EMBO/SourceData huggingface dataset:
Figure 1Bioluminescence imaging and quantification of brain metastases (H) of BALB/c nude mice xenografted with control or IFITM1-knockout H1299 cells (populations of knockout cells) by intracardiac injection (1×106 cells). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) (H, quantification of brain metastasesBioluminescence imaging overall survival (I) of BALB/c nude mice xenografted with control or IFITM1-knockout H1299 cells (populations of knockout cells) by intracardiac injection (1×106 cells). #, experimental endpoint (I). , the log-rank test (I, survival(J) Bioluminescence imaging and quantification of brain metastases and overall survivals of BALB/c nude mice xenografted with control or IFITM1-overexpressing H460 cells by intracardiac injection (1×105 cells). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) the log-rank test survival in J(N) Kaplan-Meier survival curves of lung cancer patients in the TCGA dataset (n=415) stratified by IFITM1 mRNA expression. HR, hazard ratio. the log-rank test survival in N)
Figure 2(A) Representative immunofluorescence images and quantification of microglial (TMEM119+) or macrophage (TMEM119-/F4/80+) density per microscopic field in the brain on day 1 or 7 after the intracardiac injection of control or IFITM1-overexpressing LLC-BrM cells (tdTomato, 1×106) into C57BL/6 mice (90, 90, and 120 fields for normal, day 1, and day 7, respectively; normal, 3 mice; day 1, 3 mice; day 7, 4 mice). Normal brain tissue was used as the control.(C) Flow cytometric analysis of violet-labeled LLC-BrM cells phagocytosed by microglia in the brains of LLC-BrM cell-allografted BALB/c nude mice on day 1 after intracardiac injection (1×106 control or IFITM1-knockout cells). Representative flow cytometry pseudocolor density plots and the gating strategy are shown, and the bar graph shows the percentage of violet+ microglia. (The n-values denote the number of mice per group.)(E) LLC-BrM cells (tdTomato) were surrounded by activated microglia (Iba1+, green) and IFNγ (white) in the brain on day 1 or 7 after intracardiac injection of control or IFITM1-overexpressing cells (1×106) into C57BL/6 mice. IFNγ intensities in the fields enclosed in the yellow dotted lines (ROIs) (90, 90, and 120 fields for normal, day 1, and day 7, respectively) are shown. The ROI was set as the entire area of the metastatic lesion for day 7 and as the area of single metastatic cell for day 1. ImageJ was used to quantify the immunofluorescence intensity in the ROI. Normal brain tissue was used as the control. ROI, region of interest.(G) Results of ELISA for mouse IFNγ in the brain on day 1 after the intracardiac injection of control or IFITM1-knockout LLC-BrM cells (1×106) into BALB/c nude mice. PLX5622 (1,200 PPM) was administered in chow from day -3 to day 1. BLZ945 (200 mg/kg) was administered by oral gavage from day -2 to day 1. Minocycline (33 mg/kg) was administered intraperitoneally on days -1, 0, and 1. Clodronate liposomes (100 μl/10 g) were administered intraperitoneally on day -1. Normal brain tissue was used as the control. (The n-values denote the number of mice per group.)
Figure 3(A) ELISA results of mouse TNFα in supernatants obtained from the indicated coculture systems at the indicated time points. Primary mouse microglia, 6×104 cells from 16 BALB/c mice. H460, 6×104 cells. CM, conditioned medium (from 6×104 H460 cells) (1 independent experiment). We would like to point out that TNFα, instead of IFNγ, was used as a marker of microglial activation in vitro(E) ELISA results of mouse IFNγ in the brain on day 1 after intracardiac injection of the indicated LLC-BrM cells (control, C3 knockout, IFITM1 overexpression, or IFITM1 overexpression in combination with C3 knockout, 1×106) into C57BL/6 mice. Normal brain tissue was used as the control. (The n-values denote the number of mice per group.)(G) Control and IFITM1-overexpressing LLC-BrM cells (1×106, tdTomato) were surrounded by activated microglia (left, green, Iba1+) and complement C3 (left, white) in the brain on day 1 after intracardiac injection into C57BL/6 mice (normal, 3 mice; day 1, 3 mice; day 7, 4 mice). Complement C3 intensities in the fields enclosed in the yellow dotted lines (ROIs) are shown (right; 90, 90, and 120 fields for normal, day 1, and day 7, respectively). The ROI was set as the area of a single metastatic cell. ImageJ was used to quantify the immunofluorescence intensity in the ROI. Normal brain tissue was used as the control. ROI, region of interest.We would like to point out that TNFα, instead of IFNγ, was used as a marker of microglial activation in vitro (H) Results of ELISA for mouse TNFα in the culture supernatant of primary mouse microglia (6×104, from 16 C57BL/6 mice), primary mouse BMDMs (6×104, from 3 C57BL/6 mice), or primary mouse BrM-BMDMs (6×104, from 37 C57BL/6 mice) stimulated with conditioned medium from control LLC-BrM cells (1×104) and IFITM1-knockout LLC-BrM cells (1×104). BMDMs, primary bone marrow-derived macrophages. BrM-BMDMs, primary bone marrow-derived macrophages from the brain metastatic lesions of LLC-BrM cell-isografted C57BL/6 mice on day 20 after the intracardiac injection of LLC-BrM cells (1×105 cells). CM, conditioned medium (3 independent experiments).(L) Flow cytometric analysis of apoptotic LLC-BrM cells (6×104) in contact coculture with primary mouse microglia (6×104, from 16 C57BL/6 mice), which were activated by conditioned medium from control or C3-knockdown H460 cells (1×106). CM, conditioned medium (3 independent experiments).
Figure 4(A) Schematic of efficacy of adoptive CD8+ T cell immunotherapy in suppressing brain metastasis (top far left), representative western blots of IFITM1 expression in control or IFITM1-overexpressing LLC-BrM cells (bottom far left), bioluminescence imaging (middle left), quantification of brain metastases (middle right) and overall survivals (far right) of BALB/c nude mice allografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (1×105 cells). Primary mouse naïve CD8+ T cells from 12 C57BL/6 mice or not were injected intravenously on days -1, 6, and 13 (1×104 cells at each time point). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) (B) Schematic of efficacy of adoptive CD8+ or CD4+ T cell immunotherapy in suppressing brain metastasis (far left), bioluminescence imaging (middle left), quantification of brain metastases (middle right) and overall survivals (far right) of BALB/c nude mice allografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (1×105 cells). Primary mouse naïve CD8+ or CD4+ T cells from 12 C57BL/6 mice were injected intravenously on days -1, 6, and 13 (1×104 cells at each time point). (The n-values denote the number of mice per group.) (quantification of brain metastases ANOVA with uncorrected Fisher's LSD test or the log-rank test (survival(G) Bioluminescence imaging and quantification of brain metastases on day 20 in C57BL/6 mice isografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (3×105 cells). Mice were systemically primed-boosted with homologous LLC-BrM cell lysates (1×106 cells for day -21 and 1×105 cells for day -7) before injection of cancer cells. Then, IgG or an anti-CD8α antibody (2 μg/mouse) was injected intravenously on day 3 alone or in combination with minocycline treatment (33 mg/kg) on day 1. Minocycline was administered intraperitoneally. (The n-values denote the number of mice per group.) one-way (G)
Figure 5(C) Immunofluorescence staining of H1299 cells with anti-IFITM1 and anti-MHC-I antibodies. H1299 cells (6×104) were stimulated with 1,000 U/ml IFNγ for 48 hr and were then treated with the anti-MHC-I antibody for 1 hr. The colocalization percentages of IFITM1 (red) and MHC-I (green) are shown (100 cells for each condition). : The data are presented as the mean ± s.e.m. values. P values were determined by an unpaired two-tailed Student's t-test with Welch's correction (C(D) Co-IP of endogenous IFITM1 and MHC-I proteins in H1299 cells (1×106) after IFNγ stimulation (1,000 U/ml) for 48 hr (3 independent experiments).(I) Immunoblot analysis of MHC-I expression in the cytoplasm, on the cell membrane, and in the nucleus of control and IFITM1-overexpressing H1299 cells (6×104) (3 independent experiments).(K) qPCR analysis of MHC-I mRNA expression in control and IFITM1-overexpressing H1299 cells (6×104) with or without IFNγ stimulation (1,000 U/ml) for 24 hr (3 independent experiments). (L) qPCR analysis of MHC-I mRNA expression in control and IFITM1-knockdown H1299 cells (6×104) with or without IFNγ stimulation (1,000 U/ml) for 24 hr (3 independent experiments).(M) Cleaved caspase 3 (green)-positive LLC-BrM cells (tdTomato) were surrounded by CD8+ T cells (white) in the brain metastatic lesions of LLC-BrM cell-isografted C57BL/6 mice on day 20 after intracardiac injection of control or IFITM1-overexpressing LLC-BrM cells (1×106 cells). The positively stained areas of single CD8+ T cells, clustered CD8+ T cells, cleaved caspase 3+ cancer cells, and tdTomato+ cancer cells in the ROI were calculated by ImageJ. The ratios of the areas between the clustered and single CD8+ T cells are shown as the numbers of CD8+ T cells in the ROI, and the ratios of the areas between cleaved caspase 3+ cancer cells and tdTomato+ cancer cells are shown as the percentages of cleaved caspase 3+ cancer cells in the ROI (90 fields per group, 3 mice per group). Yellow dotted lines (ROIs), brain metastatic lesions; ROI, region of interest.(O) Kaplan-Meier survival curves of lung cancer patients in the TCGA dataset stratified by IFITM1 and MHC-I mRNA expression. HR, hazard ratio. ANOVA with uncorrected Fisher's LSD test, or the log-rank test (O).
Figure 6(F) Bioluminescence imaging, quantification of brain metastases, and overall survival of C57BL/6 mice isografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (3×105 cells). Mice were systemically primed-boosted with homologous LLC-BrM cell lysates (1×106 cells for day -21 and 1×105 cells for day -7) before injection of cancer cells, and IgG or an anti-PD-1 antibody (100 μg/mouse) was then injected intravenously on days 3, 6, and 9. (The n-values denote the number of mice per group.) unpaired two-way (quantification of brain metastases in F) ANOVA with uncorrected Fisher's LSD test, or the log-rank test (survival in F).(H) Bioluminescence imaging and quantification of brain metastases in C57BL/6 mice isografted with control or IFITM1-knockout LLC-BrM cells by intracardiac injection (3×105 cells). IgG (30 μg/mouse, days 3, 6, and 9), an anti-PD-1 antibody (30 μg/mouse, days 3, 6, and 9), or UV-irradiated oncolytic virus (106 pfu/mouse, days 0, 2, and 4) was injected intravenously alone or in combination as indicated. (The n-values denote the number of mice per group.) one-way (quantification of brain metastases in H)
Figure 1Transposon screen validation of UBX5. ubx5∆ restores the growth of tdp1wss1. Cells were grown in YEPD and spotted on a medium supplemented with 1 mM auxin (for tdp1-degron degradation) and 5 μg/mL Camptothecin (CPT). Plates were incubated for 2 days at 30°C.Ubx5 levels are decreased in tdp1wss1. (E) HA-Ubx5 protein levels at steady state are compared by immunoblotting in different mutants. Mutant strains carrying the additional ubx5∆ mutation were complemented with a plasmid expressing HA-tagged Ubx5 from the endogenous pUBX5 promoter. 1mM auxin was added where indicated for 6 h to degrade tdp1-degron, when present. Pgk1 was used as a loading control.
Figure 2The UBX∆ mutant of Ubx5 is sufficient to suppress FA and HU sensitivities of wss1∆. ubx5∆wss1∆ mutants were complemented with plasmids encoding HA-Ubx5-WT or HA-Ubx5 mutants overexpressed from the pGAL promoter; cells were grown in SC-trp and either treated with 40 mM FA for 15 min or spotted directly on 20 mM HU. Plates were prepared either with glucose (no HA-Ubx5 expression) or galactose (HA-Ubx5 expression) as the source of sugar. Plates were incubated for 2.5 days at 30°C. o/e, overexpression.Protein levels of overexpressed HA-Ubx5 mutants shown in (C) were analyzed by immunoblotting (as in Fig 1E).
Figure 3(B) The ubx5∆ mutation rescues growth defects caused by Flp-nick galactose-induction in the tdp1∆ wss1∆ top1∆ mutant. Indicated strains were grown in YEP-2% raffinose prior to plating on YEP-2% glucose or YEP-2% galactose plates. Plates were incubated for 3 days at 30°C.(E) Ubx5 occupancy at the Flp-bound FRT site. Levels of Ubx5-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in cells grown as described in (C). The graph shows enrichment of qPCR signals over the unrelated intergenic region. Data are presented as means ± SDs for n = 5 independent biological replicates. p-values for +200 bp were defined by 2-way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; ** p < 0.01; **** p < 0.0001). In addition to the indicated mutations, all strains are bar1∆ Ubx5-TAP. No FRT strain and raffinose (raf) samples were used as negative controls.
Figure 4Cdc48 occupancy at the Flp-bound FRT site. Levels of Cdc48-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in asynchronous cells before and after Flp-cc induction with galactose. The graph shows enrichment of qPCR signal over the unrelated intergenic region. Data are presented as means ± SDs of 3 to 4 independent biological replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; **** p < 0.0001). In addition to the indicated mutations, all strains are Cdc48-TAP. No FRT strain was used as a negative control.Ubiquitination of the FRT locus. Asynchronous cultures were grown in 2% raffinose (raf) or additionally supplemented with 3% galactose (gal) to induce flp-H305L expression. Cells were also collected during glucose repression of flp-H305L expression at time points 30 min (glu 30') and 120 min (glu 120'). Ubiquitin antibody was used for ChIP-qPCR analysis following formaldehyde crosslinking. Data are shown as the mean ± SDs of three to six independent replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; *** p < 0.001; **** p < 0.0001). In addition to all indicated mutations, all strains are bar1∆ Ddi1-TAP.
Figure 5ubx5∆ restores wss1∆ resistance to HU and FA but not upon additional deletion of DDI1. Strains were grown in YEPD and either treated with 40 mM FA for 15 min or directly spotted on plates containing 100 mM of HU or 5 μg/mL CPT. Plates were incubated for 2 days at 30°C.Endogenous HA-Ddi1 levels are still elevated upon additional ubx5∆ mutation. Levels in total cell extracts were compared by immunoblotting. For quantification, anti-HA signal was normalized to Pgk1 and then normalized to WT (see (D)). Quantification of HA-Ddi1 levels shown in (C). The anti-HA signal was compared to Pgk1 and normalized to WT. Quantifications of 5 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparisons test and WT as a control (*p < 0.05; ** p < 0.01).Catalytic activity of the protease Ddi1 is essential for survival of the ubx5∆ wss1∆ yeast mutant. Mutants carrying wild-type (WT) Ddi1, D220A catalytic mutant or ddi1∆ in combination with ubx5∆, wss1∆ or double ubx5∆ wss1∆ were grown in YEPD. Cells were treated with 40 mM formaldehyde (FA) for 15 min or directly spotted on plates supplemented with 100 mM hydroxyurea (HU). Plates were incubated for 2 days at 30°C.
Figure 6Ubx5 loss restores HU-induced Rpb1 degradation in wss1∆ and requires Ddi1. Rpb1 levels were defined in cells growing in the presence of 200 mM HU and 100 μg/mL cycloheximide (CHX) at the indicated time points. Rpb1 and Pgk1 levels in total cell extracts were probed by immunoblotting and quantified using fluorescent secondary antibodies. See (B) for quantifications. Images show immunoblotting using chemiluminescence.Rpb1 chromatin enrichment following HU treatment. (C) Chromatin fractions were isolated from cells treated for 2 h with 200 mM HU and 100 μg/mL CHX. Pgk1 and H3 were used as controls to monitor the fractionation. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).Rpb1 chromatin enrichment following HU treatment. (D) Quantifications of Rpb1 and H3 levels in chromatin fractions. Relative Rpb1 to H3 levels were set to 1 in the respective WT samples. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).
Figure 1G. Relative expression levels of shade-regulated lncRNAs by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 20 µE, B = 20 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).
Figure 2Relative expression levels of PUAR under WL, SH, and dark (DK) conditions determined by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. The seedlings were grown under DK for 4 d or transferred to WL for 6 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).Relative expression levels of PUAR in Col-0, phyB-9, phyA-211, and pif7-1 by RT-qPCR under Red (R = 100 µE), Far-red (FR = 10 µE), or SH conditions. For R and FR conditions, the seedlings were grown under R or FR for 4 d. For SH conditions, the seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).Hypocotyl phenotypes of Col-0, PUARox, PUAR-S1ox, The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates). J. Hypocotyl phenotypes of Col-0, PHYAox, and amiR-puar. The seedlings were grown under white light for 3 d, and then maintained under white light or transferred to shade for 4 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).
Figure 3Relative expression levels of PHYA in Col-0, pif7-1, PUARox, PUAR-S1ox, by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).PHYA protein levels in Col-0, pif7-1, PUARox, PUAR-S1ox, by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.Relative expression levels of PHYA in Col-0 TATA-mut, and amiR-puar by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).PHYA protein levels in Col-0, TATA-mut, and amiR-puar by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.G-H. Hypocotyl phenotypes of Col-0, phyA-211, PUARox, PUAR-S1ox, PUARox/phyA-211, GFPox, PHYAox, PUAR-S1ox, and PHYAox*PUAR-S1ox. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).