Fig 2: Regulation of SREBF2 by ARMC5 in H295R cells.(A) Western blotting of lysates from H295R cells transfected with negative control siRNA (siControl) or siRNA targeting to ARMC5 (siARMC5) for 48 hours with indicated antibodies. (B) Gene expression of the indicated genes in H295R-TetON-hARMC5 (ARMC5[WT]) or H295R-TetON-hARMC5(R362L) (ARMC5[R362L]) transfected with negative control siRNA (C) or siRNA targeting to ARMC5 (A) for 48 hours followed by treatment of doxycycline for 48 hours (n = 3, each). *P < 0.05, **P < 0.01, ***P < 0.001 by Tukey-Kramer test. (C) Gene expression of the indicated genes in H295R cells transfected with negative control siRNA (Cont), siRNA targeting to ARMC5 (ARMC5), and/or siRNA targeting to SREBF2 (SREBF2) for 72 hours (n = 3, each). **P < 0.01, ***P < 0.001 by Dunnett’s test, compared with siControl. See complete unedited blots in the supplemental material.

Fig 3: ARMC5 interacted with N-terminus of SREBFs through the Armadillo repeat.(A) Western blotting of lysates from the differentiated 3T3-L1–TetON-FLAG-Srebf1(N) cells treated with or without 10 μg/mL doxycycline for 30 hours with an anti-FLAG antibody. (B) Venn diagrams representing the number of identified proteins by LC-MS/MS of the samples immunoprecipitated with anti-FLAG antibody from the nuclear extracts of the differentiated 3T3-L1–TetON-FLAG-Srebf1(N) cells treated with or without 10 μg/mL doxycycline. Total spectrum counts of LC-MS/MS of the indicated protein in the sample treated with doxycycline are shown. (C) Western blotting of lysates (input) or samples immunoprecipitated with anti-FLAG antibody (FLAG IP) from the HEK293T cells transfected with pcDNA3.1-FLAG, pcDNA3.1-myc, pcDNA3.1-FLAG-Srebf1(N), and/or pcDNA3.1-myc-mArmc5 with the indicated antibodies. (D) Western blotting of lysates (input) or samples immunoprecipitated with anti-myc antibody (Myc IP) from the HEK293T cells transfected with pcDNA3.1-myc, pcDNA3.1-myc-mArmc5, and pcDNA3.1-FLAG-Srebf1(N) with the indicated antibodies. (E) Western blotting of lysates (input) or samples immunoprecipitated with anti-FLAG antibody (FLAG IP) from the HEK293T cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-Srebf1(N), pcDNA3.1-FLAG-Srebf2(N), and/or pcDNA3.1-myc-mArmc5 with the indicated antibodies. (F) Schematic representation of the structure of mouse ARMC5 (WT), the N-terminal deletion mutant (ΔARM), and the C-terminal deletion mutant (ΔBTB). (G) Western blotting of lysates (input) or samples immunoprecipitated with anti-FLAG antibody (FLAG IP) from the HEK293T cells transfected with pcDNA3.1-FLAG, pcDNA3.1-myc, pcDNA3.1-FLAG-Srebf1(N), pcDNA3.1-myc-mArmc5 (WT), pcDNA3.1-myc-mArmc5ΔARM (ΔARM), and/or pcDNA3.1-myc-mArmc5ΔBTB (ΔBTB) with the indicated antibodies. See complete unedited blots in the supplemental material.

Fig 4: Regulation of cholesterol homeostasis is altered in LIMP-2 deficient cells. Analysis of LIMP-2 influence on transcriptional regulation of cholesterol homeostatic genes. a Termination of SREBP2 processing in response to abundant cholesterol in the ER (induced by LDL loading) is absent or incomplete in NPC1-silenced and LIMP-2-silenced HeLa cells, respectively. HeLa WT cells were treated with scrambled (ctrl), NPC1 or LIMP-2 siRNA, respectively, and incubated in either full medium, cholesterol-depleted medium or LDL-loaded medium. The processing of SREBP2 in response to high or low cholesterol levels was analyzed by immunoblot. b The quantification depicts the amount of nSREBP2 after LDL loading in cells treated with scrambled (ctrl), NPC1 or LIMP-2 siRNA, respectively (n = 3; data (mean ± SEM) from two experiments; *P = 0.05, t-test). c Analysis of the effect of cholesterol depletion and re-addition of LDL cholesterol for 24 h in NPC1-deficient HeLa cells on nSREBP2 formation. Cells were either treated with control siRNA or with LIMP-2 specific siRNA. The processing of the SREBP2, as well as the expression of the LDL receptor (LDLR) and LIMP-2 was followed by immunoblot. Note that additional depletion of LIMP-2 led to incomplete termination of nSREBP2 fragment and LDLR formation after cholesterol loading for 24 h. d Quantification of nSREBP2 levels. Nuclear SREBP2 fragment as percentage of total SREBP2 is depicted. (n = 2; data (mean ± SD) from one experiment). e Quantification of LDLR levels after cholesterol loading, normalized to the cholesterol-depleted samples (n = 5; data (mean ± SEM) from two experiments; *P = 0.05, **P = 0.01, unpaired two-tailed Student’s t-test). f Model of the role of LIMP-2 in lysosomal cholesterol efflux. Source data are provided as a Source Data file

Fig 5: CCL2/7 induce cholesterol synthesis in lung-colonizing TNBC cells(A) Gene Ontology (GO) analysis of the upregulated genes. RNA-seq experiments were performed in control and mouse Ccl2 (10 nM)/7 (50 nM)-treated (6 h) FOXC1-overexpressing MDA-MB-231 cells. (B) Real-time RT-PCR confirming upregulation of cholesterol synthesis-associated genes identified by RNA-seq. (C) IHC of HMGCR and HMGCS1 in human primary TNBC tumors and matched lung metastases (n = 4 pairs). (D) Western blot of the nuclear accumulation of the mature SREBP2 protein in mouse Ccl2 (10 nM)/7 (50 nM)-treated FOXC1-overexpressing MDA-MB-231 cells at different time points. Lamin A/C was used as a control. (E) IHC of SREBP2 in human TNBC lung metastases. (F) Heatmap analysis of the upregulated secreted factors in the RNA-seq analysis performed in (A). The bar graph indicates mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.