Figure 5.

Venetoclax treatment upregulates mitochondrial energy metabolism and increases NF-κB activity in NK cells

(A) Enriched GO terms of upregulated DEGs in the C3 subpopulation between the control and venetoclax-treated groups.

(B) Expression of OXPHOS-related genes in C3 NK cells.

(C and D) Representative images (C) and quantification (D) of the mitochondrial phenotype in C3 NK cells (control group, n = 27; venetoclax-treated group, n = 54; green, MitoTracker Green, an indicator of mitochondrial mass; red, MitoTracker Red CMXRos, an indicator of mitochondrial membrane potential; blue, Hoechst, a dye for cell nuclei). The results represent two independent experiments.

(E) Violin plot showing the NF-κB activation signal score and expression levels of representative NF-κB target genes in C3 NK cells.

(F and G) Expression of p-p65 (F, n = 7, biological replicates) and p-IKKα (G, n = 4, biological replicates) in venetoclax (400 nM for 18 h)-treated or untreated C3 NK cells analyzed by flow cytometry. The results represent three independent experiments.

(H) Confocal microscopy images (left) and quantification (right) of intranuclear p-p65 signal intensity in venetoclax (400 nM for 18 h)-treated or untreated C3 NK cells (control group, n = 76; venetoclax-treated group, n = 104). The results represent three independent experiments using NK cells from different donors.

(I) GO terms of genes upregulated following venetoclax treatment of total NK cells.

(J) Oxygen consumption rate (OCR) of venetoclax-treated (400 nM for 18 h) or untreated total NK cells (n = 4, biological replicates).

(K) Spare respiratory capacity calculated from (J).

(L) ATP assay was conducted to evaluate total ATP production, mitochondrial oxidative phosphorylation-derived ATP (mito-ATP), and glycolysis-derived ATP (glyco-ATP) in total NK cells treated with 400 nM venetoclax for 18 h or left untreated. Statistical analysis was performed for mito-ATP between the control and venetoclax-treated groups (n = 9–10, biological replicates).

(M) The p-p65 expression level in total NK cells treated with or without 400 nM venetoclax for 18 h was detected by flow cytometry (n = 8, biological replicates). The results represent three independent experiments using NK cells from different donors.

(N and O) Confocal microscopy images (N) and quantification of p-p65 intranuclear signal intensity (O) in total NK cells treated with or without 400 nM venetoclax for 18 h. The results represent three independent experiments using NK cells from different donors.

(P) Quantitative real-time PCR data showing changes in BCL-xL mRNA expression in CB-NK cells after 400 nM venetoclax treatment for 18 h (n = 6, biological replicates). The results represent three independent experiments using NK cells from different donors.

Statistical significance was determined by unpaired Student’s t tests (D, H, K, L, O, and P), paired Student’s t tests (F, G, and M), hypergeometric tests (A and I), and the Mann-Whitney test (E). Data are presented as mean ± SD.

See also Figure S5.