Figure 7.

Spermidine Induces TFEB Expression and Improves the Function of Old Human B Cells

(A) The protein levels of TFEB, hypusinated eIF5A, and overall eIF5A of PBMCs from healthy human donors of indicated ages were assessed using western blot. A representative plot (left) and quantifications (right) are shown. n = 8–15 donors.

(B) TFEB mRNA of human PBMCs was measured using qPCR with GAPDH as the reference gene. n = 7–9 donors.

(C) Sorted B cells from young human donors were cultured with anti-IgM/CD40L and treated with DFMO with or without 10 μM spermidine for 7 days. n = 5 donors.

(D) Sorted B cells from human donors were cultured as in (C) with spermidine and/or GC7 as indicated. The expression of hypusinated (Hyp) or non-hypusinated (AcLys or Lys) eIF5A was distinguished by two-dimensional western blot of eIF5A. Black arrow, hypusinated Lys⁵⁰, pH 5.2; red arrow, unmodified Lys⁵⁰, pH 5.1; blue arrow, acetylated Lys⁴⁷ with unmodified Lys⁵⁰, pH 5.0. The non-hypusination ratio was calculated as eIF5A dot densitometric intensity (AcLys + Lys)/(AcLys + Lys + Hyp). n = 4 donors.

(E–G) Sorted B cells from old human donors (age 77.5 ± 6.3 years) were cultured as in (C) together with spermidine and GC7. (E) The protein levels of TFEB and eIF5A hypusination were measured using western blot. (F) Autophagic flux was determined by flow cytometry as in Figure 1A. (G) Supernatant IgG was assessed using ELISA. n = 4–14 donors.

Data are represented as mean ± SEM. Unpaired two-tailed Student’s t test (A and D, comparison of young versus old). Welch’s t test (B). Paired one-way ANOVA with post hoc Tukey’s test (C). Paired one-tailed t test (D, comparisons of old versus old + Spd, old + Spd versus old + Spd + GC7 comparison, E–G). ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, and ^∗∗∗∗p ≤ 0.0001.

See also Figure S7.