Fig 1: ASCT2 drives the proinflammatory SASP via IL-1α/NF-κB feedback loop through interacting with precursor IL-1α at Lys82 of senescent HSCs. (A) Protein of p31–IL-1α and p16–IL-1α was measured by Western blot analysis with 4-OHT (n = 3 per group). (B) Detection of intracellular IL-1α by ELISA with 4-OHT (n = 3 per group). (C) mRNAs of proinflammatory SASP including IL1A, IL1B, IL6 and IL8 were measured by qPCR (n = 5 per group). (D) Co-IP of ASCT2 with IL-1α in LX2 cells, as detected by Western blot analysis (n = 3 per group). (E) Schematic of fragments of IL-1α that were fused to GST (top) and were transfection into 293T cells. Binding of ASCT2 to GST-IL-1α proteins was detected by Western blot (bottom, n = 3 per group). FL: full length; F1: domain 1–112; F2: domain 112–271; F3: NLS domain (80–88) deletion; F4: K82N mutation. (F) rIL-1α protein was added to senescent (ASCT2 depletion or etoposide treatment) LX2 cells, and IL1R1 signaling cascade indicators were analyzed by Western blotting (n = 3 per group). (G) rIL-1α protein was added to senescent (ASCT2 depletion or etoposide treatment) LX2 cells, and NF-κB protein was measured in nuclear and cytoplasmic lysates by Western blot (n = 3 per group). (H) Relative luciferase measurement of NF-κB in indicated cells (n = 5–6 per group). (I) ASCT2 depletion-senescent LX2 cells were treated with rIL-1α protein and addressed in NF-κB inhibitor PDTC, and the analysis of mRNAs of proinflammatory SASP IL1A, IL1B, IL6 and IL8 by qPCR (n = 5–6 per group). Bars indicate mean ± SEM; ∗P < 0.05, ∗∗P < 0.01; ns, no significance.
Fig 2: ASCT2-mediated glutaminolysis governs the proinflammatory SASP of senescent HSCs. (A) Schematic diagram of glutaminolysis. (B–E) Steady-state metabolite levels of glutamate, α-KG and GSH levels were measured using kits (n = 3–15 per group). (F–I) TCA cycle metabolites citrate, malate, fumarate and succinate levels were measured using kits (n = 5 per group). (J) Intracellular ATP production was determined using a kit (n = 6 per group). (K) OCR was determined and the basal respiratory rate and maximum respiration rate were quantified by seahorse assay (n = 3 per group). (L–O) mRNA levels of the proinflammatory SASP factors IL1A, IL1B, IL6, and IL8 were measured by real-time PCR after α-KG supplementation (n = 5 per group). Bars indicate mean ± SEM; ∗P < 0.05; ∗∗P < 0.01.
Fig 3: Inhibition of ASCT2 restricts proinflammatory SASP in senescent HSCs. (A) mRNA changes under the indicated conditions were observed by RNA-seq. Heat map indicates the fold changes of SASP factors in proliferating LX2 cells vs. etoposide-induced senescent LX2 cells vs. ASCT2 depletion-derived senescent LX2 cells. n = 3 per group. (B) GSEA analysis of glutamine genes and TCA cycle genes in the proliferating LX2 cells vs. ASCT2 depletion-derived senescent LX2 cells. (C) GSEA analysis of DNA replication genes and G1 checkpoints genes in proliferating LX2 cells vs. ASCT2 depletion derived-senescent LX2 cells. (D, E) mRNAs of the proinflammatory SASP: IL1A, IL1B, IL6, and IL8 were determined by qPCR in the indicated cells (n = 3–10 per group). (F, G) EdU incorporation and SA-β-Gal staining for senescence determination after 4-OHT stimulation (n = 8 per group). (H) Cell cycle distribution was determined by flow cytometry after 4-OHT stimulation (n = 3 per group). (I–L) mRNA levels of IL1B, IL6, and IL8, and protein levels of IL-8 were established by qPCR and Western blot (n = 3–5 per group). Bars indicate the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01; ns, no significance.
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