Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A) Sanger sequencing confirms compound heterozygous variants in THAP12 in both affected siblings, with a maternally inherited frameshift (c.312del, red arrow) and a paternally inherited missense variant (c.829C>A, black arrow). (B) The two variants affect conserved residues within protein domains, particularly a proline at position 277 in the DUF4371 domain, as shown in a multi-species alignment (red arrow; Hs: Homo sapiens; Mm: Mus musculus; Rn: Rattus norvegicus; Xt: Xenopus tropicalis; Dr: Danio rerio ). (C) A Sashimi plot of RNA-seq reads from patients’ primary fibroblasts across the THAP12 locus shows no major changes in exon usage or alternative splicing between probands and their parents. Read counts on the splice junction arcs indicate the number of split reads supporting each exon-exon connection in each sample. (D) Structural modelling with AlphaFold3 predicts that THAP12 forms homodimers primarily through interactions between DUF4371 domains (left). In a THAP12-DNA complex predicted using AlphaFold3, the N-terminal THAP zinc-finger domains interacts with DNA using an electropostive surface (right). (E) The maternally inherited frameshift variant (Glu105AsnfsTer2) is predicted to truncate the protein after residue 106, abolishing the DUF4371 domain and likely impairing dimerization. The N-terminal segment (residues 1-106) is shown in color, corresponding to the truncated product of the frameshift allele. The paternally inherited Pro277Thr missense variant affects a conserved residue buried within the hydrophobic core of the DUF4371 domain, likely disrupting local folding and protein stability. (F-G) THAP12 transcript levels are not significantly changed in patient fibroblasts compared to parental controls, as shown by RNA-seq and qPCR analyses. (H-I) In contrast, THAP12 protein levels in patient fibroblasts are significantly reduced in both probands, as shown by Western blot and quantification. (J) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of patient-derived fibroblasts compared to parental controls. Downregulated genes include several involved in neuronal and synaptic function (e.g., TUBB, RIMS1, GABRA3, KCNMB1 ). Significance is color-coded according to the - log10(adjusted p-value). The full list of differentially expressed genes is provided in Table S1. (K) Pathway enrichment analysis of differentially expressed genes highlights over-representation of pathways such as “Neuronal System”, “Signal Transduction”, and “Transmission across Chemical Synapses”. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). Statistical analyses in panels F, G, and I used unpaired two-tailed Student’s t-test: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Sequencing, Variant Assay, RNA Sequencing, Alternative Splicing, Residue, Western Blot, Derivative Assay, Transduction, Transmission Assay, Two Tailed Test

Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A-B) Dorsal views of Tg[ elavl3 :GFP] larvae at 2 dpf (A) and 5 dpf (B) showing a reduced brain size in thap12 CRISPant compared to sham-injected controls. The lack of a clear midbrain-hindbrain boundary (dotted line) is indicated by asterisks. (C-E) Transverse sections immunostained for elavl3 show reduced brain size in thap12 CRISPant embryos at 2 dpf ( C ) and at 5 dpf ( D, E ). (F) Dorsal view of 3 dpf brains immunostained for acetylated tubulin showing reduced density of axonal tracts in thap12 CRISPant , especially at the level of the commissure (asterisks). (G-H) Quantification of brain area from whole-brain imaging of Tg[elavl3:GFP] larvae at 2 dpf (G) and 5 dpf (H) confirms a significant reduction of brain size in thap12 CRISPant . (I-J) thap12 CRISPant larvae show reduced numbers of elavl3 + neurons from immunolablled cross-sections (I) and commissural axonal tracts (J). (K) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of microdissected larval brains from 4dpf thap12a -/- compared to wild-type siblings. Upregulated genes include several involved in apoptosis (e.g. tp53 ) and cell cyle (e.g. ccng1 ). Significance is color-coded according to the -log10(adjusted p-value). (L) Pathway enrichment analysis identifies p53 signaling, cell cycle, and metabolic stress as significantly enriched pathways in mutants compared to wild-type siblings. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). (M) Acridine orange staining reveals increased cell death in the brain of thap12 CRISPant at 1 dpf. (N) Anti-phospho-H3 immunostaining shows a reduced number of proliferating cells in thap12 CRISPRant larvae at 1 dpf. Quantification are shown in panel O and P. (Q-T) Injection of human wild-type THAP12 mRNA, but not Pro277Thr mutant mRNA, rescues reduced proliferation in thap12 CRISPant larvae at 1 dpf (Q-R) and partially rescues brain size at 2 dpf (S-T) . The lack of a clear midbrain-hindbrain boundary (dotted line) described in panel A is indicated by asterisks. Scale bars are shown on each panel. Statistical analyses in panel G-J and O-P used unpaired two-tailed Student’s t-test, and in panels R and T used one-way ANOVA: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant. fb: forebrain; mb: midbrain; hb: hindbrain; mhb: midbrain-hindbrain boundary (dotted line); bs: brainstem; ey: eye; re: retina; tec: tectum; teg: tegmentum; hl: hypothalamus .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Injection, Imaging, RNA Sequencing, Staining, Immunostaining, Mutagenesis, Two Tailed Test

Journal: Neuro-Oncology

Article Title: Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography

doi: 10.1093/neuonc/nor228

Figure Lengend Snippet: Experimental setup for 2PLSM studies. (A) Experimental flowchart for 2PLSM LT (long-term) and ST (short-term) studies. (B) 2PLSM image of SMA-560 experimental glioma (green) and attracted murine HPC (red signal dots; arrows). (C) Combined volume view of a 2PLSM Z-stack (50 µm depth). HPC are indicated as a representative example by two arrows. (D) Combined volume view of a 2PLSM Z-stack under data analysis. Defined HPC cells can be recognized as white dots and are marked here by arrows). Background signals are red. (E) 3D reconstruction after complete data analysis. Attracted HPC are white, GFP-positive glioma cells are green.

Article Snippet: All acquired 2PLSM data were analyzed using Bitplane Imaris (Fig. B–E, Supplementary material, Fig. S2 ) after acquiring single images as outlined representatively for T269 gliomas attracting human HPC (Fig. A–C).

Techniques:

Journal: Neuro-Oncology

Article Title: Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography

doi: 10.1093/neuonc/nor228

Figure Lengend Snippet: Real-time monitoring glioma growth by 2PLSM. (A) Schematic overview of experimental setup (see text for details). (B) Schematic overview of surgical procedure (see text for details). (C) Representative 2PLSM of SMA-560 experimental glioma (days 5 and 14 after implantation, magnitude 10×, scale bar = 300 µm).

Article Snippet: All acquired 2PLSM data were analyzed using Bitplane Imaris (Fig. B–E, Supplementary material, Fig. S2 ) after acquiring single images as outlined representatively for T269 gliomas attracting human HPC (Fig. A–C).

Techniques:

Journal: Neuro-Oncology

Article Title: Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography

doi: 10.1093/neuonc/nor228

Figure Lengend Snippet: Imaging glioma-mediated attraction of HPC by 2PLSM. (A) 2PLSM image of a T269 glioma (magnification 10×; green/FITC channel) (scale bar = 300 µm). (B) 2PLSM image of a T269 glioma (magnification 40×; green/FITC channel) (scale bar = 100 µm). (C) Combined volume view of a 2PLSM Z-stack (50 µm depth) of a T269 glioma (magnification 40×; green/FITC channel) and attracted human HPC (red/TRITC channel) (scale bar = 100 µm). (D) Morphology of gliomas was assessed by H&E (left), CD45 immunostaining (top right) and nuclear counterstaining with DAPI (down right). (E) In vitro coculture of GFP-positive SMA-560 glioma cells (green) and PKH26-labelled murine HPC (red) (scale bar = 100 µm). (F) Summation of attracted HPC in 5 xyz locations after a LT time schedule. ST time schedule of future scans (as shown in G) is highlighted with a grey frame in the left corner of the diagram. Mice numbers 1–3, murine SMA560/murine HPC. Mice numbers 4–6, LNT229 glioma cells/human HPC (see text for details). (G) Summation of attracted HPC in 5 xyz locations as acquired during the ST time schedule. Mice numbers 1–3, murine SMA560/murine HPC. Mice numbers 4–6, LNT229 glioma cells/human HPC. Mice numbers 7–8, T269 glioma/human HPC (see text for details).

Article Snippet: All acquired 2PLSM data were analyzed using Bitplane Imaris (Fig. B–E, Supplementary material, Fig. S2 ) after acquiring single images as outlined representatively for T269 gliomas attracting human HPC (Fig. A–C).

Techniques: Imaging, Immunostaining, In Vitro

Journal: Nature Communications

Article Title: Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

doi: 10.1038/s41467-022-33951-0

Figure Lengend Snippet: a PPIs identified in the XL-LC-MS/MS dataset. Lysosomal proteins (blue dots), non-lysosomal proteins (gray dots), and PPIs (gray lines) are indicated. b Matching of PPIs to the STRING database. c Numbers of proteins from distinct subcellular localizations interacting with lysosomal proteins. d , e Interaction networks of the V-ATPase ( d ) and the flotillin ( e ) complex. f Co-IP of ATP6V1D and FZD9 ( n = 2). ATP6V1B2 and ATP6V1A1 are known members of the V-ATPase complex; LAMP2 is a lysosomal membrane protein. Control: empty beads. g Co-IPs of FLOT1, FLOT2, and GNB4 ( n = 2). RRAGA is a lysosomal membrane-associated protein. Control: empty beads. h , i Site frequency distribution for identified FLOT1 ( h ) and FLOT2 ( i ) cross-links. Site frequency represents the percentage of cross-links detected in bins of 20 residues each. The region indicated by red dots represents the PHB domain. CY cytoplasm, CK cytoskeleton, NC nucleus, ER endoplasmic reticulum, GA Golgi apparatus, PM plasma membrane, LS lysosome, OT others, NK not known, IN input, SN supernatant, W wash, EL eluate, X empty lane. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: goat anti LIMP2 (Cat# AF1966-SP,1:2000), mouse anti ACT2 (Cat# A5316, 1:4000), and rabbit anti LAMTOR1 (Cat # HPA002997, 1:1000) from Sigma-Aldrich; mouse anti CANX (Cat# 66903-1-AP, 1:20,000), mouse anti FLOT2 (Cat# 66881-1-Ig, 1:1500), mouse anti FZD9 (Cat# 67023-1-Ig, 1:1500), rabbit anti ATP6V1D (Cat# 14920-1-AP, 1:1000), rabbit anti CTSD (Cat# # 21327-1-AP, 1:1000), rabbit anti FLOT1 (Cat# 15571-1-AP, 1:2000), rabbit anti GNB4 (Cat# 11978-2-AP, 1:2000), and rabbit anti SDHA (Cat# 14865-1-AP, 1:800) from Proteintech; goat anti LIMP2 (Cat# AF1966-SP, 1:2000) from R&D system; mouse anti ATP6V1B2 (Cat# SC166045, 1:1000) from Santa Cruz; mouse anti FLOT1 (Cat# 610821, 1:200), mouse anti FLOT2 (Cat# 610383, 1:200), and mouse anti GM130 (Cat# 610822) from BD Biosciences; mouse anti GAPDH (Cat# 5174, 1:2500), rabbit anti EEA1 (Cat# 2411, 1:200), rabbit anti FLOT1 (Cat# 18634, 1:200), and rabbit anti RRAGA (Cat# 4357, 1:1000) from Cell signaling; rabbit anti LPHN2 (Cat# NBP2-58704, 1:100) and rabbit anti LPHN3 (Cat# NLS1138, 1:200) from Novus Biologicals; goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat# A-11029, 1:400), rabbit anti ATP6V1A1 (Cat# PA5-29191,1:2000), and rabbit anti LPHN1 (Cat # PA5-77475, 1:200) from Thermo Fisher Scientific; rabbit anti TUBA (Cat# 600-401-880, 1:2000) from Rockland; mouse anti LAMP2 (Cat# H4B4, 1:1000 for WB, 1:100 for IF) from Hybridoma Bank. rabbit anti DSSO (Self-made , 1:5000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Membrane, Control, Clinical Proteomics

Journal: Nature Communications

Article Title: Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

doi: 10.1038/s41467-022-33951-0

Figure Lengend Snippet: a Western blot analysis of lysosome-enriched fractions ( n = 3). CTSD is a lysosomal luminal and LAMP2 a lysosomal transmembrane protein. b Identified cross-links, predicted secondary structures (PSIPRED), and coiled-coil probabilities (PCOILS) for FLOT1 and FLOT2. c Heterodimeric model of FLOT1-FLOT2 interaction (ColabFold). The model satisfies the distance constraints of all cross-links. d FLAG-IP from HEK293 cells transfected with either full-length FLAG-FLOT1 and FLAG-FLOT2 or versions lacking amino acid residues 200–300 (Δ100) ( n = 2). WCL whole-cell lysate, EL eluate, PHB prohibitin homology domain, SPFH stomatin/PHB/flotillin/HflK/C domain, WND window, E glutamic acid, A alanine, PZD3 postsynaptic density protein-95/discs large/zonula occludens-1, IN input, WA wash, WT wild-type, Δ 100 mutant with a deletion of 100 amino acids (residues 200–300 of FLOT1/FLOT2). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: goat anti LIMP2 (Cat# AF1966-SP,1:2000), mouse anti ACT2 (Cat# A5316, 1:4000), and rabbit anti LAMTOR1 (Cat # HPA002997, 1:1000) from Sigma-Aldrich; mouse anti CANX (Cat# 66903-1-AP, 1:20,000), mouse anti FLOT2 (Cat# 66881-1-Ig, 1:1500), mouse anti FZD9 (Cat# 67023-1-Ig, 1:1500), rabbit anti ATP6V1D (Cat# 14920-1-AP, 1:1000), rabbit anti CTSD (Cat# # 21327-1-AP, 1:1000), rabbit anti FLOT1 (Cat# 15571-1-AP, 1:2000), rabbit anti GNB4 (Cat# 11978-2-AP, 1:2000), and rabbit anti SDHA (Cat# 14865-1-AP, 1:800) from Proteintech; goat anti LIMP2 (Cat# AF1966-SP, 1:2000) from R&D system; mouse anti ATP6V1B2 (Cat# SC166045, 1:1000) from Santa Cruz; mouse anti FLOT1 (Cat# 610821, 1:200), mouse anti FLOT2 (Cat# 610383, 1:200), and mouse anti GM130 (Cat# 610822) from BD Biosciences; mouse anti GAPDH (Cat# 5174, 1:2500), rabbit anti EEA1 (Cat# 2411, 1:200), rabbit anti FLOT1 (Cat# 18634, 1:200), and rabbit anti RRAGA (Cat# 4357, 1:1000) from Cell signaling; rabbit anti LPHN2 (Cat# NBP2-58704, 1:100) and rabbit anti LPHN3 (Cat# NLS1138, 1:200) from Novus Biologicals; goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat# A-11029, 1:400), rabbit anti ATP6V1A1 (Cat# PA5-29191,1:2000), and rabbit anti LPHN1 (Cat # PA5-77475, 1:200) from Thermo Fisher Scientific; rabbit anti TUBA (Cat# 600-401-880, 1:2000) from Rockland; mouse anti LAMP2 (Cat# H4B4, 1:1000 for WB, 1:100 for IF) from Hybridoma Bank. rabbit anti DSSO (Self-made , 1:5000).

Techniques: Western Blot, Transfection, Mutagenesis

Journal: Nature Communications

Article Title: Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

doi: 10.1038/s41467-022-33951-0

Figure Lengend Snippet: a HeLa cells were stained with antibodies for FLOT1, FLOT2, and LAMP2 followed by microscopy imaging ( n = 3). Lower panels display a zoom-in of the regions indicated in the full picture. Mander’s coefficients show the average degree of overlap of one protein population relative to another. Data are presented as mean values + SD ( n = 3, cells examined over one experiment). Profile plots indicate the degree of colocalization for individual vesicles. b Analysis of FLOT1/FLOT2 in early endosomes ( n = 1). Following pulsed SPIONs treatment, endosomes were enriched at the indicated chase times. Pre-CTSD serves as a marker for early endosomes. c Western blot analysis of early endosome-enriched fractions with marker proteins for different subcellular compartments ( n = 1): Cytosol (ACTG2 and GAPDH), cytoskeleton (TUBA), endoplasmic reticulum (CANX), and mitochondria (SDHA). d Experimental workflow for early endosome enrichment and XL-LC-MS/MS analysis. Created with BioRender.com. e Overview of the cross-linking dataset obtained from early endosome-enriched fractions. f Overlap of unique FLOT1/FLOT2 cross-links for XL-LC-MS/MS analyses of early endosome- and lysosome-enriched fractions. g Western blot analysis of BN-PAGE-separated FLAG-IP eluates with/without cross-linking by DSSO ( n = 5). IN/EL refers to individual FLAG-IP fractions. h Western blot analysis of SDS-PAGE separated FLAG-IP ELs with/without DSSO cross-linking for WT and FLOT1/FLOT2 residue 200–300 deletion mutants (Δ100) ( n = 3). Scale bar = 5 µM; WCL whole cell lysate, IN input, WA wash, EL eluate, MCOE Mander’s coefficient, Dst. distance, LS lysosomes, EE early endosomes, XL cross-linking, SPIONs superparamagnetic iron oxide nanoparticles, M marker, WT wild type. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: goat anti LIMP2 (Cat# AF1966-SP,1:2000), mouse anti ACT2 (Cat# A5316, 1:4000), and rabbit anti LAMTOR1 (Cat # HPA002997, 1:1000) from Sigma-Aldrich; mouse anti CANX (Cat# 66903-1-AP, 1:20,000), mouse anti FLOT2 (Cat# 66881-1-Ig, 1:1500), mouse anti FZD9 (Cat# 67023-1-Ig, 1:1500), rabbit anti ATP6V1D (Cat# 14920-1-AP, 1:1000), rabbit anti CTSD (Cat# # 21327-1-AP, 1:1000), rabbit anti FLOT1 (Cat# 15571-1-AP, 1:2000), rabbit anti GNB4 (Cat# 11978-2-AP, 1:2000), and rabbit anti SDHA (Cat# 14865-1-AP, 1:800) from Proteintech; goat anti LIMP2 (Cat# AF1966-SP, 1:2000) from R&D system; mouse anti ATP6V1B2 (Cat# SC166045, 1:1000) from Santa Cruz; mouse anti FLOT1 (Cat# 610821, 1:200), mouse anti FLOT2 (Cat# 610383, 1:200), and mouse anti GM130 (Cat# 610822) from BD Biosciences; mouse anti GAPDH (Cat# 5174, 1:2500), rabbit anti EEA1 (Cat# 2411, 1:200), rabbit anti FLOT1 (Cat# 18634, 1:200), and rabbit anti RRAGA (Cat# 4357, 1:1000) from Cell signaling; rabbit anti LPHN2 (Cat# NBP2-58704, 1:100) and rabbit anti LPHN3 (Cat# NLS1138, 1:200) from Novus Biologicals; goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat# A-11029, 1:400), rabbit anti ATP6V1A1 (Cat# PA5-29191,1:2000), and rabbit anti LPHN1 (Cat # PA5-77475, 1:200) from Thermo Fisher Scientific; rabbit anti TUBA (Cat# 600-401-880, 1:2000) from Rockland; mouse anti LAMP2 (Cat# H4B4, 1:1000 for WB, 1:100 for IF) from Hybridoma Bank. rabbit anti DSSO (Self-made , 1:5000).

Techniques: Staining, Microscopy, Imaging, Marker, Western Blot, Liquid Chromatography with Mass Spectroscopy, SDS Page, Residue

Journal: Nature Communications

Article Title: Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

doi: 10.1038/s41467-022-33951-0

Figure Lengend Snippet: a Western blot analysis of FLAG-FLOT1/FLOT2 in early endosome-enriched fractions (SPIONs) and intact endosome-IP fractions ( n = 1). b Data-independent acquisition (DIA)-based protein abundance fold-change ratios of SPIONs/SPIONs+IP fractions ( n = 3, biologically independent samples over three independent experiments). Significant differentially regulated proteins are indicated (cut-offs: q -value: <0.05, fold change: >1.5). c STRING-based PPI analysis of selected protein categories overrepresented in the SPIONs+IP fraction. Node size corresponds to the protein abundance and line thickness to the PPI confidence score. SPIONs superparamagnetic iron oxide nanoparticles, IP immunoprecipitation, IN input, SN supernatant, FT flow through, WA wash, EL eluate, PPI protein–protein interaction. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: goat anti LIMP2 (Cat# AF1966-SP,1:2000), mouse anti ACT2 (Cat# A5316, 1:4000), and rabbit anti LAMTOR1 (Cat # HPA002997, 1:1000) from Sigma-Aldrich; mouse anti CANX (Cat# 66903-1-AP, 1:20,000), mouse anti FLOT2 (Cat# 66881-1-Ig, 1:1500), mouse anti FZD9 (Cat# 67023-1-Ig, 1:1500), rabbit anti ATP6V1D (Cat# 14920-1-AP, 1:1000), rabbit anti CTSD (Cat# # 21327-1-AP, 1:1000), rabbit anti FLOT1 (Cat# 15571-1-AP, 1:2000), rabbit anti GNB4 (Cat# 11978-2-AP, 1:2000), and rabbit anti SDHA (Cat# 14865-1-AP, 1:800) from Proteintech; goat anti LIMP2 (Cat# AF1966-SP, 1:2000) from R&D system; mouse anti ATP6V1B2 (Cat# SC166045, 1:1000) from Santa Cruz; mouse anti FLOT1 (Cat# 610821, 1:200), mouse anti FLOT2 (Cat# 610383, 1:200), and mouse anti GM130 (Cat# 610822) from BD Biosciences; mouse anti GAPDH (Cat# 5174, 1:2500), rabbit anti EEA1 (Cat# 2411, 1:200), rabbit anti FLOT1 (Cat# 18634, 1:200), and rabbit anti RRAGA (Cat# 4357, 1:1000) from Cell signaling; rabbit anti LPHN2 (Cat# NBP2-58704, 1:100) and rabbit anti LPHN3 (Cat# NLS1138, 1:200) from Novus Biologicals; goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat# A-11029, 1:400), rabbit anti ATP6V1A1 (Cat# PA5-29191,1:2000), and rabbit anti LPHN1 (Cat # PA5-77475, 1:200) from Thermo Fisher Scientific; rabbit anti TUBA (Cat# 600-401-880, 1:2000) from Rockland; mouse anti LAMP2 (Cat# H4B4, 1:1000 for WB, 1:100 for IF) from Hybridoma Bank. rabbit anti DSSO (Self-made , 1:5000).

Techniques: Western Blot, Data-independent acquisition, Quantitative Proteomics, Immunoprecipitation

Journal: Nature Communications

Article Title: Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

doi: 10.1038/s41467-022-33951-0

Figure Lengend Snippet: a Co-IP of FLOT1 with possible cargo proteins detected by DIA-LC-MS/MS analysis of FLOT1-/FLOT2-positive early endosomes ( n = 1). b Average DIA protein abundance for LPHN1, LPHN2, LPHN3, CLTA, EEA1, and RAB5C. Data are presented as mean values + SD ( n = 3, biologically independent samples over three independent experiments). Significance based on Student’s unpaired two-sided t test; range: ∗ P < 0.05, ∗∗ P < 0.01; exact P values: LPHN1 (0.022), LPHN2 (0.004), LPHN3 (0.044), CLTA (0.047), EEA1 (0.035), RAB5C (0.008). c Co-IP of FLOT1 and FLOT2 with LPHN1, LPHN2, and LPHN3. LDLR serves as marker for plasma membrane and clathrin-mediated endocytosis. d Immunostaining of HeLa cells for FLOT1 and FLOT2 in combination with LPHN1, LPHN2, and LPHN3 ( n = 3). Lower panels show a zoom-in for regions indicated in full-size images. Mander’s coefficients were determined to assess signal overlap between individual populations. Data are presented as mean values + SD ( n = 3, cells examined over one experiment). Profile plots indicate the degree of colocalization for individual vesicles. Scale bars = 5 µM; Cntrl. control, IN input, WA wash, EL eluate. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: goat anti LIMP2 (Cat# AF1966-SP,1:2000), mouse anti ACT2 (Cat# A5316, 1:4000), and rabbit anti LAMTOR1 (Cat # HPA002997, 1:1000) from Sigma-Aldrich; mouse anti CANX (Cat# 66903-1-AP, 1:20,000), mouse anti FLOT2 (Cat# 66881-1-Ig, 1:1500), mouse anti FZD9 (Cat# 67023-1-Ig, 1:1500), rabbit anti ATP6V1D (Cat# 14920-1-AP, 1:1000), rabbit anti CTSD (Cat# # 21327-1-AP, 1:1000), rabbit anti FLOT1 (Cat# 15571-1-AP, 1:2000), rabbit anti GNB4 (Cat# 11978-2-AP, 1:2000), and rabbit anti SDHA (Cat# 14865-1-AP, 1:800) from Proteintech; goat anti LIMP2 (Cat# AF1966-SP, 1:2000) from R&D system; mouse anti ATP6V1B2 (Cat# SC166045, 1:1000) from Santa Cruz; mouse anti FLOT1 (Cat# 610821, 1:200), mouse anti FLOT2 (Cat# 610383, 1:200), and mouse anti GM130 (Cat# 610822) from BD Biosciences; mouse anti GAPDH (Cat# 5174, 1:2500), rabbit anti EEA1 (Cat# 2411, 1:200), rabbit anti FLOT1 (Cat# 18634, 1:200), and rabbit anti RRAGA (Cat# 4357, 1:1000) from Cell signaling; rabbit anti LPHN2 (Cat# NBP2-58704, 1:100) and rabbit anti LPHN3 (Cat# NLS1138, 1:200) from Novus Biologicals; goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat# A-11029, 1:400), rabbit anti ATP6V1A1 (Cat# PA5-29191,1:2000), and rabbit anti LPHN1 (Cat # PA5-77475, 1:200) from Thermo Fisher Scientific; rabbit anti TUBA (Cat# 600-401-880, 1:2000) from Rockland; mouse anti LAMP2 (Cat# H4B4, 1:1000 for WB, 1:100 for IF) from Hybridoma Bank. rabbit anti DSSO (Self-made , 1:5000).

Techniques: Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Marker, Clinical Proteomics, Membrane, Immunostaining, Control

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 1. Expression of TRPC6 protein in human glioma tissue. A) Representative immunoblots of total lysates extracted from human glioma (six samples shown) and normal brain (six samples shown) tissues probed with anti–transient re ceptor potential canonical (TRPC)6 (106 kDa) or anti-TRPC3 (97 kDa) antibodies. GAPDH served as the protein loading control. B and C) Quantification of TRPC6 and TRPC3 protein levels in glioma (n = 33) and normal brain (n = 17) tissues from the immunoblots. Each dot repre sents the band density of TRPC6 and 3 proteins normalized to that of the corre sponding GAPDH in each sample. n = number of samples. Means and 95% confidence intervals are represented by open lines and error bars. **P < .001 vs normal, calculated using the two-sided Student t test. D) Representative immu nohistological staining samples of human glioma or normal brain tissues with anti- TRPC6 antibody. a–i) Three glioblastoma multiforme (GBM, grade 4) samples; j–l) anaplastic oligodendrocytoma (grade 3 oligodendrocytoma); m–o) grade 2 astro cytoma; and p–r) normal (para-tumor) tissues. Sections were stained by the 3,3′-diaminobenzidine (DAB) staining method to detect TRPC6 protein in the tissues and were counterstained with hematoxylin to stain the nucleus. Serial sections of the same samples were used for hematoxylin and eosin (HE) staining. Magnified ×40 panels represented the white rectangles in the ×10 panels. Scale bar = 100 µm. E) Bar graph representa tion of TRPC6 immunohistological scores in glioma of different grades. The per centage of sections with different scores (strong, moderate, and weak) in each grade is shown. Grade 4 glioma (GBM), n = 18; grade 3, n = 7; grades 1 and 2 combined, n = 8. n = number of samples. F) Expression of TRPC6 mRNA in glioma and normal tissues detected by in situ hybridization. Immunostaining by anti- TRPC6 antibody (DAB, ×40) on serial sections of the same samples used for immunohistological staining. Sense probe (×40) was used as a negative con trol, and the antisense probe (×40) was used to detect the TRPC6 mRNA. Scale bar = 100 µm.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Expressing, Western Blot, Control, Staining, In Situ Hybridization, Immunostaining

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 2. Functional activity of transient receptor potential canonical 6 (TRPC6) channels in glioma cells. Fura-2 AM imaging showing induc tion and attenuation of transient elevation of [Ca2+]i in U87 cells. R refers to the ratio of the emission at 500 nm induced by 340 and 380 nm exci tation, captured at 6-second intervals. DR/R was calculated as (R 2 Rbaseline)/Rbaseline, Rbaseline being the mean value of R during pretreatment. The recording time in minutes is shown. Squares represent the mean values. Error bars = 95% confidence intervals. Corresponding bar charts show the area of DR/R curve in the Fura-2 AM imaging, 3 minutes after addition of platelet-derived growth factor-BB (PDGF-BB) or 10 minutes after addition of cyclopiazonic acid (CPA). In all bar charts, the y-axis shows the absolute value of the area divided by 100. Means and upper 95% confidence intervals are shown. All P values were calculated using the two-sided Student t test. Ctrl = control; n = number of cells. A) Induction with 50 ng/mL PDGF-BB (Ctrl, solid square) and attenuation with 20 µM SKF96365 (SKF, open square). Medium was supplemented with calcium (Ca2+). B) Bar chart corresponds to (A). Ctrl, n = 48; SKF, n = 32. *P = .014 vs Ctrl. Data from three independent experiments are shown in (A) and (B). C) Induction with PDGF-BB in cells infected with adenovirus-expressing wild-type TRPC6 (WTC6, solid square) and at tenuation in cells expressing the dominant-negative form of TRPC6 (DNC6, open square). Duration of infection was 48 hours. Medium was supplemented with Ca2+. D) Bar chart corresponds to (C). Cells infected with adenovirus expressing green fluorescent protein (GFP) served as the control. GFP, n = 48; DNC6, n = 51; WTC6, n = 45. **P = .001, GFP vs DNC6, and **P < .001, WTC6 vs DNC6. Data from at least three indepen dent experiments are shown in (C) and (D). E) Induction with PDGF-BB in cells infected with GFP (solid square) or DNC6 (open square) in Ca2+- free medium (0 Ca2+) and Ca2+-containing medium (2 mM Ca2+). Smaller peaks represent the internal Ca2+ release induced by PDGF-BB, and larger peaks represent the extracellular Ca2+ entry on addition of 2 mM

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Functional Assay, Activity Assay, Imaging, Derivative Assay, Control, Infection, Expressing, Dominant Negative Mutation

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 3. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity or expression on glioma cell growth. A and B) Number of U251 and U87 cells infected with adenovirus expressing green fluorescent protein (GFP), DNC6, or WTC6. Cell numbers were normalized to those at day 0. Means and upper 95% confidence intervals from three independent experiments performed in dupli cate are shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. C) Representative images of U251 and U87 colonies infected with adeno virus expressing GFP, DNC6, or WTC6. Colonies stained with crystal violet after 14 days of seed ing are shown. D) Quantification of U251 and U87 colonies (shown in C) by measuring the optical density at 595 nm (OD595). OD595 values of DNC6 and WTC6 groups were normalized to that of GFP group. Means and upper 95% confi dence intervals are shown. Data are representa tive of four independent experiments, performed in duplicate. *P < .05, **P < .01, GFP or WTC6 vs DNC6. E) U251 colonies infected with lentivi rus-based scrambled short hairpin RNA (shRNA) (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). Colonies stained with crystal violet after 14 days of seeding were shown. Ctrl = control. F) Differential interference contrast (DIC) and fluo rescent (GFP) images of typical single colonies formed by U251 cells infected with the indi cated lentiviral constructs—scrambled shRNA (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). The duration of infection was 72 hours before seeding. The lentivirus was GFP tagged. Ctrl = control. Images were taken 14 days after seed ing. Scale bar = 100 µm. Merge panels show the merged DIC and GFP images. G) Quantification of the U251 colonies (shown in E) by measuring the OD595. Means and upper 95% confidence intervals are shown. Data are representative of four independent experiments performed in duplicate. *P = .032, **P < .001 vs Ctrl. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Expressing, Infection, Virus, Staining, shRNA, Control, Construct

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 4. Effect of inhibition of transient receptor potential canonical 6 (TRPC6) activity or expres sion on G2 phase arrest. A and B) Flow cytometric analysis of U251 DNA content after infection with adenovirus expressing green fluorescent protein (GFP), DNC6, and WTC6 for 72 hours or the indi cated lentivirus-based short hairpin RNA (shRNA) constructs (Ctrl, C6-1, and C6-2), 5–10 days after infection. DNA content is shown as 2n and 4n in the x-axis. 2n = cells in G0/G1 phase, and 4n = cells in the G2/M phase. Data are representative of four independent experiments. Ctrl = control, scrambled shRNA (Ctrl); C6-1 = shTRPC6-1 and C6-2 = shTRPC6-2. C) Quantitative real-time poly merase chain reaction and immunoblot analysis (inset) showing knockdown of CACNA1G in U251 cells by lentivirus-based shRNA constructs against the CACNA1G (CACNA1G RNA interference [RNAi]). Control (Ctrl) RNAi was nonsense shRNA. Data are representative of five independent exper iments. TUBA1A served as the protein loading control. Error bar = upper 95% confidence inter val. **P < .001 vs Ctrl RNAi. D) Flow cytometric analysis of U251 cell cycle after infection with the lentivirus-based shRNA construct (CACNA1G RNAi) (shown in C), 5–10 days after infection. Data shown are a representative of six independent experiments. E) Mitotic index of U251 cells after infection with adenovirus expressing GFP, DNC6, and WTC6 for 72 hours. Mitotic index (%) is the percentage of mitotic cells in total cells. Means and upper 95% confidence interval from three independent experiments are shown. **P = .005, GFP vs DNC6, and *P = .022, WTC6 vs DNC6. F) Effect of TRPC6 knockdown on the level of phos phorylated cyclin-dependent kinase 1 at tyrosine15 (P-CDK1 [Y15]). Immunoblot analysis of total U251 cell lysates using the indicated antibodies. Proteins were extracted approximately 10 days after infection. C6-1 and -2 lanes show approxi mately 40% and 60% decrease in TRPC6 level and increased in P-CDK1 (Y15) level by two- or four fold, respectively. TRPC3 blots show that TRPC6 knockdown did not have an effect on its homolog. TUBA1A served as the protein loading control. Data are representative of three independent ex periments. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Expressing, shRNA, Construct, Control, Western Blot, Knockdown

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 6. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on radiosensitization of glioma cells. A) Representative crystal violet staining of the colonies formed by U251 cells 11 days after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) 24 hours before irradiation. Ctrl = con trol. B and C) Surviving fraction of U251 cells after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) for 24 hours (B), or infected with adenovirus-based green fluorescent protein (GFP) or DNC6 at multiplicity of infec tion = 2, for 72 hours (C) before irradiation. Ctrl = control. Data were representative of five independent experiments, performed in dupli cate. Error bars = 95% confidence interval. **P = .006, SKF vs Ctrl at 2 Gy; **P = .005, SKF vs Ctrl at 4 Gy; **P < .001, GFP vs DNC6 at 2 Gy; **P = .002, GFP vs DNC6 at 4 Gy. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Staining, Irradiation, Infection, Control

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 7. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on the development of xenografted human glioma. A) The tumor volumes in green fluorescent protein (GFP)-, DNC6-, and WTC6-infected groups (n = 7 per group) were determined every 5 days for 30 days after implantation of U87 cells. Means and upper 95% confidence inter vals were shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. P values were calculated using the two-sided Student t test. B) Representative photographs of nude mice bearing xenografted tumors on day 30 of implantation. C) Scatter diagram of individual tumor volume of xeno grafted tumors on day 30 of implantation. Each symbol represents a single tumor. Error bars represent the interquartile range. *P = .014, GFP vs DNC6, **P < .001 WTC6 vs DNC6. P values were calculated using the two-sided Student t test. D) Survival curve was plotted by Kaplan– Meier analysis. Mice bearing glioma from DNC6-infected U87 cells survived longer than GFP- or WTC6-infected U87 cells. The number of mice (n) in each group is indicated. Each curve was compared using the two-sided log- rank test. P < .001, DNC6 vs GFP or WTC6. Number of mice at risk at each time point is also shown. E) Hematoxylin and eosin staining of representative coronal sections of the mouse brains 20 days after implantation. The tumors were derived from GFP-, DNC6-, and WTC6- infected U87 cells, respectively.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Staining, Derivative Assay