Journal: bioRxiv

Article Title: SORLA-driven endosomal trafficking regulates the oncogenic fitness of HER2

doi: 10.1101/299586

Figure Lengend Snippet: ( a ) Western blot analysis of SORLA and HER2 in bladder cancer cell lines (5637 and T24) and three patient-derived cell cultures. α-tubulin is a loading control. ( b ) Confocal microscopy imaging of endogenous SORLA or SORLA-GFP (green) with endogenous EEA1, VPS35, LAMP1, Rab11, Rab5 (magenta) or Rab7-mRFP (magenta; Rab7 was overexpressed due to lack of reliable Rab7 antibodies for staining) in MDA-MB-361 (top panel) or JIMT-1 cells. ( c ) Endogenous HER2 (magenta) and EEA1 (green) staining in SKBR3, HCC1419 and HCC1954 cells. ( d ) Co-localization between SORLA-GFP and HER2 in JIMT-1 cells (n = 130 cells from 7 independent experiments). Details of colocalization analysis can be found in the methods section. ( e ) Live-cell confocal imaging of AlexaFluor-568-labeled trastuzumab (Tz-568; red) and SORLA-GFP (green) in the cytoplasm of MDA-MB-361 cells, 250 ms frames. ( f ) Confocal imaging of different SORLA-GFP fusion proteins in MDA-MB-361 cells. ECD: extracellular domain; TM: transmembrane domain; CD: cytosolic domain.

Article Snippet: Lab Vision autostainer (Thermo Fisher Scientific) with SORLA antibody (rabbit polyclonal, Atlas Antibodies; 1:300 dilution) was used and primary antibodies were detected with PowerVision Poly-HRP anti-mouse/anti-rabbit IHC system (Leica BioSystems).

Techniques: Western Blot, Derivative Assay, Confocal Microscopy, Imaging, Staining, Labeling

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were arrested in G0 by contact inhibition and 48 h serum starvation (-FCS 48 h), in G1 by 24 h serum starvation (-FCS 24 h), in S phase by 24 h treatment with 1 mM hydroxyurea (HU) and in G2/M by 24 h treatment with 200 ng/ml nocodazole. Asynchronous (asynchr.) cells are shown for comparison. The cells were fixed and stained with propidium iodide. The DNA content was analyzed by flow cytometry to verify synchronization. Cells in G0 and G1 have a DNA content of 2n, cells in S phase between 2n and 4n, and cells in G2/M a 4n DNA content. The percentages of cells in each cell cycle phase are indicated. (B) FAM111A and FAM111B expression in cell-cycle arrested cells was analyzed by immunoblot. Cyclin E and cyclin B1 were analyzed as markers for S and G2/M phases, respectively. GAPDH was used as a loading control. ( C) Subconfluent RPE-1 cells were treated with an E2F inhibitor (HLM0006474, 40 μM) or vehicle (DMSO). Cell lysates were harvested at the indicated times (hours post-treatment, hpt) and analyzed by immunoblot. (D to F) WT RPE-1 cell, E2F1 KO, and E2F3 KO cells were synchronized by serum starvation for 24 h and infected with MCMVs expressing full-length or mutant M117 (MOI 5). Cell lysates were harvested at the indicated times post-infection and analyzed by immunoblot. The MCMV immediate-early 1 (IE1) protein and β-actin were used as infection and loading controls, respectively. Representative blots out of multiple experiments are shown. hpt: hours post treatment.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Inhibition, Comparison, Staining, Flow Cytometry, Expressing, Western Blot, Control, Infection, Mutagenesis

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) RPE-1 cells were infected (MOI 5 TCID 50 /cell) with recombinant MCMV-GFP encoding FAM111A-specific (A sh1, A sh2), FAM111B-specific (B sh1, B sh2), or scrambled (scr) control shRNA. Cell lysates were harvested 24 and 28 hpi and analyzed by immunoblot. (B) RPE-1 cells (MOI 0.2), (C) ARPE-19 (MOI 1) and (D) RPTEC (MOI 1) were infected with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. (C) Rhesus fibroblasts were infected (MOI 1.5 TCID 50 /cell) with the same viruses as above and analyzed by immunoblot or infected at MOI 1 TCID 50 /cell (D) with the same viruses as above. Viral replication kinetics were determined by titration of virus released into the supernatant. Mean ±SEM of triplicates are shown. DL, detection limit. (E, F) Cell lysates of WT RPE-1 cells, empty vector (EV)-transduced RPE-1 cells, two FAM111B KO RPE-1 clones (cl 1-9 and 3-18), and one FAM111A KO RPE-1 (cl 3-10) clone were analyzed by immunoblot. (G) Multistep replication kinetic of WT MCMV in the same RPE-1 as in E. Supernatants of infected cells were harvested at the indicated times post infection and titrated. Mean ±SEM of triplicates are shown. DL, detection limit.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Control, shRNA, Western Blot, Titration, Virus, Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) 10.1 mouse fibroblasts were infected (MOI 1 TCID 50 /cell) with recombinant MCMVs encoding HA-tagged WT or mutant FAM111B. Cell lysates were analyzed by immunoblot. (B) 10.1 cells were infected (MOI 0.02) with the same viruses as above to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown. DL, detection limit. (C) NIH-3T3 fibroblasts transduced with a lentiviral vector encoding tet-inducible FAM111B were induced with 2 µg/ml doxycyline or left untreated. Cell lysates were harvested and analyzed by immunoblot. (D) Transduced NIH-3T3 cells were treated with doxycycline 1h before (−1 hpi) or 4 h after (+4 hpi) infection with WT MCMV (MOI 0.02) to analyze multistep replication kinetics. Mean ±SEM of triplicates are shown.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Infection, Recombinant, Mutagenesis, Western Blot, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells

doi: 10.1101/2024.08.02.606359

Figure Lengend Snippet: (A) Immunofluorescence of 10.1 fibroblasts mock-infected or infected with MCMV-eGFP-FAM111B (MOI 1 TCID 50 /cell). Cells were fixed 24 hpi and stained with an antibody recognizing the MCMV E1 protein (red), a marker for vRCs. Nuclei were stained with DAPI. Scale bar, 25 µm. (B) Immunofluorescence of FAM111B KO or WT RPE-1 cells mock-infected or infected with WT MCMV (MOI 1 TCID 50 /cell). Cells were fixed 40 hpi and stained with antibodies recognizing FAM111B (green) or MCMV E1 (red). Nuclei were stained with DAPI or Hoechst 33342. Arrow heads indicate FAM111B in vRCs. Scale bar, 25 µm.

Article Snippet: Polyclonal rabbit antibodies against FAM111B (HPA038637, Sigma-Aldrich), FAM111A (EPR14407, Abcam), Flag (F7425, Sigma-Aldrich), E2F3 (C-18, Santa Cruz) and E2F4 (C-20, Santa Cruz) were used.

Techniques: Immunofluorescence, Infection, Staining, Marker

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating D50 MCT8-COs and obtaining single-cell suspension for sc-RNA seq analysis. B . UMAP plot showing the cell clusters identified by principal component analysis in the indicated COs C . UMAP plots showing the distribution of markers for NPCs and neurons. D . Dot plot showing the relative expression levels of the gene markers used to identify each cell population. E . UMAP plot showing the cell types identified by manual curation in control COs. F . Dot plot showing the relative expression levels of SLC16A2, THRB, and THRA in each cell population of the dorsal projection trajectory. G . Interpretation of sequential gene expression changes during dorsal projection trajectory progression. H . UMAP plot showing the cell types identified by manual curation in MCT8-COs. I . Histogram of the relative number of cells in control and MCT8-COs. J . Volcano plots showing the distribution of differentially expressed genes in MCT8-deficient vs. control COs. iPSCs, induced pluripotent stem cells; COs, cortical organoids; NPCs, neural precursor cells; IPCs, intermediate precursor cells; DPT, dorsal projection trajectory; UMAP, Uniform Manifold Approximation and Projection. The X 2 test for multiple comparisons and pairwise cell proportions. **P < 0.01; ***P < 0.0001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Suspension, RNA Sequencing, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of obtaining spatial transcriptomic data. B . Image showing the cell segmentation on the indicated COs, based on transcript localization data. Scale bar: 100 µm. C . Heatmap showing the relative expression levels of the genes used to identify the indicated neural cell types. D . Heatmap of the differentially expressed genes in the indicated neural cells between control vs. MCT8-COs. E . Same as in B , except the cells are classified into the indicated types. F . Distribution of distances between the indicated cells and groups. G . Histogram of the distribution of cell densities. Considering a radius of 100 µm, if only one cell was in contact with another cell, it was considered as sparse density. If one cell was in contact with five or more cells, it was considered a dense density. H . Violin plots show the expression levels of the indicated genes, considering the indicated cellular densities. I. Ligand-receptor interaction between the indicated neural cells in Control and MCT8-deficient COs. Differentially expressed genes threshold: p-value < 0.05.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating NPCs from iPSCs and the subsequent differentiation of the NPCs into neurons. B . Brightfield and confocal fluorescence images showing iPSCs-derived NPCs stained for SOX2 (magenta), NESTIN (yellow), MCT8 (magenta), and Dapi (blue; nuclear). C . Relative mRNA levels of the indicated genes in Control and MCT8 NPCs after 1, 6, and 12 days of neurodifferentiation. D . Brightfield images showing NPCs after six days of neurodifferentiation. E . Principal component plot illustrating differences between control and MCT8-deficient NPCs. F . Volcano plots showing the distribution of differentially expressed genes in control vs. MCT8-NPCs; each point represents the average of 5 control and 5 MCT8-deficient samples of pooled NPCs for each transcript. G . Heatmap depicting the top 20 differentially expressed genes related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. H . Venn comparison of differentially expressed genes belonging to the gene set related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. (blue) and sc-RNA seq analysis (green) and between control vs. MCT8-IPCs identified by sc-RNA seq analysis (purple); common differentially expressed genes were identified (grey box). I . Brightfield images of control and MCT8-NPCs after twelve days of neurodifferentiation; TUJ1 staining in green and Dapi (blue; nuclear). J . The upper two panels are MCT8 staining in red, TUJ1 in green, and Dapi (blue); the lower panels are RBFOX3 staining in green, NEUROD1 in red, and Dapi in blue. K . MCT8-NPCs after being treated with 60nM T3 during the twelve days of neurodifferentiation. TUJ1 staining is green, and SOX2 is red. L . SOX2 staining in red and Dapi in blue on the indicated cells and treatments. Scale bars: B: 50 µm; D, I, J: 100 µm. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 1.5 in the Partek Flow platform. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 2 pooled 6-well plates of NPCs from either control or MCT8-NPCs; Two-tailed Student’s test for comparing D2 deiodination and relative mRNA expression between D1, D6 and D12 of neurodifferentiation; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Fluorescence, Derivative Assay, Staining, Control, RNA Sequencing, Comparison, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of treating iPSCs-derived NPCs with radioactive T4 I125 to measure T3 I125 production. B . Representative chromatograms of the medium after control and MCT8-NPCs were incubated with T4 I125 for 24 hours. C . Quantitation of the DIO2 deiodination in control and MCT8 NPCs; n = 5 DIO2 assays. D . Volcano plots showing the distribution of differentially expressed genes in control + 1nM T4 vs. control NPCs; each point represents the average of five control + 1nM T4 and five control samples of pooled NPCs for each transcript. E . Interpretation of the findings in A-D . F . Schematic of the generation and timing of COs generation, starting with iPSCs to a culture of embryoid bodies, followed by neural induction, neuroepithelial bud expansion, and maturation. G . Quantitation of DIO2 deiodination in control COs during their first 20 days in culture. n = 4 DIO2 assays per timepoint, each consisting of 4 pooled COs from control COs. H . Relative SOX2 mRNA levels in control COs during their first 20 days in culture. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 4 pooled COs from control COs; I . Schematic representation of the experiment: COs are treated with 1nM T4 from D7 to D50 and then dissociated into a single-cell suspension for sc-RNA seq. J . UMAP plot showing the cell types identified. K . Histogram of the relative number of cells in T4-COs and control COs. L . Histograms of the relative number of cells in clusters of NPCs. The identification number of each cell cluster is indicated at the bottom right corner of each rectangle. M . Volcano plots showing the distribution of differentially expressed genes in T4-CO vs. control COs. H . Gene set enrichment analysis reveals gene ontology terms enriched in T4 TX COs. O . Histograms of the relative number of cells undergoing the indicated cell cycle phase in clusters one and nine of control and NPCs. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; ***P < 0.001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Derivative Assay, Control, Incubation, Quantitation Assay, Expressing, Suspension, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of the protocol to isolate mNPCs, propagate them in culture, and differentiate into neurons. B, C. Brightfield images showing representative neurospheres containing mNPCs ( B ) and the mNPCs cultured in collagen-coated plasticware ( C ). D-F . Confocal images showing mNPCs expressing Nestin (green) and Sox2 (red) ( D ), and Mct8 (red) ( E ). The insets in E depict two dividing mNPCs that exhibited higher intensity of Mct8 immunofluorescence. F . Quantitation of the DIO2 deiodination in mNPCs; n = 6 DIO2 assays. G. Brightfield images showing representative mNPCs after two days of neurodifferentiation. H - J . Relative mRNA levels of the indicated genes in mNPCs under the indicated conditions; n = 4. K . Brightfield images showing representative mNPCs after four days of neurodifferentiation. Note the neuronal process extension. L . Tuj1+ cells under the indicated conditions and the quantitation of the percentage of Tuj1+ cells. Values are the mean ± SD of 5 replicates. Scale bars: B: 300 µm, C-L: 25 µm. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Cell Culture, Expressing, Immunofluorescence, Quantitation Assay, Two Tailed Test, Derivative Assay