goat anti pdgfr β  (R&D Systems)

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of CCR2-Cre ERT2 ;ZsGreen mouse. b , Representative confocal images showing lineage + and lineage - macrophages in the lungs of CCR2-Cre ERT2 ;ZsGreen mice after tumour organoid engraftment. DAPI (blue), ZsGreen (lineage-traced monocytes, green), CD64 (grey), and RFP (red, tumour). Scale bar, 100 μm. Insets (bottom) are enlargements of the dashed boxes (top). Scale bars, 50 μm. Images representative of n = 3 mice. c , Flow cytometry analysis of alveolar macrophages in control and clodronate liposome-treated lungs. d , Quantification of alveolar macrophages in control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. e , Representative confocal images of macrophages and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), F4/80 (grey) and RFP (red, tumour). Scale bar, 100 μm. Images representative of n = 3 mice. f , Dotplot of key chemokine receptors in neutrophils and γδ T cells. Each dot is coloured based on the average expression and sized based on the percentage of cells expressing that gene, as shown. g,h , Representative confocal images of neutrophils ( g ), γδ T cells ( h ) and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), Ly6G (green) and RFP (red) ( g ). DAPI (blue), GL3 (green) and RFP (red) ( h ). Scale bar, 100 μm. Images representative of n = 3 mice. i , Flow cytometry analysis of neutrophils from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. Ly6G (neutrophils), CD11B (leukocytes/granulocytes). j , Quantification of neutrophils from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. k , Flow cytometry analysis of γδ T cells from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. GL3 (TCR gamma/delta), CD3ε (T cells). l , Quantification of γδ T cells from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Flow Cytometry, Control, Two Tailed Test, Expressing, Activation Assay

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Representative confocal images confirming an absence of detectable Areg expression in homeostatic lungs ( Confetti ). Right panels are the magnifications of left panel. DAPI (blue), pro-SPC (green), Areg (grey), YFP (yellow) and RFP (red). Scale bar, 50 µm. Image representative of n = 2 mice. b , Violin plot showing the expression of Areg and EGFR across epithelia, mesenchymal and immune cell populations. c , Experimental scheme to investigate the effect of Areg on wildtype mesenchymal cells. d , Representative brightfield images showing morphological changes of lung mesenchymal cells treated with Areg after 7 days in 3D Matrigel culture. Images are representative of n = 3 independent experiments. Scale bar, 50 µm. e , Representative confocal images showing the expression of reprogrammed fibroblast markers in lung mesenchyme cultures in ( c ). Top panel: DAPI (blue) and Acta2 (red). Bottom panel: DAPI (blue) and Pdgfrβ (red). Scale bar, 50 µm. f , Experimental scheme to investigate the effect of Areg in AMs. g , Flow cytometry analysis of AMs numbers after Areg treatment. h , Flow cytometry analysis showing Msr1 expression in AMs from ( f ). i , Quantification of the MFI for Msr1 in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. j , Flow cytometry analysis showing MHCII expression on AMs from ( f ). k , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. l , Experimental scheme to investigate the effect of Areg on RFP + tumour cells isolated from Red2Kras lungs. m , Bright-field image from ( l ). n , Representative confocal image of RFP + tumour organoids from ( l ). DAPI (blue), Krt8 (grey), pro-SPC (green) and RFP (red). Images representative of n = 3 independent experiments. Scale bar, 100 µm. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ; tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing reprogrammed fibroblasts and lineage-labelled cells in ( o ). Right panels are magnifications of left panel for each condition. DAPI (blue), ZsGreen (green), Pdgfrβ (grey), and RFP (red). Scale bar, 50 µm. Images of representative n = 3 independent experiments.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Expressing, Flow Cytometry, Control, Two Tailed Test, Isolation

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Schematic illustrating 3D organoid co-cultures of wildtype AMs and mesenchymal cells with lineage-labelled RFP + cells isolated Red2Kras lungs. b , Representative flow cytometry plots showing the populations of macrophages (CD45 + EpCAM − ), tumour cells (CD45 − EpCAM + ), and mesenchymal cells (CD45 − EpCAM − ) gated from CD31 − populations and stablished in ( a ). c , Flow cytometry analysis of Msr1 expression in AMs from ( a ). d , e , Quantification of the MFI for Msr1 ( d ) and relative cell numbers ( e ) of AMs obtained from ( a ). Data are mean ± s.e.m. Each dot represents one independent experiment. 1 ( n = 4 ), 2 ( n = 4 ) and 3 ( n = 4 ). P values were calculated using two-tailed unpaired t-test. f , Experimental scheme to investigate the effect of Areg on AMs co-cultured with wildtype mesenchymal and AT2 cells. g , Flow cytometry analysis showing the proportion of AMs from ( f ). h , Quantification of frequency of AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. i , Flow cytometry analysis showing MHCII expression in AMs from ( f ). j , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. k , Experimental scheme to investigate the effect of Areg/Ereg on AMs co-cultured with wildtype mesenchymal and AT2 cells. l , Quantification of the relative cell numbers of AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg/Ereg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. m , Quantification of MFI for MHCII in AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. n , qPCR analysis of Arg1 , Ym-1 , and Tnfa expression in AMs co-cultured with wildtype mesenchyme and AT2 cells treated with PBS, Areg alone, or Areg/Ereg. Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ), Areg ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ;tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing lineage-labelled DATP-like mutant cells in 3D organoid co-cultures from ( o ). DAPI (blue), ZsGreen (green), Sox9 (grey), and RFP (red, tumour). Scale bar, 50 µm. q , Quantification of the percentage of Sox9 expressing cells within RFP + mutant organoids assessed in ( p ). Data are presented as mean ± s.e.m. Each dot represents one organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using Mann–Whitney test. r , Representative confocal images showing lineage-labelled cells in 3D organoid co-cultures with wild-type mesenchymal cells and treated with Gefitinib or DMSO. DAPI (blue), LpCat1 (green) and RFP (red, tumour). Scale bar, 100 µm. s , Quantification of the percentage of LpCat1 expressing cells within RFP + mutant organoids. Data are presented as mean ± s.e.m. Each dot represents one organoid from three independent experiments. DMSO ( n = 3 ), Gefitinib ( n = 3 ). P values were calculated using Mann–Whitney test. t , Schematic illustrating 3D organoid cultures of mutant AT2 cells isolated from Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. u , Representative confocal images showing the expression of DATP-like cell state marker in Red2Kras RFP + mutant organoids from ( t ). DAPI (blue), Sox9 (green) and RFP (red, tumour). Scale bar, 50 µm. v , Percentage of DATP-like cell states expressing Sox9 in Red2Kras RFP + mutant organoids from ( u ). Data are presented as mean ± s.e.m.; Each dot represents an individual organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Isolation, Flow Cytometry, Expressing, Two Tailed Test, Cell Culture, Control, Mutagenesis, MANN-WHITNEY, Marker

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a . Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of Pdgfra-Cre ERT2 ;ZsGreen;iDTR mice. b-d . Representative confocal images for fibrotic fibroblasts ( b ), macrophage ( c ) and mutant epithelial cells ( d ) in PBS-treated and DTR-treated lungs. DAPI (blue), Pdgfrβ (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( b ). DAPI (blue), Cd68 (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( c ). DAPI (blue), Sox9 (grey), and RFP (red, tumour) ( d ). Images are representative of n = 2 mice. Scale bar, 100 μm. e , Experimental design for EGFR tyrosine kinase inhibitor administration. Red2Kras mice received two doses of tamoxifen and were treated with DMSO or Gefitinib (80 mg/kg) at the indicated timepoints. Lung tissues were collected for histological analyses. f , Representative tile scans of entire lung lobe cross-sections from Red2Kras animals treated with DMSO or Gefitinib. DAPI (blue) and RFP (red, tumour). Scale bar, 1,000 µm. g , Percentage of RFP + mutant cell area relative to lobe area assessed in ( f ). Data are as mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ). h,i Representative confocal images for reprogrammed fibroblasts, macrophages and lineage-labelled cells in DMSO or Gefitinib-treated lungs. DAPI (blue), Tnc (grey) and RFP (red, tumour) ( h ). Images representative of n = 2 (DMSO) and n = 2 (Gefitinib) mice. DAPI (blue), F4/80 (grey) and RFP (red, tumour) ( i ). Images representative of n = 2 (DMSO) and n = 3 (Gefitinib) mice. Scale bar, 50 µm. j , Flow cytometry analysis for AMs from Red2Kras lungs treated with DMSO or Gefitinib. k , Quantification of the percentage of AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 3 ) and Gefitinib ( n = 3 ). P values were calculated using two-tailed unpaired t-test. l , Flow cytometry analysis for MHC-II expression on AMs from DMSO- or Gefitinib-treated Red2Kras lungs. m , Quantification of the MFI for MHC-II in AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ) and Gefitinib ( n = 3 ). n , Representative confocal images for neutrophils and lineage-labelled cells in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Ly6G (green) and RFP (red, tumour). Images representative of n = 3 biologically independent mice. Scale bar, 50 µm. o-q , Representative confocal images of the different epithelial states in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Sox9 (grey) and RFP (red, tumour) ( o ). DAPI (blue), Cd177 (grey) and RFP (red, tumour) ( p ). DAPI (blue), Ager (grey), LpCat1 (yellow) and RFP (red, tumour) ( q ). Scale bar, 50 µm. r , Quantification of the percentage of each cell state within the RFP + mutant cell population in DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ).

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Mutagenesis, Flow Cytometry, Two Tailed Test, Expressing