), AKT (#4691, CST), p-p70S6K (#9234, CST), p70S6K (#2708, CST), p-mTOR (#5536, CST), mTOR (#2983, CST), and GAPDH (#5174, CST).

Techniques: Clinical Proteomics, Activation Assay, Western Blot, Co-Culture Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: Plasma Exosomal LncRNA XIST Impairs CD4 + T Cell Autophagy and Promotes Activation via the miR-98/RICTOR/Akt-mTOR Axis in Systemic Lupus Erythematosus

doi: 10.2147/JIR.S574715

Figure Lengend Snippet: SLE plasma exosomes restore autophagy inhibition and activation in SLE CD4 + T cells after lncRNA XIST knockdown. (A) qRT-PCR analysis of lncRNA XIST levels in SLE CD4 + T cells transduced with sh-lncRNA XIST (shXIST) or control (shNC), followed by co-culture with autologous SLE plasma exosomes (Sexo) (n = 5). (B) Western blot detection of autophagy markers LC3B-II and SQSTM1/p62 (n = 5). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls. (C) Flow cytometric analysis of autophagic CD4 + T cells (n = 8). (D) Flow cytometry for activation markers CD69, CD70, and CD40L (n = 10). (E) ELISA quantification of IL-6, IL-10, and TNF-α in the culture supernatant (n = 9). (F) qRT-PCR analysis of miR-98 and RICTOR expression (n = 8). (G) Western blot analysis of proteins involved in the Rictor/Akt/mTOR/p70S6K pathway (n = 4). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls. n represents the number of independent SLE CD4 + T cell donors and their corresponding autologous exosomes. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: The following primary antibodies were used: CD63 (ab193349, Abcam), Alix (ab186429, Abcam), LC3B (#2775, CST), SQSTM1/p62 (#5114, CST), Rictor (#2140, CST), p-AKT (#4058, CST), AKT (#4691, CST), p-p70S6K (#9234, CST), p70S6K (#2708, CST), p-mTOR (#5536, CST), mTOR (#2983, CST), and GAPDH (#5174, CST).

Techniques: Clinical Proteomics, Inhibition, Activation Assay, Knockdown, Quantitative RT-PCR, Transduction, Control, Co-Culture Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Inflammation Research

Article Title: Plasma Exosomal LncRNA XIST Impairs CD4 + T Cell Autophagy and Promotes Activation via the miR-98/RICTOR/Akt-mTOR Axis in Systemic Lupus Erythematosus

doi: 10.2147/JIR.S574715

Figure Lengend Snippet: Exosomal lncRNA XIST inhibits autophagy and promotes activation in HC CD4 + T cells. (A) qRT-PCR validation of lncRNA XIST overexpression in HC plasma exosomes (Hexo) loaded with lncRNA XIST compared to negative control (lncRNA NC) (N = 4). (B) qRT-PCR analysis of lncRNA XIST in HC CD4 + T cells after co-culture with Hexo loaded with lncRNA XIST (Hexo-oeXIST) or negative control (Hexo-oeNC) (n = 20). (C) Western blot detection of autophagy markers LC3B-II and SQSTM1/p62 (n = 12). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls. (D) Flow cytometry analysis of autophagic CD4 + T cells (n = 8). (E) Flow cytometry for surface activation markers CD69, CD70, and CD40L (n = 9). (F) ELISA quantification of IL-6, IL-10, and TNF-α (n = 9). (G) qRT-PCR detection of miR-98 and RICTOR expression (n = 21). (H) Western blot analysis of Rictor and key signaling proteins in the Akt/mTOR/p70S6K pathway (n = 10). Representative immunoblots are shown, with densitometric quantification normalized to the corresponding loading controls. N indicates the number of independently engineered exosome preparations; n indicates the number of independent HC CD4 + T cell donors. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: CD63 (ab193349, Abcam), Alix (ab186429, Abcam), LC3B (#2775, CST), SQSTM1/p62 (#5114, CST), Rictor (#2140, CST), p-AKT (#4058, CST), AKT (#4691, CST), p-p70S6K (#9234, CST), p70S6K (#2708, CST), p-mTOR (#5536, CST), mTOR (#2983, CST), and GAPDH (#5174, CST).

Techniques: Activation Assay, Quantitative RT-PCR, Biomarker Discovery, Over Expression, Clinical Proteomics, Negative Control, Co-Culture Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing