Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative images from IHC staining on human SCLC biopsies for given markers. One row = one tumour. Scale bar=25 μm. (b) Venn diagram depicting number of human SCLC biopsies (n=119 total) staining positive for ASCL1, NEUROD1, POU2F3, or lacking all markers (“Subtype-Neg”). (c) Representative images from co-IF staining on human SCLC biopsies for DAPI (nuclei, blue), ASCL1 (yellow), NEUROD1 (purple) and POU2F3 (green). Individual channels (top) and an overlay without DAPI (bottom) are shown. Scale bars=50 μm (n=28 biopsies stained). (d) Representative images from co-IF staining on patient-derived-xenografts (PDX, n=2 distinct models) for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple) and POU2F3 (red). Individual channels (left) and an overlay without DAPI (right) are shown. Yellow arrows and insets (a-b) emphasize co-expressing cells (bottom). Scale bars=75 μm.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Schematic depicting method to induce SCLC from basal cells in the RPM GEMM. (b) Survival of RPM mice infected with indicated cell type-specific Ad-Cre viruses. Number of mice indicated in the figure. Dashed lines indicate historical data . Mantel-Cox log-rank test comparing each cohort to K5-Cre (purple); **** p<0.0001; ns=not significant, p>0.05. (c) Hematoxylin and eosin (H&E) staining of RPM K5-Cre tumours. Representative whole lung lobes (left) and individual tumour morphology (right) depicted. Scale bars=1 mm (left) or 50 μm (right). (d) Representative IHC images from RPM tumours initiated by indicated Ad-Cre viruses for ASCL1, NEUROD1, and/or POU2F3. Scale bars=50 μm for main images, 10 μm for high magnification insets. (e) Quantification of number of POU2F3 + tumours (H-score >50) vs total tumour number per lung per mouse for indicated cell-type-specific or CMV-Cre adenoviruses. Each dot represents lungs of one mouse. Number of mice indicated in the figure. Error bars represent mean ± SEM. (f) H-score quantification of IHC on K5-Cre RPM tumours vs other cells of origin for indicated proteins. Each dot represents one tumour. For each marker, n=11-101 tumours quantified from n=4-19 mice per Ad-Cre group. Median (red bar) and upper and lower quartiles (dotted line) are indicated. (g) Representative co-immunofluorescent (co-IF) staining for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple), and POU2F3 (red) in RPM K5-Cre tumours. Tumour regions outlined with dashed line. High magnification insets of co-expressing cells (yellow arrows) are in the upper left corner of overlays. Scale bars=75 μm. (h) UMAP of scRNA-seq data from n=5 tumours initiated from NE cells (Cgrp-Cre, purple) from n=5 RPM mice, or n=4 tumours initiated from basal cells (K5-Cre, orange) from n=2 RPM mice. Cells coloured by sample in the same UMAP on right. (i) UMAP in (h) annotated by Leiden cluster (left) (Supplementary Table 1). Proportion of cells in Cgrp vs K5 tumours per Leiden cluster, represented as % of all cells per sample (right). (j) FeaturePlots depicting expression of indicated genes in UMAP, as in (h) (top). Split violin plot depicting mRNA expression of indicated genes by scRNA-seq for all cells in Cgrp (purple, left) and K5 (orange, right) tumours (bottom). Each dot is one cell. Student’s unpaired t-tests. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, ns=not significant, p>0.05. Unless otherwise noted, statistical tests are one-way ANOVA with Tukey’s correction (e,f). **** p<0.0001, ** p<0.006, * p<0.02, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Infection, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative IHC images from RPM K5-Cre tumours for KRT5. A Sox2 LSL/LSL ;Nkx2-1 fl/fl ;Lkb1 fl/fl mouse lung squamous tumour is included as a positive control. (b) Representative IHC for YAP1 in RPM tumours initiated by indicated Ad-Cre viruses (left) and corresponding H-score quantification (right). Median (red bar) and upper and lower quartiles (dotted lines) indicated. One-way ANOVA with Tukey’s correction (e,f). *** p<0.0003, ** p<0.003. (c) Split violin plot depicting expression of SCLC archetype signatures per tumour cell by scRNA-seq in CGRP (purple, left) and K5 (orange, right) tumours in . (d) Split violin plot depicting transcriptional signatures of human ASCL1, NEUROD1, or POU2F3 + tumours derived from scRNA-seq data or (e) expression of ASCL1, NEUROD1, and POU2F3 target gene signatures derived from ChIP-seq data , , , (Supplementary Table 2) by scRNA-seq for all cells in CGRP (purple, left) and K5 (orange, right) tumours. Each dot is one cell. (f) Split violin plot depicting NE score per tumour cell by scRNA-seq in CGRP (purple, left) and K5 (orange, right) tumours. All scale bars=50 μm. Unless otherwise noted, statistical tests are Student’s unpaired t-tests. **** p<0.0001, ** p<0.01, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Positive Control, Expressing, Derivative Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Schematic depicting isolation, growth and transformation of basal cell-derived organoids from RPM mice followed by implantation into the flanks of scid/beige hosts. (b) Representative brightfield images of basal organoids pre- (wildtype) and post- (transformed) CMV-Cre. Scale bars=650 μm (left, low mag) or 275 μm (right, high mag). (c) Representative H&E staining of RPM basal-organoid-derived tumours isolated from scid/beige mouse flanks with more classic (left) or variant (right) histopathology. Scale bar=50 μm. (d) UMAP of scRNA-seq data from wildtype (orange) and transformed (purple) RPM basal organoids, plus basal-organoid-derived RPM allograft tumour cells (turquoise). Allograft sample includes n=5 distinct RPM basal allograft tumours. FeaturePlots depicting expression of gene signatures derived from normal basal versus NE cells (right) (Supplementary Table 2). (e) UMAP of scRNA-seq data from RPM allograft tumours only, annotated by Leiden cluster (left) (Supplementary Table 1), FeaturePlot expression of indicated genes (top, right), and corresponding violin plot expression of indicated genes per Leiden cluster (bottom, right). Red dashed circle outlines Cluster 9 enriched for Pou2f3 . (f) UMAP in (e) annotated by SCLC fate. Fates assigned based on enriched cell fate marker gene expression per Leiden cluster. (g) Violin plot of NE score per cell grouped by SCLC fate (left) from data in (f). UMAP of scRNA-seq data in (e) coloured by NE score (right). (h) Violin plot depicting ASCL1, NEUROD1, and POU2F3 ChIP target gene enrichment , , , (Supplementary Table 2) in tumour cells from (e), grouped by SCLC fate assignment. (i) Violin plot depicting gene set enrichment of normal NE, tuft, and basal cells (Supplementary Table 2) in tumour cells from (e), grouped by SCLC fate assignment. (j) Representative co-immunofluorescent (co-IF) staining for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple), and POU2F3 (red) in RPM basal allograft tumours. High magnification insets of co-expressing cells (yellow arrows) are in the upper right corner of overlays. Scale bars=75 μm. (k) Representative IHC of RPR2 basal-derived allograft tumours for H&E and indicated SCLC subtype markers (left) with corresponding H-score quantification for ASCL1 (A), NEUROD1 (N) or POU2F3 (P) compared to RPM basal allograft tumours (right). Scale bar=50 μm. Mann-Whitney two-tailed t-test. * p<0.02, *** p<0.0005. (l) UMAP of scRNA-seq data from basal-organoid-derived RPM (turquoise, n=5 tumours) and RPR2 (maroon, n=1) allograft tumour cells. (m) UMAP in (l) annotated by Leiden cluster (Supplementary Table 1) (left). Proportion of cells from RPM vs RPR2 allograft tumours in each Leiden cluster, represented as % of all cells per sample (right). (n) UMAP of scRNA-seq data in (l) coloured by NE score (left). Violin plot of NE score per cell in RPM vs RPR2 basal allograft tumour cells (right). Student’s unpaired t-test. ** p<0.01. Box-whisker overlays on all violin plots indicate median and upper and lower quartile. Unless otherwise indicated, statistical tests are one-way ANOVA with Tukey’s correction. **** p<0.0001, * p<0.03, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Isolation, Transformation Assay, Derivative Assay, Staining, Variant Assay, Histopathology, Expressing, Marker, MANN-WHITNEY, Two Tailed Test, Whisker Assay

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Recombination PCR for RPM basal organoids for indicated alleles pre- and post-treatment with TAT-Cre recombinase or Ad-CMV-Cre at two concentrations (2.5e7 or 5e7 pfu). *Organoids subject to spinoculation with CMV-Cre virus. Red font indicates condition used for subsequent allografting. (b) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA) for DAPI (nuclei, blue), KI67 (proliferation, orange), and ASCL1 (NE cell, green). Positive controls for KI67 and ASCL1 (bottom panel) is an RPR2 SCLC lung tumour. Quantification via CellProfiler of KI67 positivity per organoid from indicated conditions (right). Number of organoids quantified per group is labeled. One-way ANOVA with Tukey’s correction. *** p<0.003, ** p<0.005, ns=not significant, p>0.05. Error bars represent mean ± SD. (c) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA): Left) DAPI (nuclei, blue), NEUROD1 (neuronal cell, green), DNP63 (basal cell, yellow) and KRT8 (luminal basal cell, red). Positive control (+) for NEUROD1 is an RPM olfactory neuroblastoma tumour and for basal markers is an SNL GEMM lung tumour . Right) DAPI (nuclei, blue), FOXJ1 (ciliated cell, purple), CCSP (SCGB1A1, club cell, green) and KRT8 (luminal basal cell, red). Positive control (+) control for FOXJ1 and CCSP is airway from a normal mouse lung, and for KRT8 is an SNL GEMM lung tumour. (d) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA) for DAPI (nuclei, blue) and POU2F3 (tuft cell, green). Positive control (+) is an RPMA olfactory neuroblastoma tumour . All co-IF scale bars=150 μm.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Virus, Labeling, Positive Control, Control

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) UMAP of scRNA-seq data from wildtype (orange) and transformed (purple) RPM basal organoids (left) and annotated by Leiden cluster (right) (Supplementary Table 1). (b) Dot plot expression of genes marking major lung cell types, stem-like, proliferative, and tumour cells for wildtype versus transformed RPM organoids, grouped by Leiden cluster as assigned in (a). Colour indicates level of gene expression and dot size represents frequency of expression per cluster. (c) UMAP of RPM organoids pre- and post-CMV-Cre coloured by cell cycle phase. (d) UMAP as in of RPM organoids pre- and post-CMV-Cre and RPM basal allograft tumour coloured by cell cycle phase (left). Proportion of cells from WT and transformed (RPM) organoid samples and RPM allograft tumour in each phase, represented as % of all cells per sample (right). (e) Dot plot expression of genes marking indicated cell types, stem-like, proliferative, and tumour cells for RPM basal allograft tumour by Leiden cluster derived from scRNA-seq in . (f) Violin plot expression of SCLC archetype signatures in RPM basal allograft tumour cells by scRNA-seq as in , grouped by Leiden cluster. (g) Recombination PCR for RPR2 basal organoids for indicated alleles pre- and post-treatment with TAT-Cre recombinase or Ad-CMV-Cre at two concentrations (2.5e7 or 5e7 pfu). *Organoids subject to spinoculation with CMV-Cre virus. Red font indicates condition used for subsequent allografting. (h) UMAP and corresponding violin plots depicting expression of indicated SCLC subtype markers and Myc-family oncogenes in RPM versus RPR2 basal organoid allografts from . (i) Violin plot expression of SCLC subtype archetype signatures or (j) ChIP target genes signatures (Supplementary Table 2) per tumour cell in RPM vs RPR2 basal allograft tumour samples from scRNA-seq data in . A=ASCL1, N=NEUROD1, and P=POU2F3. Unless otherwise noted, statistical tests are Student’s unpaired t-test. **** p<0.0001.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Transformation Assay, Expressing, Derivative Assay, Virus

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative H&E staining of RPM (top) and RPMA (bottom) basal-organoid-derived tumours isolated from scid/beige mouse flanks. Scale bar=50 μm. (b) Representative IHC images from RPM and RPMA basal-organoid-derived tumours for indicated markers (left). H-score IHC quantification for indicated proteins (right). Each dot represents one tumour. For each marker, n=6-9 tumours quantified. Scale bars=50 μm. Student’s unpaired t-tests. **** p<0.0001, ** p<0.01, ns=not significant, p>0.05. Error bars represent mean ± SD. (c) Immunoblot depicting expression of indicated markers in RPM (n=2) vs RPMA (n=3) basal allograft tumours with HSP90 as a loading control. (d) Representative co-IF staining for DAPI (nuclei, blue), NEUROD1 (purple), and POU2F3 (green) in RPMA basal allograft tumours. Scale bars=75 μm. (e) UMAP of scRNA-seq data from basal-organoid-derived RPM (purple, n=5) and RPMA (orange, n=3) allograft tumours. (f) UMAP in (e) annotated by Leiden cluster (left). Proportion of cells from RPM vs RPMA allograft tumours in each Leiden cluster (Supplementary Table 3), represented as a % of all cells per sample (right). (g) Dot plot expression of genes marking indicated cell fates, stem-like, proliferative, and tumour cells for RPM and RPMA basal-derived allograft tumour cells, grouped by Leiden cluster as assigned in (f). Colour indicates level of gene expression and dot size represents frequency of expression per cluster. Genotypes indicate enrichment but not exclusive expression of each cluster. (h) UMAP of scRNA-seq data in (e) coloured by SCLC fate (left). Fates assigned based on enriched fate marker gene expression per Leiden cluster. Proportion of cells from RPM and RPMA allograft tumour samples in each fate, represented as % of all cells per sample (right). (i) UMAP of scRNA-seq data in (e) coloured by NE score according to legend (left). Violin plot of NE score per cell grouped by SCLC fate or genotype as indicated on the x-axis (right). (j) UMAP of scRNA-seq data in (e) coloured by ASCL1, NEUROD1, and POU2F3 ChIP target gene scores , , , (Supplementary Table 2) where red/dark purple is high and orange is low. Upper right insets are violin plots depicting expression of target gene scores, grouped by genotype. Student’s unpaired t-tests. **** p<0.0001. (k) UMAPs (top) and violin plots (bottom) depicting gene set enrichment of normal NE, tuft, and basal cells (Supplementary Table 2) in tumour cells from (h), grouped by fate assignment. (l) Violin plot expression of SCLC subtype archetype signatures (Supplementary Table 2) per tumour cell in RPM vs RPMA basal allograft tumour samples from data in (h), grouped by SCLC fate. A=ASCL1, N=NEUROD1, and P=POU2F3. Box-whisker overlays on all violin plots indicate median and upper and lower quartile. Unless otherwise indicated, statistical tests are one-way ANOVA with Tukey’s correction. **** p<0.0001, ** p<0.001, * p<0.01, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Staining, Derivative Assay, Isolation, Marker, Western Blot, Expressing, Control, Whisker Assay

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Survival of RPP mice infected with K5-Cre or Cgrp-Cre compared to RPM mice infected with K5-Cre. Dashed line indicates historical data . Number of mice indicated in the figure. Mantel-Cox log-rank test; **** p<0.0001, ns=not significant, p >0.05. (b) H-score quantification of ASCL1 and NEUROD1 in RPP GEMM tumours initiated by indicated Ad-Cre viruses. Each dot is one tumour. N=35-40 tumours from n=5-8 mice per cohort. One-way ANOVA with Tukey’s correction. ns=not significant, p>0.05. (c) Immunoblot analysis of human SCLC cell line, H1048, for indicated markers after LentiCRISPRv2 infection with non-targeting ( sgNTC ) or sgPTEN sgRNAs. (d) Immunoblot analysis of H1048 for indicated markers in parental cells versus cells with ectopic myristoylated-AKT (myrAKT) with HSP90 as a loading control.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Infection, Western Blot, Control

Journal: JCI Insight

Article Title: Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie Allan-Herndon-Dudley syndrome

doi: 10.1172/jci.insight.174645

Figure Lengend Snippet: ( A ) Schematic of the protocol for COs’ maturation using platelet-derived growth factor AA and insulin-like growth factor 1. ( B – E ) Whole-mount immunolabeling of D65 WT COs containing differentiated NEUROD1 + neurons ( B ), GFAP + neural precursor cells and GFAP-AQP4 + astrocytes ( C and D ), and OLIG2 + oligodendrocyte precursor cells and O4 + premyelinating oligodendrocytes ( E ); in blue is the nuclear staining with DAPI. B : scale bar 75 μm; C : scale bar 75 μm; inset in C : scale bar 25 μm; D : scale bar 40 μm; E : scale bar 75 μm; inset in E : scale bar 40 μm. Arrows in D indicate AQP4 staining in the membrane of GFAP + cells. The arrow in the inset in E points to the cellular body of a representative oligodendrocyte; arrowheads point to its cellular processes. ( F and G ) Volcano plots showing the distribution of DEGs in WT vs. Mut1 ( F ) and in WT vs. Mut2 ( G ); each point represents the average of 3 (WT) or 2 (MCT8-deficient) samples consisting of 4 pooled COs for each transcript. ( H ) Venn comparison of WT vs. Mut1, WT vs. Mut2, and Mut1 vs. Mut2 DEGs. A common cluster (cluster E) between WT vs. Mut1 and WT vs. Mut2 containing 949 DEGs was identified. ( I ) Venn comparison of the 949 DEGs in cluster E shows similar transcriptomic changes in both MCT8-deficient COs. ( J ) Heatmap depicting the top 63 DEGs from cluster E. ( K ) KEGG pathway analysis of the top 63 DEGs in J . ( L – O ) Heatmaps of T3-regulated genes involved in cerebral cortex development ( L ), neural cell migration ( M ), astrocytes and myelination ( N ), and neurotransmitter receptors, transcription factors, potassium channels, and extracellular matrix protein ( O ). DEG thresholds (FDR ≤ 0.25 and fold-change ± 4) were identified by gene set–specific analysis in the Partek Flow platform.

Article Snippet: Immunofluorescence studies were performed as previously described , and COs were incubated with primary and secondary antibodies using the following dilutions: mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), anti-Ki67 1:1,000 (MilliporeSigma; MAB4190), anti-PVIM 1:400 (MBL; D076-3), anti-PH3 1:400 (Cell Signaling Technology; 9701S), anti-SATB2 1:400 (Abcam; ab92446), anti-Vimentin (Novus; NB300-223), anti-MCT8 antibody 1:400 (Atlas; HPA003353), anti-NEUROD1 1:400 (Bio-Techne; AF2746), anti-GFAP 1:200 (Novus; 05198), anti-AQP4 1:200 (Novus; 52872), anti-Olig2 (Abcam; ab109186), anti-O4 (Bio-Techne; MAB1326), Alexa Fluor 488–conjugated goat anti-mouse IgG 1:200 (Vector Laboratories; DK-2488), Alexa Fluor 594–conjugated horse anti-rabbit IgG 1:200 (Vector Laboratories; DK-1594), and Alexa Fluor 488–conjugated horse anti-rabbit IgG 1:200 (Vector Laboratories; DI-1788).

Techniques: Derivative Assay, Immunolabeling, Staining, Membrane, Comparison, Migration