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. Author manuscript; available in PMC: 2024 Jul 8.
Published in final edited form as: Nat Cell Biol. 2023 Oct 2;25(10):1506–1519. doi: 10.1038/s41556-023-01241-6

Extended Data Fig. 8 |. Reelin-Vldlr signaling regulates SCLC-induced astrocyte migration.

Extended Data Fig. 8 |

a. Chromatin accessibility at two sites in the Reln locus assessed by ATAC-seq in shCtrl (control) and shNfib 16 T and KP1 cells, and KP22 cells overexpressing empty vector (oe empty) or mouse NFIB (oe Nfib). Chromatin immunoprecipitation (ChIP) for NFIB in 16 T, KP1, and KP22 cells. b. Immunoassay for NFIB in shCtrl and shReln N2N1G cells. HSP90 serves as a loading control. c. Quantification of (b) (n = 3 independent experiments). d. Representative images showing immunofluorescence staining of Reelin (red) and GFP (green, cancer cells), or Vldlr (gray) and GFAP (red) on an N2N1G brain allograft section. Blue: DAPI DNA stain. Scale bar, 50 μm. Similar results were observed from 3 biologically independent samples. e. Immunoassay for Reelin in shCtrl and shRELN NCI-H69 cells. HSP90 serves as a loading control (n = 1). f. Representative images showing immunofluorescent staining of human astrocytes (hA, marked by Vimentin) for the reactivation marker GFAP (green) when co-cultured (CC) with shCtrl or shRELN NCI-H69 cells. Blue: DAPI to stain DNA. Scale bar, 20 μm. g. Quantification of fluorescent intensity for GFAP normalized to Vimentin (n = 3 independent experiments) as in (f). h. Representative images showing rare immunofluorescent staining of GFAP (green) in Vimentin+ (red) hA treated with or without recombinant human Reelin. Blue: DAPI DNA stain. Scale bar, 20 μm. Similar results were observed from 2 independent experiments. i. Cell viability (AlamarBlue assay) measured in shCtrl and shRELN NCI-H69 cells cultured with hA compared to without hA (n = 3 independent experiments). j. Chemotaxis of mouse astrocytes (mA) in control medium or medium with recombinant mouse Reelin protein and the mouse Reelin-blocking antibody CR-50. k. Relative viability of mA in shCtrl and shReln N2N1G-conditioned medium with or without the addition of recombinant mouse Reelin compared to control medium (n = 3 independent experiments). l. Same as (i) for hA and human Reelin. m. Immunoassay for VLDLR in shCtrl and shVLDLR hA. HSP90 serves as a loading control (n = 1 experiment). n. Chemotaxis assay for shCtrl and shVLDLR hA with or without hSCLC-conditioned medium (n = 3 independent experiments). o. Viability of shDAB1 hA cells at 48 hours. Data show mean with SD. n = 3 independent experiments. P values calculated via two-sided t-test when comparing two groups and via one-way ANOVA when comparing 3–4 groups.