EMBO/SourceData huggingface dataset:
Figure 6A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.
Figure 1C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).
Figure 2Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients (A) and data summaries of mononucleotide change signatures (B), of indices of genomic instability and mutation burden (C), of clinical featuresMolecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients overall survival (F).
Figure 3H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from (A-G).
Figure 4Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.
Figure 5B Kaplan-Meier survival plot.F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].
Figure 6Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.
Figure 7C Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group).E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.
Figure 8A Kaplan-Meier survival plot with survival table.F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).
Figure 9B Volcano plot showing some top KPM versus PMC differentially expressed genes.C KPM and PMC expression of classic mesothelioma markers (top) and top KPM versus PMC overexpressed genes (bottom).
Figure 1(A) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4).(B) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates.(C) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test).(D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+cells; CD1d and CCR6 expression in NCR-ILC3s and CD1d and CD4 expression in NCR-CCR6+ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates.(E) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).
Figure 2(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).(B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5).(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.
Figure 3(A-B) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.(A-B) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.(C) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody-mediated CD1d crosslink (n=3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *p<0.05 two-tailed unpaired t-test.(D) Fold change in IL22 mRNA expression in ILC3s after antibody-mediated CD1d crosslink and/or IL-23 stimulation (n=4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **p<0.01 two-tailed unpaired t-test.(E) IL-22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL-23 (n=3). *p<0.05 two-tailed unpaired t-test(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.
Figure 1(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi). (B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue). (C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 100nm.
Figure 2(A) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green). (B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test). Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.(E) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red). (F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),
Figure 3(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green). Data information: Scale bars indicate 5μm for all images.(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.
Figure 4(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green). Data information: Scale bars indicate 5μm for all images.(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green). Data information: Scale bars indicate 5μm for all images.(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.
Figure 1A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.
Figure 2A, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.
Figure 4Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.
Figure 1b. Multiplexed Error Robust Fluorescent in situ hybridization (MERFISH) and DAPI stained nuclei in the mouse hippocampus. Each gene is represented by a different color. For the entire hippocampus (left), only the mRNA spots are shown with a scale bar of 500 microns. On the zoomed-in section (right), each gene is represented by a different color dot, and the DAPI intensity is displayed in white. The scale bar is 20 microns.
Figure 6a. Normalized expression of Tox in CA1sp1, Leng8 in CA3sp3, Hecw1 in DG3, and Thra in Astro1 show variable expression throughout the hippocampus. Scale bar is 500 microns. spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell (sub)type neighborhoods.
Figure 1A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. B: HIF-2α levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA. C: CA9 levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA. D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors. Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown. Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.
Figure 2A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle). B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. C: Correlation between metastatic and peri-metastatic HIF-1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,57) = 150.7 and P< 0.0001; slope ≠ 0 by F-test. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test.. D: Correlation between metastatic and peri-metastatic HIF-2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-2α expression. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test. n, number of nodules.