primary antibodies  (Proteintech)

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) TFEB (top, red) and TFE3 (middle, red) immunofluorescence in control TH-positive (green) iPSC-DaNs. White box indicates magnified area (far-right). Scale bar: 50 µm. (B) TFEB and TFE3 mRNA expression in control iPSC-DaNs by (i) qRT-PCR, or (ii) bulk RNA-Seq. qRT-PCR: N= 3 iPSC-DaN lines, 3 differentiations; RNA-Seq: N=4 iPSC-DaN lines. (C) TFE3 and TFEB protein expression in iPSC-DaNs and SH-SY5Y. ACTB was used as a protein loading control. N=3 iPSC-DaN lines, 3 differentiations and 3 SH-SY5Y samples. (D) Dot plots showing mean expression value and percentage of cells expressing TFE3/Tfe3 and TFEB/Tfeb across (i) cell types in human post-mortem midbrain snRNA-seq and (ii) cell lineages in mouse brain scRNA-seq . Gene expression is presented as log 2 (CPM+1) and log e (CP10K+1) for the human and mouse datasets and averaged only over the cells expressing the given genes. Dots are color- and size-coded by mean expression and fraction of expressing cells, respectively. Bar plots on top show the total cell number in each category. OLG_Lin: oligodendrocyte lineage; ASC_Lin: astrocyte lineage and stem cells; NEURON_Lin: neuronal lineage; EPC_Lin: ependymal cells; VASC_Lin: vasculature cells; IMMUNE_Lin: immune cells. (E) TFEB and TFE3 (red) immunofluorescence in MAP2-positive control i 3 cortical neurons (green, top) and Iba1-positive control iPSC-microglia (green, bottom). White boxes indicate magnified area. Non-magnified scale bars: 50 µm, magnified scale bars: 20 µm. (F) TFEB and TFE3 mRNA expression, normalised to GAPDH expression, in 3 control iPSC-DaN lines, 4 i 3 cortical neuron differentiations, and 2 control iPSC-microglia lines. Ordinary one-way ANOVA with Tukey’s multiple comparison’s test between cell-types for each gene. (G) TFEB and TFE3 protein expression in iPSC-microglia, iPSC-DaNs, and i 3 neurons. Actin used as a protein loading control. All bar graphs represent mean ± SD.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Immunofluorescence, Control, Expressing, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Positive Control

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) Dual qRT-PCR screening in iPSC-cortical neurons to identify hit targets for TFEB derepression in neurons. One screen utilises a well-characterised epigenetic compound library, and the second screen uses a diverse 3,000 bioactive compound set. (B) Fold change TFEB mRNA expression following treatment with epigenetic screening compounds, relative to DMSO. Dotted line indicates fold change at +3 S.D. DMSO. TFEB-EGFP was used as a positive control. (C) TFEB and TFE3 mRNA expression in control iPSC-DaNs treated with LAQ824, relative to DMSO. Paired t-test applied to each gene. N=3 iPSC-DaN lines, 3 differentiations. (D) (i) Representative western blot and (ii) quantification of TFEB and TFE3 protein levels in control iPSC-DaNs treated with LAQ824, relative to DMSO. Paired t-test applied to each protein. N=3 iPSC-DaN lines, 3 differentiations. (E) CLEAR gene mRNA expression in control iPSC-DaNs treated with LAQ824, relative to DMSO treatment. Paired t-test applied to each gene. N=3 iPSC-DaN lines, 2 differentiations. (F) (i) Representative western blot and (ii) quantification of lysosomal protein expression in control iPSC-DaNs treated with LAQ824, relative to DMSO. N=3 iPSC-DaN lines, 2 differentiations. Paired t-test applied to each protein. (G) (i) Representative image and quantification of DQ-BSA (yellow) (ii) spot number and (iii) corrected spot intensity in control iPSC-DaNs treated with LAQ824, relative to DMSO. NeuO (green) identifies live neurons. Paired t-test. N=3 iPSC-DaN lines, 3 differentiations. Scale bar: 20 µm. (H) TFEB mRNA expression concentration-response in iPSC-Ngn2 neurons treated with Torin1, LAQ824, Tunicamycin, CQ and Staurosporine, relative to DMSO. A non-linear curve was fit to the data with error bars (±SEM). N= 2 differentiations. All bar graphs represent mean ± SD.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Quantitative RT-PCR, Drug discovery, Expressing, Positive Control, Control, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) (i) Representative images of TFEB (red) immunocytochemistry in control iPSC-DaNs treated with DMSO or ACY-738. Quantification of TFEB relative fluorescence units (RFUs) in the (ii) whole cell or (iii) nucleus following treatment. Scale bars: 50 µm. N=3 iPSC-DaN lines, 3 differentiations. (B) TFEB and TFE3 mRNA expression in control iPSC-DaNs following ACY-738 treatment, normalised to DMSO. N=3 iPSC-DaN lines, 3 differentiations. (C) (i) Representative western blot and (ii) quantification of TFEB and TFE3 protein levels in control iPSC-DaNs treated with ACY-738, relative to DMSO. N=3 iPSC-DaN lines, 3 differentiations. (D) (i) Representative images of TFE3 immunocytochemical staining and (ii) quantification of % cells displaying nuclear TFE3 in control iPSC-DaNs treated with DMSO or ACY-738. Paired t-test. Scale bars: 20 µm. N=3 iPSC-DaN lines, 2-3 differentiations. (E) CLEAR gene mRNA expression in control iPSC-DaNs treated with ACY-738, normalised to DMSO. N=3 iPSC-DaN lines, 3 differentiations. (F) (i) Representative western blot and (ii) quantification of lysosomal protein expression in control iPSC-DaNs treated with ACY-738, relative to DMSO. N=3 iPSC-DaN lines, 2 differentiations. (G) (i) Representative images and quantification of DQ-BSA (ii) spot number and (iii) intensity in control iPSC-DaNs treated with ACY-738, relative to DMSO. Scale bars: 20 µm. N=3 iPSC-DaN lines, 2 differentiations. Paired t-test carried out for all graphs. All graphs represent mean ± SD.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Immunocytochemistry, Control, Fluorescence, Expressing, Western Blot, Staining

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) 3 control iPSC-DaN lines were transduced with control lentiviral vector (mApple) or TFEB-iRFP670 or TFE3-iRFP670 overexpressing lentiviruses at D20. D45 iPSC-DaNs were harvested and underwent RNA-Seq. (B) Overlap in differentially expressed genes upregulated and downregulated by TFEB or TFE3 overexpression. (C) Top 20 biological pathway gene ontologies enriched following (i) TFEB or (ii) TFE3 overexpression using differentially upregulated genes. Dot size and colour indicates number of differentially expressed genes and p-adjusted value, respectively. (D) Visual representation of organelle enrichment for upregulated genes by (i) TFEB or (ii) TFE3 overexpression modified from SubcellulaRVis . Enriched organelles are coloured by scale from green (FDR=0.05) to red (FDR=0). Organelles not enriched (FDR > 0.05) are coloured white. Organelle name and FDR is displayed next to the organelle.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Control, Transduction, Plasmid Preparation, RNA Sequencing, Over Expression, Modification

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) mRNA expression of (i) mitochondria-associated transcription factors, and (ii) mitochondrial genes following TFEB-iRFP670 (blue) or TFE3-iRFP670 (red) overexpression relative to iRFP670 (grey) transduced iPSC-DaNs. RM one-way ANOVA with Dunnett’s multiple comparisons carried out for each gene. N=3 iPSC-DaN lines, 3 differentiations. (B) (i) Representative western blot and (ii) quantification of mitochondrial protein expression in control iPSC-DaNs overexpressing TFEB or TFE3, relative to control lentivirus. RM one-way ANOVA with Dunnett’s multiple comparisons carried out on each protein. N = 3 iPSC-DaNs lines, 3 differentiations. (C) (i) Representative images of TOM20 staining and quantification of mitochondrial (ii) width:length and (iii) area in control iPSC-DaNs overexpressing TFEB-iRFP670, relative to iRFP670 transduced iPSC-DaNs. Scale bars: 20 µm. Paired t-test. N=3 iPSC-DaNs, 2 differentiations. (D-E) (i) Representative Seahorse flux analyzer plot of normalised oxygen consumption rate (OCR) and quantification of (ii) basal respiration and (iii) spare capacity in (D) control and (E) SNCA-Trp iPSC-DaNs overexpressing TFEB-iRFP670, relative to iRFP670. Paired t-test. N=3 iPSC-DaN lines, 2-3 differentiations. Paired t-test. N= 3 lines per genotype, 3 differentiations. All graphs represent mean ± SD.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Expressing, Over Expression, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

doi: 10.1101/2025.04.22.649949

Figure Lengend Snippet: (A) CLEAR gene mRNA expression following TFEB-iRFP670 and TFE3-iRFP670 overexpression, relative to iRFP670 in control iPSC-DaNs. RM one-way ANOVA with Dunnett’s multiple comparisons test for each gene. N=3 iPSC-DaN lines, 2 differentiations. (B) (i) Representative western blot and (ii) quantification of lysosomal protein expression in control iPSC-DaNs following TFEB-iRFP670, TFE3-iRFP670 or iRFP670 overexpression in iPSC-DaNs. Paired RM one-way ANOVA with Dunnett’s multiple comparisons was carried out for each protein. (C) (i) Representative images and (ii-v) quantification of MDW-941 spot number and corrected spot intensity of control iPSC-DaNs overexpressing TFEB-iRFP670 or TFE3-iRFP670 relative to iRFP670 transduced iPSC-DaNs. Paired t-test. N=3 iPSC-DaN lines, 3 differentiations. (D) (i) Representative images and (ii-v) quantification of DQ-BSA spot number and corrected spot intensity in control iPSC-DaNs overexpressing TFEB-iRFP670 or TFE3-iRFP670 relative to iRFP670 transduced iPSC-DaNs. Scale bars: 20 µm. Paired t-test. (E) (i) Confirmation of TFE3 mRNA knockdown. T-test. N=2 sgRNA guides per condition, 2 differentiations, (ii) Western blot confirmation of TFE3 protein knockdown in i 3 neurons transduced with Scrambled sgRNA guides or TFE3 sgRNA guides. (F) CLEAR gene mRNA expression in i 3 neurons following TFE3 knockdown, relative to scrambled sgRNA transduction. T-test for each gene. N=2 sgRNA guides per condition, 2 differentiations. (G) (i) Representative images and quantification of DQ-BSA (ii) spot number and (iii) corrected spot intensity in i 3 neurons following TFE3 knockdown, relative to scrambled sgRNA guides. T-test. N=2 sgRNA guides per condition, 2 differentiations. All bar charts represent mean ± SD.

Article Snippet: Primary antibodies (TFEB (Cell Signalling) – 1:500, TFEB (Bethyl) – 1:1000, TFE3 (abcam) – 1:1000, TH (abcam, ab76442) – 1:1000, TH (Millipore, AB152) – 1:500, MAP2 (Millipore, ab92434) – 1:2000, FOXA2 (R&D systems, AF2400) – 1:250, Iba1 (abcam, ab5076) – 1:1000, LMX1A (abcam, ab139726) – 1:1000, TUJ1 (Biolegend, 801202) – 1:500, LAMP1 (Santa Cruz) – 1:500, TOM20 (Santa Cruz) – 1:1000) were incubated overnight at 4°C.

Techniques: Expressing, Over Expression, Control, Western Blot, Knockdown, Transduction