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. 2021 Oct 1;30(2):606–620. doi: 10.1016/j.ymthe.2021.07.015

Figure 4.

Figure 4

PKLR delivered by hepatocyte-derived EVs regulates NK cell function

(A) Pathway clustering analysis of identified proteins in hepatocyte-derived EVs based on the Dtabase for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation. (B) Pathway clustering analysis of metabolism-related proteins identified in (A) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. (C) Correlation analysis between canonical glycolytic proteins identified in (B) and FBP1 in normal liver tissues collected by TCGA and the Genotype-Tissue Expression (GTEx) project. (D) A scatterplot showing the positive correlation between PKLR and FBP1 levels in normal liver tissues collected by TCGA and the GTEx project. Transcripts per kilobase of exon model per million mapped reads (TPM) represents the normalized sequencing depth of RNA sequencing (RNA-seq). (E) Western blot analysis of whole-cell lysates (WCLs) and EVs, confirming that PKLR but not FBP1 protein is present in hepatocyte-derived EVs. (F) Western blot analysis showing FBP1 knockout (KO) in HHL5 cells results in decreased protein expression of PKLR in both WCLs and EVs. (G) Real-time qPCR analysis of PKLR mRNA levels in NK cells treated with EVs collected from hepatocytes, with or without FBP1, relative to no EV-treated control, using primers targeting multiple PKLR 3′ UTR or coding sites. (H) Representative histograms (upper) and quantifications (lower) of indicated makers in NK cells incubated with EVs secreted from HHL5 cells with PLKO.1 or PKLR shRNAs. Western blots confirming the efficiencies of PKLR knockdown were shown in (J). (I) Representative histograms (upper) and quantification (lower) of indicated makers in NK cells incubated with EVs secreted from HHL5 cells using FBP1 WT or FBP1 KO clones in the presence or absence of PKLR overexpression. Western blots confirming the efficiencies of FBP1 KO and PKLR re-expression were shown in (J). (J) Western blots confirming the efficiencies of PKLR knockdown as shown in (H) and the efficiencies of FBP1 KO and PKLR re-expression as shown in (I). (K) Analysis of glucose uptake and lactic acid production in NK cells incubated with EVs secreted from HHL5 cells with or without PKLR shRNAs or with EVs secreted from HHL5 cells using FBP1 WT or FBP1 KO clones in the presence or absence of PKLR overexpression. Data are the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by unpaired Student’s t test. See also Figure S5.