Fig 2: Genes involved in the generation of protrusions also control the formation of metastases.(A) Diagram of the approach to investigate the formation of liver metastases (met.) after intravenous injection of SCLC cells. (B–C) Quantification of the number of metastases 4 and 5 weeks after intravenous injection of N2N1G and 16T mSCLC cells, respectively, with control knock-down or knock-down of Gap43 with two independent shRNAs. For N2N1G, tumors at the surface of the liver were quantified on the liver surface, as shown in Figure 4—figure supplement 2D. Too many tumors were present with the 16 T cell line and the control shRNA, and quantification was thus performed by measuring liver weight. N = 4–5 mice per condition in one biological replicate. Mean + /- s.d. unpaired t-test. (D) Representative hematoxylin and eosin (H and E) images of liver sections of mice in (B–C). Scale bars, 5 mm. (E–H) As shown in (A–D) for Fez1 knock-down. See Figure 4—figure supplement 2E for representative images with N2N1G cells for the quantification in (F–G) of tumors at the surface of the liver. Arrows point to metastases. N = 4–5 mice per condition in one biological replicate. Mean + /- s.d. is shown, unpaired t-test. (I) Diagram of the approach to investigate early steps in liver metastasis, 2 days after intravenous injection. (J–M) Quantification of the number of GFPpositiveN2N1G and 16T mSCLC cells 2 days after intravenous injection. See Figure 4—figure supplement 2F-ID for representative flow cytometry. N = 5 mice per condition in one biological replicate. Mean + /- s.d., unpaired t-test.

Fig 3: FEZ1 regulates HSPA8 localization(A) Representative IF images of CHME3s expressing Flag control or HSPA8-Flag stained for total HSPA8 (red), exogenous HSPA8 (Flag, green), or the nucleus (Hoechst, blue). Scale bar, 10 µm.(B, D, and F) Representative IF images of intracellular distribution of HSPA8 in either CHME3s treated with two different negative controls (NC1 and NC2) or FEZ1-specific siRNAs (B) or three different FEZ1 KO CHME3 pools (FEZ1 KO 1, 3, and 4) (D), or CHME3 expressing Flag control, FEZ1-Flag, or S58A-Flag (F). Nuclei were stained with Hoechst. Scale bar, 10 µm.(C, E, and G) Quantitative analysis of HSPA8 staining in nuclei of CHME3s in (B, D, and F), respectively. Number of cells analyzed is indicated, n = 3; red line, mean; bars, SD. One-way ANOVA was used to calculate statistical significance with Tukey (C and G) and Dunnett (E) post-hoc tests.(H and I) Representative (n = 2) WB analysis of nuclear and cytoplasmic fractions from control and FEZ1 KO CHME3s (cropped to remove irrelevant samples) (H) or Flag, FEZ1-Flag, and S58A-Flag expressing CHME3s (I).See also Figure S3.

Fig 4: FEZ1 and HSPA8 regulate nuclear localization of DNA-PK(A, C, and E) Representative IF images of DNA-PK staining in three different FEZ1 KO CHME3 pools (FEZ1 KO 1, 3, and 4) (A), or CHME3s expressing Flag control, or FEZ1-Flag or S58A-Flag (C), or HSPA8 KO CHME3 pools (HSPA8 KO 1, 2, and 4) (E). Nuclei were stained with Hoechst (blue). Scale bar, 10 µm.(B, D, and F) Quantitative analysis of nuclear DNA-PK staining in samples from (A, C, and E), respectively.(G–L) Responses to CT-DNA in CHME3 microglia. (G) WB analysis showing decreased phosphorylated FEZ1 at S58 (pFEZ1) as well as increased levels of phosphorylated IRF3 (pIRF3) and ISGs (MxA, MxB, and ISG56) in CHME3s treated with CT-DNA. (H) Quantification of the pFEZ1 levels and ISGs from samples in (G) n = 3; bars, SD. (I–L) Representative images (I and K) and quantitative analysis (J and L) of HSPA8 (I and J) or DNA-PK (K and L) staining in nuclei of CHMEs either untreated or treated with CT-DNA. One-way ANOVA followed by Dunnett post-hoc test (B and F) or Tukey post-hoc test (D), and t test (J, H, and L) was used to calculate statistical significance. (B, D, F, J, and L) Number of cells analyzed is indicated; n = 3; red line, mean; bars, SD.See also Figures S4 and S5.

Fig 5: HSPA8 interacts with FEZ1 and regulates ISG expression(A) WB confirmation of FEZ1 binding to endogenous HSPA8 in complexes isolated on GFP-TRAP agarose from CHME3s or CHME3s stably expressing GFP control, GFP-FEZ1, or GFP-S58A.(B) Quantification of HSPA8 levels in protein complexes from (A).(C) Reciprocal GFP pulldowns from CHME3 lysates expressing GFP or GFP-HSPA8 along with either FEZ1-Flag or S58A-Flag.(D) Quantification of FEZ1 and FEZ1 S58A levels in protein complexes from (C).(E) CoIP analysis showing interaction between endogenous FEZ1 and HSPA8 in CHME3 control or FEZ1 KO cells.(F) Quantification of HSPA8 levels in protein complexes from (E).(G) Representative WB (n = 2) showing decreases ISG levels in CHME3 expressing either GFP-HSPA8 (left panels) or HSPA8-Flag (right panels). Exo., exogenous; End., endogenous.(H) KO of HSPA8 (HSPA8 1, 2, or 4, or HSPA8 pooled), but not non-targeting (NT) gRNAs, increases ISG levels in CHME3s.(I) Quantification of ISG levels relative to HSPA8 in cells from (H). Data are presented as the ratio to the difference between control and treatment groups. A Student’s t test was used in (F) and one-way ANOVA followed by Tukey post-hoc test in (B, D, and I) to calculate statistical significance. (B, D, F, and I) n = 3; red line, mean; bars, SD.See also Figure S2.