Fig 2: SREBP1 Mediated Regulation of MRP1 Expression Induced by Endoplasmic Reticulum Stress. (a) After treating cells with TG (30 nM), the mRNA levels of MRP1 were assessed by real‐time qPCR. (b) TG (30 nM) and exogenous XBP1 cDNA significantly increased the promoter activity of MRP1. (c) There are two putative binding sites for SREBP1 on the MRP1 promoter. (d) Following 48‐h transfection of SREBP1 cDNA, Western blot results revealed the expression of full‐length SREBP1, cleaved (active) SREBP1, and MRP1. (e) H1299 and A549 cells were transfected for 48‐h with SREBP1 siRNA or control siRNA. Western blot results revealed the expression of full‐length SREBP1, cleaved SREBP1 (active), and MRP1. (f) After transfection cells with SREBP1 siRNA, cells were further treated with TG (30 nM) for an additional 12 h. Western blot analysis revealed the expression of p‐IRE1α, XBP1s, SREBP‐P, SREBP‐N, and MRP1. (g) 48 h before Western blot experiments, cells were co‐transfected with IRE1α cDNA and SREBP1 siRNA. Western blot analysis revealed the expression of IRE1α, XBP1s, SREBP‐P, SREBP‐N, and MRP1. (h) Western blot analysis of cytosolic and nuclear extracts of SREBP1from TG (30 nM) or untreated cells. (i) H1299 cells were co‐transfected with MRP1 promoter reporter construct 1412 and SREBP1 cDNA plasmid. 48 h after transfection, luciferase activity was determined and normalized using the dual luciferase reporter system (***p < 0.001 for difference from control cells). (j) H1299 cells were co‐transfected with MRP1 promoter reporter construct 1412 and SREBP1 siRNA. Transfection of SREBP1 siRNA significantly reduced the promoter activity of MRP1. (k) The cells were co‐transfected with MRP1 promoter and SREBP1 siRNA in the presence and absence of TG (30 nM) (Left panel) or co‐transfection with IRE1α cDNA (Right panel). Luciferase activity was determined and normalized using the dual luciferase reporter system (**p < 0.01, ***p < 0.001 for difference from control cells, ### p < 0.001 for the difference from TG or transfected with IRE1α cDNA cells by ANOVA with Dunnett's correction for multiple comparisons). (l) Schematic representation of two designated constructs of MRP1 promoter (1412‐Mut and 408). (m) Cells were co‐transfected either MRP1 promoter reporter construct 1412 or mutated MRP1 promoter reporter (1412‐Mut) with SREBP1 cDNA. After 48‐h transfection, luciferase activity was determined and normalized using the dual luciferase reporter system. (***p < 0.001 for difference of wild‐type MRP1 promoter, #### p < 0.0001 for difference of mutated MRP1 promoter). (n) The cells were either co‐transfected MRP1 promoter reporter construct 1412 or the short length MRP1 promoter construct 408 with SREBP1 cDNA. Luciferase activity was determined and normalized using the dual luciferase reporter system. (o) qRT‐PCR were taken to analyze the CUT&Tag results. qRT‐PCR products were run on a gel (Right panel). qRT‐PCR results were quantified and are indicated (Left panel) (***p < 0.001 for difference from IgG‐added cells). (p) After transfecting with SREBP1 siRNA alone or co‐transfected with IRE1α cDNA for 48 h, cells were treated with etoposide (40 μM) in the presence or absence of TG (30 nM). MTT assay was used to detect cell viability. (****p < 0.001 for difference from control cells, #### p < 0.0001 for the difference from etoposide‐treated cells, $$$$ p < 0.001 for difference from SREBP1 siRNA and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons).

Fig 3: The mRNA and protein expression of CAV-1, MFSD2A and SREBP1 on hCMEC/D3 cells (x¯ ± S.D, n = 3). (a–c) The mRNA expression of CAV-1, MFSD2A and SREBP-1 by RT-qPCR analysis. (d–g) The protein expression of CAV-1, MFSD2A and SREBP-1 by Western blot analysis. A. Control group; B. Model group; C. Nimodipine positive control group; D. L-Z 6: 4 Low-dose group; E. L-Z 6: 4 Medium-dose group; F. L-Z 6: 4 High-dose group. (h) Fuorescence expression of CAV-1 on hCMEC/D3 cells. (i) Fluorescence expression of MFSD2A on hCMEC/D3 cells. * p < 0.05, ** p < 0.01 vs. control, # p < 0.05, ## p < 0.01 vs. model.

Fig 4: Dysregulated cholesterol metabolism after PCZ + PIT treatment.(A) GSEA transcriptomic analysis of treatment response in LU-NB-2 PDX cells after 48 h treatment using KEGG pathways, average of n = 2*4 per condition (biological*technical replicates). (B) Transcription factor (TF) activity inferred with CollecTRI after combination treatment. (C) Volcano plots displaying expression of the downstream targets of transcription factors SREBF1 and SREBF2 after PCZ + PIT combination treatment. (D) Western blot analysis of treatment response after 48 h. Samples within one biological replicate (experiment) were run in parallel to ensure the capture of multiple markers of similar size (n = 4-5 biological replicates). (E) Quantification of protein expression of SREBP1 (n = 4), SREBP2 and cleaved SREBP2 (n = 5 each), followed by MYCN and cleaved PARP (bottom row) (n = 4, one-way ANOVA followed by Tukey’s multiple comparison test). (F) Images of immunofluorescence of SREBP2 in LU-NB-2 cells after 48 h PCZ-PIT combination treatment (n = 3 biological replicates). (G) Colocalization analysis of LU-NB-2 cells, mean intensity of SREBP2 fluorescence signal in the nucleus. Data presented as median violin plot; each dot represents a cell. For each condition, 382–432 DAPI+ cells were counted (i.e., three biological replicates (R)* two conditions: control and PIT 2.2 µM + PCZ 1.6 µM treated cells). Kolmogorov–Smirnov analysis for testing the assumption of normality, followed by two-way ANOVA and Bonferroni´s nonparametric test for data with non-normal distribution. Scale bar 20 µm. GSEA gene set enrichment analysis, PCZ prochlorperazine, PIT pitavastatin, TF transcription factor. Source data are available online for this figure.

Fig 5: The mRNA and protein expression of CAV-1, MFSD2A and SREBP1 in the brain of rats. (x¯ ± S.D, n = 3). (a–c) The mRNA expression of CAV-1, MFSD2A and SREBP-1 by RT-qPCR analysis. (d–g) The protein expression of CAV-1, MFSD2A and SREBP-1 by Western blot analysis. (h) Fluorescence expression of CAV-1 on brain microvascular endothelial cells (Scale bar, 50 μm). (i) Fluorescence expression of MFSD2A on brain microvascular endothelial cells (Scale bar, 50 μm). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. MCAO group.