Fig 2: Involvement of transcription factor ID3 in M-CD5L polarization. THP1 macrophages were left untreated (−) or were transfected with siRNA targeting ID3 (ID3) or a non-targeting negative control (Ct), and the following functional tests were performed: (A) RT-qPCR (left) and western blot representative image (right) of ID3 mRNA and protein silencing. RT-qPCR data show mean of five independent experiments. Detection of HIST3H3 was used as a measure of equal loading in western blot experiments. (B) The amount of mRNA encoding CD80, CD163, and Mer tyrosine kinase (MERTK) was measured by RT-PCR. Data show mean of five independent experiments. (C) Analysis of phagocytosis of apoptotic HepG2 cells by flow cytometry. CFSE-apoptotic HepG2 cells were co-cultured with THP1 macrophages for 1 h at a ratio of 1:2 (THP1: apoptotic cells). Graphs show mean ± SEM of five independent experiments. (D) Amounts of TNF, IL1β, and IL6 measured by ELISA in culture supernatants after 4 h (for TNF) or 24 h (for IL1β and IL6) of stimulation with 10 ng/mL LPS. Graphs show mean ± SEM of four independent experiments performed in triplicates. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001; Student’s t-test.

Fig 3: NRF3 impairs breast cancer cell invasion through the AKT/ID3 axis. (A) .The most ten downregulated genes under NRF3 overexpression obtained from the data of RNA seq, relative expression level, normalized by log10 (FPKM). (B) qRT-PCR was used to validate the RNA seq data, relative expression level, normalized by GADPH. (C) Western blot for ID3, AKT, p-AKT, NF-κB p65, and p-NF-κB p65 protein levels in MDA-MB-231 cells overexpressed with NRF3 or not. β-actin was as a loading control. (D) Western blot (left panel) for ID3, AKT and p-AKT protein levels in MDA-MB-231 cells treated with LY294002 or not. β-actin was as a loading control. (E) Western blot for ID3, E-cadherin and MMP-2 protein levels in MDA-MB-231 cells over-expressed with ID3 or not. β-actin was used as a loading control. (F) Cell invasion assays for MDA-MB-231 cells with overexpressed NRF3, ID3 alone or in combination by transwell with matrigel. Representative images at 40× (left panel), total view for whole well (middle panel) and the quantification for cell number invaded per view (right panel). Error bar indicated SD (*P<0.05 and **P<0.01). Three independent experiments were performed for all assays.Abbreviations: NRF3, nuclear factor, Erythroid 2 Like 3; AKT, AKT serine/threonine Kinase; ID3, inhibitor of DNA binding 3; FPKM, Reads Per Kilobase of exon model per Million mapped reads; MMP, matrix metallopeptidase; TIMP, TIMP metallopeptidase inhibitor.

Fig 4: EGF promotes trophoblast cell invasion by downregulating KISS1 expression. a, b, HTR-8/SVneo cells were pretreated with vehicle control (DMSO), 5 µM AG1478 (a), or 5 µM LY294002 (b) for 1 h, and then treated with 50 ng/mL EGF. After treatments, cells were seeded onto Matrigel-coated transwell inserts. After 48 h of incubation, noninvading cells were wiped from the upper side of the filter, and the nuclei of the invading cells were stained with crystal violet. c HTR-8/SVneo cells were transfected with 1 µg control vector (pCMV) or vector containing human ID3 cDNA (pCMV-ID3) for 48 h. After transfections, cells were treated with 50 ng/mL EGF and the levels of cell invasiveness were examined by transwell invasion assay. d HTR-8/SVneo cells were treated with 1 µM kisspeptin-10 (KP-10) and the levels of cell invasiveness were examined by transwell invasion assay. e HTR-8/SVneo cells were transfected with 1 µg control vector (pCMV) or vector containing human KISS1 cDNA (pCMV-KISS1) for 48 and 72 h. The protein levels of KISS1 were examined by western blot. Numbers under the western blots represent the densitometry quantifications. f HTR-8/SVneo cells were transfected with 1 µg control vector (pCMV) or vector containing human KISS1 cDNA (pCMV-KISS1) for 48 h. After transfections, cells were treated with 50 ng/mL EGF and the levels of cell invasiveness were examined by transwell invasion assay. The invasion assay results are expressed as the mean ± SEM of at least three independent experiments. Values without a common letter are significantly different (p < 0.05)

Fig 5: Human induced pluripotent stem cell (iPSC)‐derived astrocytes confirm a cell‐intrinsic vanishing white matter (VWM) phenotype. (A–F) Immunostaining showed expression of astrocyte markers glial fibrillary acidic protein (GFAP; A, D), nestin and Id3 (B, E), and CD44 and SOX9 (C, F) in both control (A–C) and VWM (D–F) human astrocyte cultures. To quantify proliferation of the astrocytes, bromodeoxyuridine (BrdU) labeling was performed. (G, H) Representative control (G) and VWM (H) astrocyte lines are shown. Quantification of proliferation was assessed using the percentage of BrdU‐labeled cells of the total population of cells (4,6‐diamidino‐2‐phenylindole [DAPI]) in control human iPSC (hiPSC)‐derived astrocytes. (I) VWM astrocytes showed increased proliferation compared to control astrocytes. (J, K) Immunostaining for Olig2 and myelin basic protein (MBP) of primary wild‐type embryonic day 18 mouse oligodendrocyte precursor cells cultured for 7 days in media conditioned by hiPSC‐derived astrocytes in representative examples of VWM and control cultures is shown. Maturation of the oligodendrocyte precursor cells was assessed using a ratio of MBP‐positive cells of the total Olig2‐positive population, in the presence of media conditioned by control and VWM iPSC‐derived astrocytes. Astrocyte conditioned media were treated with either vehicle or hyaluronidase (HAse). (L) Oligodendrocyte maturation was impaired in VWM compared to control cultures, and HAse rescued oligodendrocyte maturation comparable to control level. Scale bars = 100μm. *p < 0.05, **p < 0.01.