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: GIPC2 regulates SREBP1 activity.a GSEA comparing gene sets involved in the adipocytokine signaling pathway, lipid homeostasis, positive regulation of transcription by RNA polymerase and transcription coregulator binding between GIPC2-overexpressing UC-MSCs treated with or without β-elemene after 7 days of adipogenic induction. b Network plot illustrating transcription factors (TFs) enriched with differentially expressed genes (DEGs) in GIPC2-overexpressing UC-MSCs treated with or without β-elemene, with green nodes representing TFs and blue nodes representing target DEGs. c qRT-PCR analysis of SREBP1 in GIPC2-overexpressing UC-MSCs following β-elemene or PKM2-IN-1 treatment. d, e WB and immunofluorescence analyses evaluating SREBP1 expression in GIPC2-overexpressing UC-MSCs following β-elemene treatment. f qRT-PCR analysis of ACLY, FASN, FDPS, FDFT1, and HMGCS1 in GIPC2-overexpressing UC-MSCs following β-elemene treatment. Transcriptomic analysis was performed on three samples per group (n = 3) in (a). All experiments were conducted in triplicate unless otherwise indicated. Statistical analysis: one-way ANOVA (c), two-sided t-test (d–f). Error bars: mean ± s.e.m.; Scale bar, 100 μm (e), 10 μm (e, enlarged image). a, d–f “GIPC2” indicates UC-MSCs overexpressing GIPC2, and “β-elemene” indicates GIPC2-overexpressing UC-MSCs with β-elemene. c “GIPC2” indicates UC-MSCs overexpressing GIPC2, “β-elemene” indicates GIPC2-overexpressing UC-MSCs with β-elemene, and “PKM2-IN-1” indicates GIPC2-overexpressing MSCs with PKM2-IN-1.

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: GIPC2 promotes adipogenic differentiation in MSCs via the PKM2-SREBP1 axis.a WB analysis of the adipogenic markers PPAR-γ, C/EBP-α, and FABP4 in GIPC2-overexpressing MSCs treated with Fatostatin. b ORO staining of Fatostatin-treated GIPC2-overexpressing UC-MSCs to assess lipid droplet formation after 15 days of adipogenic induction. c WB analysis of nuclear proteins isolated from UC-MSCs undergoing adipogenic differentiation for 15 days was performed to assess the nuclear expression of SREBP1 and PKM2 during the differentiation process. d Mechanism schematic diagram of GIPC2 promoting adipogenic differentiation in MSCs via the PKM2-SREBP1 axis. Statistical analysis: one-way ANOVA (c), two-sided t-test (a, b). Error bars: mean ± s.e.m.; Scale bar, 50 μm (b). a, b “GIPC2” indicates UC-MSCs overexpressing GIPC2, and “Fatostatin” indicates GIPC2-overexpressing MSCs with Fatostatin.