Fig. 5

PHLPP1-dependent neutral lipid accumulation involves ChREBP and AMPK

(A) Scatterplot of enriched transcription factors in set 3: Differentially altered gene-set from set 3 (cut-off: FC > 1.5, FDR 0.05) was analyzed by “Enrichr” tool to analyze the possible transcription factors involved in the gene alterations using ChIP-X enrichment analysis (ChEA) gene set library (The brighter the color, the more significant the transcription factor).

(B and C) Plot and table indicating the –log₁₀ p value (B) and terms odds ratio, and combined score (C) used by the Enrichr software to represent the output (scatterplot). p values are computed based on alterations in the number of genes that are known primary or secondary targets of transcription factors; Odds ratio indicates a measure of association between input and outcome; combined score: log(p) ∗z, p is p value, and z = deviation from the expected rank.

(D) Neutral lipid staining by Oil Red O in ChREBP overexpressing cells along with quantification (top); Images are taken at |×20×| magnification. Each image is a representative image of four different experiments with a minimum of eight different fields in each experiment. Statistical analysis was performed using two-tailed t-test (∗p < 0.05, vs Vector).

(E and F) Total cellular FFA (E) and cholesterol levels (F) upon ChREBP overexpression. Statistical analysis was performed using two-tailed t-test (∗p < 0.05, vs Vector).

(G) Protein levels of foam cell markers in ChREBP overexpressing cells. Densitometric analysis is shown in Figure S5A.

(H and I) Pull-down assay: HA-PHLPP1 and Flag-ChREBP interaction in HEK293T cells (H) and endogenous interaction (I) between PHLPP1 and ChREBP in RAW 264.7 cells;

(J) Malachite green assay: phosphate release upon the incubation of Flag-ChREBP with SFB-PHLPP1 in vitro. Statistical analysis was performed using two-tailed t-test (∗p < 0.05, vs Vector).

(K) AMPK Thr¹⁷² phosphorylation in ChREBP or PHLPP1 overexpressing RAW 264.7 cells. Densitometric analysis is shown in Figure S5B.

(L) Graph showing enriched ChREBP binding to FASN promoter upon PHLPP1 overexpression in ChIP-qPCR. Statistical analysis was performed using two-tailed t-test (∗p < 0.05 vs vector).

(M) Effect of AICAR and Act-3 on CD36 and FASN protein levels in PHLPP1 overexpressing cells. Densitometric analysis is shown in Figures S5C and S5D.

(N) Oil Red O quantification of neutral lipids in PHLPP1 and ChREBP over-expressed RAW 264.7 cells treated with AICAR and Act3. Images from three different experiments with a minimum of eight different fields in each experiment were used to quantify the data. Statistical analysis was performed using one-way ANOVA followed by Bonferroni′s post hoc analysis (∗p < 0.05 versus vector-untreated; ^δp < 0.05, ^δδp < 0.01 versus corresponding control-untreated; ^ζp < 0.05 versus corresponding sample OxLDL-treated). Data are a representative of three independent experiments. Numerical data are expressed as mean ± SEM. See also Figures S4 and S5.