Journal: bioRxiv

Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma

doi: 10.1101/2024.07.11.603140

Figure Lengend Snippet: ( A ) Screenshot of IGV genome browser (GRCH37/hg19) visualization of bigwig files generated from anti-MITF CUT&RUN sequencing in SKMEL28 cells that are wild-type or MITF -KO (delta 6). Three groups of anti-MITF peaks were identified based on the presence or absence of an anti-MITF peak in MITF -KO cells. The frequency of each peak type; “MITF-homodimer peaks”, “persistent-paralog peaks” and “gained paralog peaks”, are labeled. Peaks were called using MACS2 software using FDR < 0.05 with IgG serving as background control ( B ) Density plot of MITF homodimer peaks (upper panel) and Gained-paralog peaks (lower panel) showing anti-MITF (blue) and anti-H3K27Ac (Green) CUT&RUN signal in SKMEL28 WT and MITF -KO cells. Peaks are sorted on anti-MITF-signal in wild-type SKMEL28 cells ( C ) Volcano plot showing 2136 DEGs with qval <0.5 among which 1516 genes with log2FC≥|1| fold change in expression ΔMITF-X6 vs. EV-SkMel28 . Hypergeometric analysis was used to determine the association of MITF-homodimer peaks and gained-paralog peaks with DEG, p-values are as labeled, significant associations are indicated with black rectangles. ( D ) IGV screenshot illustrating a MITF-homodimer peak near the MITF-activated gene TYRP1 and ( E ) a gained-paralog peak near the MITF-inhibited gene ZEB1. Additional tracks include ATAC-Seq (Black), and anti-H3K27Ac CUT&RUN (Green) in WT and MITF -KO cells. Bigwig files were generated using deeptools. ( F ) Enrichment of transcription factor motifs using HOMER at MITF-homodimer peaks (upper panel) and gained-paralog peaks (lower panel). ( G ) Density plot of anti-MITF CUT&RUN-seq in WT and two clones of MITF -KO cells and anti-TFE3 CUT&RUN-seq in MITF -KO and double MITF:TFE3 -KO SKMEL28 cells at gained-paralog peaks. ( H ) Venn diagram illustrating the overlap of genes occupied by gained TFE3 peaks and upregulated in MITF -KO cells compared to WT SKMEL28 cells (depicted by the orange circle), with the genes downregulated in double TFE3:MITF -KO compared to MITF -KO cells (depicted by the black circle). Additionally, box plot visualization displays the expression profiles (Log normalized counts) of the overlapping genes (n=425).

Article Snippet: SKMEL28 (HTB-72), A375, RPMI-7951 (HTB-66), SKMEL3 (HTB-69), and SKMEL24 (HTB-71) cell lines was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the appropriate medium as recommended by the manufacturer.

Techniques: Generated, Sequencing, Labeling, Software, Control, Expressing, Clone Assay

Journal: bioRxiv

Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma

doi: 10.1101/2024.07.11.603140

Figure Lengend Snippet: (A) Density plot depicting ATAC-Seq (black) and anti-H3K27Ac (green) CUT&RUN-seq in WT and MITF -KO SKMEL28 cells, alongside ATAC-Seq (black), anti-H3K27Ac (green), and H3K9Ac (purple) CUT&RUN-seq in A375 cells. The density heatmap is ordered by MITF-homodimer peaks (upper panel) and gained-TFE3 peaks (lower panel) as identified in WT and MITF -KO SKMEL28 cells, respectively. ( B ) Summary plots illustrating normalized ATAC-Seq read counts at MITF-homodimer (blue line) and Gained-TFE3 peaks (Red line) for “Melanocytic” (MM001), “Intermediate” (MM074), and two “mesenchymal-like” (MM029, MM099) human patient derived melanoma cell lines . ( C ) Genome browser screenshot of enhancers activated by MITF-homodimers (Blue highlight) near the melanocytic gene GPR143 and enhancers activated by gained TFE3-peaks (red highlight) near the mesenchymal-like gene ZEB1. Bigwigs of anti-MITF CUT&RUN-seq and ATAC-Seq from SKMEL28 and MITF -KO cells as well as ATAC-Seq traces from Melanocytic (blue), Intermediate (black) and mesenchymal-like (red) are shown.

Article Snippet: SKMEL28 (HTB-72), A375, RPMI-7951 (HTB-66), SKMEL3 (HTB-69), and SKMEL24 (HTB-71) cell lines was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the appropriate medium as recommended by the manufacturer.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma

doi: 10.1101/2024.07.11.603140

Figure Lengend Snippet: ( A ) Screenshot of IGV genome browser (GRCH37/hg19) visualization of bigwig files generated from anti-MITF CUT&RUN sequencing in SKMEL28 cells that are wild-type or MITF -KO (delta 6), as well as anti-MITF, anti-TFE3, and IgG CUT&RUN sequencing in A375 cells that are wild-type or TFE3 -KO. Gained paralog peaks in MITF-KO cells near the ZEB1 gene are highlighted in blue, and yellow asterisks indicate TFE3 occupancy at gained paralog peaks in A375 cells. ( B ) Density heatmap illustrating anti-MITF, anti-TFE3, and IgG CUT&RUN bigwig signals in A375 cells that are wild-type or TFE3 -KO at genomic regions bound by MITF-homodimer and gained-TFE3 peaks in SKMEL28 wild-type and MITF -KO cell lines, respectively. ( C ) Volcano plot showing differentially expressed genes (DEGs) with qval < 0.5, among which 3344 genes were downregulated (blue) and 3206 genes upregulated (red) with log2 fold change (log2FC) ≥ |1| in TFE3 -KO vs. A375 cells. ( D ) Gene Set Enrichment Analysis (GSEA) was used to determine differentially regulated gene ontology and signaling pathways using DEGs from TFE3 -KO and wild-type A375 cell lines. Enriched ontology terms and pathways are labeled. The enrichment score is indicated by green lines, the number of genes by vertical black lines, and gene expression changes (positive or negative) by the red to blue heatmap (high to low). ( E ) Screenshot of IGV genome browser (GRCH37/hg19) visualization of bigwig files generated from anti-MITF and anti-TFE3 CUT&RUN sequencing in A375 cells that are wild-type (blue) or TFE3 -KO (orange) near TFE3-activated genes associated with epithelial to mesenchymal transition and neural crest cell migration in cancer. ( F ) Validation of RNA-Seq by qPCR Analysis. Bar chart displaying the expression levels of genes identified as directly TFE3-dependent in A375 cells. The comparison is made between wild-type (WT) and TFE3 knockout ( TFE3 -KO) cells. The y-axis represents the relative expression levels, while the x-axis lists the validated genes. Error bars indicate the standard deviation from triplicate experiments.

Article Snippet: SKMEL28 (HTB-72), A375, RPMI-7951 (HTB-66), SKMEL3 (HTB-69), and SKMEL24 (HTB-71) cell lines was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the appropriate medium as recommended by the manufacturer.

Techniques: Generated, Sequencing, Protein-Protein interactions, Labeling, Gene Expression, Migration, Biomarker Discovery, RNA Sequencing, Expressing, Comparison, Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma

doi: 10.1101/2024.07.11.603140

Figure Lengend Snippet: ( A ) Nuclear and cytoplasmic protein lysates were extracted from MITF-high SKMEL28 cells and MITF-low RPMI-7951 and A375 cell lines. The localization and expression of TFE3 was analyzed using Western blot. LaminA/B and GAPDH served as loading controls for the nuclear and cytoplasmic fractions, respectively. ( B ) Scatterplot representing the comparative cell invasion of SKMEL28, A375, and RPMI-7951 through Matrigel-coated Boyden chambers over 24 hours. Individual dots represent biological experiments (n=3) with three technical replicates. Statistical analysis was performed using the Student’s t-test. **P-value < 0.01. ( C ) Nuclear and cytoplasmic protein lysates were extracted from patient-derived xenograft (PDX) cell lines characterized as MITF-high (PDX10, PDX15) or MITF-low (PDX16, PDX34). The localization and expression of TFE3 was analyzed using Western blot. LaminA/B and GAPDH served as loading controls for the nuclear and cytoplasmic fractions, respectively. ( D ) Scatterplot representing the comparative cell invasion of MITF-high and MITF-low PDX cell lines through Matrigel-coated Boyden chambers over 24 hours. Individual dots represent biological experiments (n=4) with four technical replicates. Statistical analysis was performed using the Student’s t-test. **P-value < 0.01. ( E ) Knockdown of TFE3 using siRNA in A375 and RPMI cells, as well as in ( F ) PDX16 and PDX32, was performed and cell invasion was assessed over 24 hours using Matrigel-coated Boyden chambers. Scatterplots of the results indicate a profound effect of TFE3 on cell invasion for all cell lines tested. Individual dots represent biological experiments (n=3) with three technical replicates. Statistical analysis was performed using the Student’s t-test. **P-value < 0.01. ( G ) Tail vein metastatic colonization assay design. Tumor size measured via photon flux (radiance). ( H ) Comparison of metastasis size at 5 weeks post inoculation. Statistical analysis using Student’s t-test. **** P-value <0.0001. ( I ) Kaplan Meier curve of metastasis free survival. Statistical analysis using Log-Rank Test.

Article Snippet: SKMEL28 (HTB-72), A375, RPMI-7951 (HTB-66), SKMEL3 (HTB-69), and SKMEL24 (HTB-71) cell lines was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the appropriate medium as recommended by the manufacturer.

Techniques: Expressing, Western Blot, Derivative Assay, Knockdown, Comparison

Journal: bioRxiv

Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma

doi: 10.1101/2024.07.11.603140

Figure Lengend Snippet: ( A ) Gel image of RT-PCR showing two distinct bands separated by 65 base pairs (bps), corresponding to different transcript variants of TFE3. Both bands are present in equal ratios within and between the SKMEL28 and MITF -KO cell lines, indicating consistent expression levels of the TFE3 transcript variants across these cell lines. Primers targeting actin mRNA were used as a loading control to ensure equal RNA input and efficient cDNA synthesis across samples. ( B ) Schematic of two TFE3 variants, labeled as full-length and short-length TFE3. The full-length TFE3 contains a degron sequence in exon 2, represented by a yellow rectangle. Both TFE3 variants share the same transcriptional start site, but have different ribosome entry sites, leading to two distinct protein products with molecular weights of 72 kDa and 82 kDa. ( C ) Immunoblotting of anti-TFE3 on bulk protein lysates of SKMEL28 cells, two MITF -KO cell lines (delta 2 and delta 6; ), and one double MITF/TFE3 -KO cell line. Immunoblotting of anti-GAPDH was used as a loading control. The upper ∼82 kDa band represents the full-length TFE3, while the lower ∼72 kDa band represents the short-length TFE3. ( D ) Immunoblotting of anti-TFE3 from SKMEL28 and MITF -KO (delta 6) nuclear and cytoplasmic cell fractions +/- fetal calf serum (FBS) for 24 hours. Immunoblotting of LAMIN A/B and GAPDH were used as nuclear and cytoplasmic loading controls, respectively. ( E ) Immunoblotting of anti-TFE3 from bulk protein lysates of SKMEL28, SKMEL24, and SKMEL3 cell lines, -/+ 1 μM MLN4924. ( F ) Immunoblotting of anti-TFE3 from SKMEL28 nuclear and cytoplasmic cell fractions, treated with -/+ 1 μM MLN4924 for 24 hours. Immunoblotting of LAMIN A/B and GAPDH were used as nuclear and cytoplasmic loading controls, respectively. ( G ) Immunoblotting of TFE3 in bulk (left) or nuclear and cytoplasmic cell fractions (right) for SKMEL28 cells -/+ 500 nM Torin1 for 24 hours. ( H ) Screenshot of IGV genome browser (GRCH37/hg19) visualization of bigwig files generated from anti-MITF, anti-H3K27Ac, and anti-H3K4Me3 CUT&RUN-seq and ATAC-seq at the FNIP2 locus in SKMEL28 cells that are wild-type or MITF -KO (delta 6). ( I ) Schematic representation of MITF-mediated regulation of TFE3 protein stability and nuclear localization in MITF-high and MITF-low conditions.

Article Snippet: SKMEL28 (HTB-72), A375, RPMI-7951 (HTB-66), SKMEL3 (HTB-69), and SKMEL24 (HTB-71) cell lines was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in the appropriate medium as recommended by the manufacturer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, cDNA Synthesis, Labeling, Sequencing, Western Blot, Generated

Journal: Frontiers in Integrative Neuroscience

Article Title: Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus

doi: 10.3389/fnint.2022.934764

Figure Lengend Snippet: NG2 and BCAN form coats surrounding axons in the human MD thalamus. Photomicrograph of a luxol blue stained section depicting the subregions of the human mediodorsal nucleus sampled for quantification of axonal coats (A) . Myelinated fiber bundles were sampled from the parvocellular (PVC), the magnocellular (MC), caudodorsalis (CD) regions of the MD. (B) Low magnification (10×) confocal image of NG2 labeling (red) and BCAN labeling (green) in the mediodorsal nucleus of the human thalamus. Labeling for both CSPGs was observed in structures resembling myelinated fiber bundles. Higher magnification images (C) revealed that these CSPGs labeled tube-like structures apparently surrounding openings that were consistent with the diameters of single axons. Scale bar equals 30 μm. Confocal micrographs of dual immunofluorescence labeling revealed that these CSPG structures resembling axonal coats, labeled with NG2 (red), surrounded SMI-312 immunoreactive axons (green). (D) Low magnification (10× objective) image showing NG2 axonal coats around SMI-312 axons in transverse and cross-section slices of the axons. (E) An intermediate magnification image depicting an example of cross-sectional SMI-312 axons surrounded by NG2 coats. Pink arrows indicate axons surrounded by NG2 coats, yellow arrows indicate axons without NG2 coats. (F) High-resolution imaging allowed for measurements of the diameter of these axons (3.95 and 3.99 μm) as well as the thickness of the NG2 axonal coats [892 and 856 nanometers; (G) ]. Scale bars equal 4 μm for D–G .

Article Snippet: NG2 – rabbit polyclonal IgG anti-CSPG4 (55027-1-AP, lot#09000034, Protein Tech Group Inc., Rosemont, IL, United States) raised against a synthetic peptide corresponding to human CSPG4/NG2, GenBank accession # NM_001897 .

Techniques: Staining, Labeling, Immunofluorescence, Imaging

Journal: Frontiers in Integrative Neuroscience

Article Title: Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus

doi: 10.3389/fnint.2022.934764

Figure Lengend Snippet: Percentage of Axons surrounded by NG2 or BCAN Coats. Stereology-based sampling was used to quantify the number of SMI-312 immunoreactive axons and axonal coats within bundles of the MD (A) approximately 65% (standard deviation ±3.3%) of axons were associated with NG2 immunoreactive coats (B) and 64% (standard deviation ±2.7%) were associated with BCAN immunoreactive coats (C) .

Article Snippet: NG2 – rabbit polyclonal IgG anti-CSPG4 (55027-1-AP, lot#09000034, Protein Tech Group Inc., Rosemont, IL, United States) raised against a synthetic peptide corresponding to human CSPG4/NG2, GenBank accession # NM_001897 .

Techniques: Sampling, Standard Deviation

Journal: Frontiers in Integrative Neuroscience

Article Title: Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus

doi: 10.3389/fnint.2022.934764

Figure Lengend Snippet: NG2 and BCAN labeling is associated with larger axons. (A) Electron microscopy image depicting a cross-sectional axon with myelin labeling and interweaved immunoreactivity for NG2. (B) Two-dimensional quantitative analysis of 24,000 axon segments revealed that BCAN and NG2 labeling within the cytoplasm was more frequently observed in larger axons. The * symbol indicates location of the axon.

Article Snippet: NG2 – rabbit polyclonal IgG anti-CSPG4 (55027-1-AP, lot#09000034, Protein Tech Group Inc., Rosemont, IL, United States) raised against a synthetic peptide corresponding to human CSPG4/NG2, GenBank accession # NM_001897 .

Techniques: Labeling, Electron Microscopy

Journal: Journal of Gastrointestinal Oncology

Article Title: NCAPG is transcriptionally regulated by CBX3 and activates the Wnt/β-catenin signaling pathway to promote proliferation and the cell cycle and inhibit apoptosis in colorectal cancer

doi: 10.21037/jgo-23-63

Figure Lengend Snippet: NCAPG silence inhibited the proliferation and cell cycle of HCT116 cells. (A,B) The mRNA and protein expressions of NCAPG and CBX3 were detected using RT-qPCR and western blot. (C,D) The transfection efficacy of sh-NCAPG was detected using RT-qPCR and western blot. (E) The viability of transfected HCT116 cells was detected using CCK-8. (F) The cell cycle of transfected HCT116 cells was detected using flow cytometry. (G) The expression of cyclin D1 and CDK4 was detected using western blot. Data are expressed as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. NCAPG, non-SMC condensin I complex subunit G; SMC, structural maintenance of chromosomes; CBX3, chromobox protein homolog 3; mRNA, messenger RNA; sh-NC, short hairpin specific to negative control; sh-NCAPG, short hairpin specific to NCAPG; CV, coefficient of variation; CDK4, cyclin-dependent kinase 4; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, Cell Counting Kit-8; SD, standard deviation.

Article Snippet: The membranes were then cultivated with primary antibodies targeting NCAPG (24563-1-AP; 1:5,000; Proteintech, Wuhan, China), cyclin D1 (60186-1-AP; 1:5,000; Proteintech), cyclin-dependent kinase 4 (CDK4) (66950-1-Ig; 1:5,000; Proteintech), Bcl2 (12789-1-AP; 1:2,000; Proteintech), Bax (50599-2-Ig; 1:2,000; Proteintech), CBX3 (ab217999; 1:2,000; Abcam), Wnt3a (26744-1-AP; 1:500; Proteintech), β-catenin (66379-1-Ig; 1:5,000; Proteintech) or β‐actin (20536-1-AP; 1:2,000; Proteintech) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Flow Cytometry, Expressing, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Standard Deviation

Journal: Journal of Gastrointestinal Oncology

Article Title: NCAPG is transcriptionally regulated by CBX3 and activates the Wnt/β-catenin signaling pathway to promote proliferation and the cell cycle and inhibit apoptosis in colorectal cancer

doi: 10.21037/jgo-23-63

Figure Lengend Snippet: NCAPG transcriptionally regulated by CBX3 activated the Wnt/β-catenin signaling pathway to regulate proliferation, cell cycle, and apoptosis in HCT116 cells. (A) The protein expression of Wnt3a and β-catenin was detected using western blot. (B) The viability of HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 was detected using CCK-8. (C) The cell cycle of HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 was detected using flow cytometry. (D) The expression of cyclin D1 and CDK4 in HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 was detected using western blot. (E) The apoptosis of HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 was detected using TUNEL. DAPI was used for staining. Magnification, ×200. (F) The expression of Bax and Bcl2 in HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 were detected using western blot. (G) The activity of cleaved caspase3 and cleaved caspase9 in HCT116 cells cotransfected with sh-NCAPG and Ov-CBX3 was detected using colorimetric caspase activity assay. Data are expressed as mean ± SD. ***P<0.001. Ov-NC, pcDNA3.1 empty vector; Ov-CBX3, plasmids carrying CBX3; CBX3, chromobox protein homolog 3; sh-NC, short hairpin specific to negative control; sh-NCAPG, short hairpin specific to NCAPG; NCAPG, non-SMC condensin I complex subunit G; SMC, structural maintenance of chromosomes; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole, CCK-8, Cell Counting Kit-8; SD, standard deviation.

Article Snippet: The membranes were then cultivated with primary antibodies targeting NCAPG (24563-1-AP; 1:5,000; Proteintech, Wuhan, China), cyclin D1 (60186-1-AP; 1:5,000; Proteintech), cyclin-dependent kinase 4 (CDK4) (66950-1-Ig; 1:5,000; Proteintech), Bcl2 (12789-1-AP; 1:2,000; Proteintech), Bax (50599-2-Ig; 1:2,000; Proteintech), CBX3 (ab217999; 1:2,000; Abcam), Wnt3a (26744-1-AP; 1:500; Proteintech), β-catenin (66379-1-Ig; 1:5,000; Proteintech) or β‐actin (20536-1-AP; 1:2,000; Proteintech) overnight at 4 °C.

Techniques: Expressing, Western Blot, CCK-8 Assay, Flow Cytometry, TUNEL Assay, Staining, Activity Assay, Caspase Activity Assay, Plasmid Preparation, Negative Control, Cell Counting, Standard Deviation