Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 μg of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean ± standard deviation of the amount of immunoblot signal normalized with the signal for β-actin, and also for the total amount of protein in each fraction (for more details see ). *** P < 0.001; ns , not statistically significant.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) Samples of a membrane fraction (70 μg of proteins) from MCF 10A (A), MCF7 (B), and MDA-MB-231 (C) cells were incubated on ice for 1 hour with either 10 mM Tris HCl pH 7.4 ( Control ), 1 M KCl in 10 mM Tris HCl pH 7.4 ( KCl ) or 0.2 M Na 2 CO 3 pH 11.3 ( Na 2 CO 3 ). After centrifugation, pelleted membranes ( P ) and extracted proteins in the supernatant ( S ) were processed by SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. Syn16 , Syntaxin 16. The position of molecular mass markers is indicated on the left. (D-F) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in pellets ( P ) and supernatants ( S ) as shown in A-C of membranes incubated in control conditions (D), in 1 M KCl (E), or in 0.2 M Na 2 CO 3 (F). Bar represents the mean ± standard deviation of the amount of immunoblot signal. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Incubation, Control, Centrifugation, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: NRK (A and B), MCF 10A (C and D), MDA-MB-231 (E and F), and MCF7 (G and H) cells were left untreated ( Control ) or treated with 5 μg/ml BFA for 60 min ( BFA ). Cells were fixed, permeabilized, and immunolabeled with rabbit polyclonal antibody to GOLPH3, mouse monoclonal antibody to GM130, and either sheep antibody to TGN38 (A and B) or sheep antibody to TGN46 (C to H). Secondary antibodies were Alexa-594-conjugated donkey anti-rabbit IgG (red channel), Alexa-488-conjugated donkey anti-mouse IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Stained cells were examined by fluorescence microscopy. Merging red, green, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Insets show 1.7x magnifications. Bar, 10 μm.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Control, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C and examined by fluorescence microscopy. The time after initiation of imaging is shown in the bottom right corner of each panel in minutes:seconds. Images are representative of 15–20 videos of up to 200 seconds of recording. In B, filled arrows indicate a vesicular structure moving from the Golgi to the periphery of the cell. In C, filled arrows indicate a vesicular structure moving from the periphery of the cell to the Golgi area, and filled arrowheads indicate a tubular structure elongating from the Golgi. Empty arrows and empty arrowheads indicate the initial position of mobile structures. Bars, 5 μm. (D-F) The number of tubule-vesicular structures moving centrifugally (D), the number of tubular structures elongating from the Golgi (E), or the number of tubule-vesicular structures moving centripetally (F), were quantified from videos corresponding to 180 seconds of imaging. Bar represents the mean + standard deviation of the observed profiles (n = 15). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Imaging, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C. The area indicated by a white dotted-line rectangle in each set of images was bleached with a 488-nm laser set to 100% power. The fluorescence recovery after photobleaching (FRAP) was tracked by laser confocal microscopy with the 488-nm laser set to 2% power. Images were acquired before bleaching ( Pre-bleaching ), immediately after bleaching ( Bleaching ), and during the recovery of the fluorescence ( Recovery ) at approximately every 0.4-sec. Images of a representative experiment performed on each cell line are shown in each set of panels. Two images of the recovery of fluorescence are depicted with the time indicated in parenthesis in seconds. Bar, 10 μm. (D) Plot of the FRAP analysis of GFP-GOLPH3 in MCF 10A (black circles; n = 10), MDA-MB-231 (white circles; n = 10), and MCF7 (white squares; n = 10) cells. P , pre-bleaching; B ; bleaching. For simplicity, error bars are not depicted. *** P < 0.001; ns , not statistically significant. The halftime ( t 1/2 ) of maximal fluorescence recovery is indicated on the right in seconds (s).

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: Samples (30 μg of proteins) of rat liver cytosol ( Cyt ), rat liver Golgi membranes, and of cytosolic ( Cyt ) and membrane ( Memb ) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase ( CIAP ) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point ( pI ) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 ( GOLPH3 ) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. LysoPtdA , lysophosphatidic acid; LysoPtdCho , lysophosphatidylcholine; PtdIns , phosphatidylinositol; PtdIns(3)P , phosphatidylinositol 3-phosphate; PtdIns(4)P , phosphatidylinositol 4-phosphate; PtdIns(5)P , phosphatidylinositol 5-phosphate; PtdEth , phosphatidylethanolamine; PtdCho , phosphatidylcholine; S1P , sphingosine 1-phosphate; PtdIns(3 , 4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3 , 5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4 , 5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3 , 4 , 5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdA , phosphatidic acid; PtdSer , phosphatidylserine; Blank , no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Incubation, Recombinant, Western Blot

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: PRSS55 deletion leads to impaired mitochondrial function in mouse testes and sperm. A in vitro sperm (marked by white cycle) migration in 10% MC4000 solution (n = 4). Capacitated sperm that migrate over 1 cm from the bottom of the capillary slide were counted under a microscope, 200 × magnification. Sperm were highlighted in white circle. B ATP levels in Prss55 −/− testes and sperm is shown as mean ± SE (n = 3). The liver in which PRSS55 is not expressed was used as unrelated control. C NAD+ , NADH levels and NAD+ /NADH ratio in wt and Prss55 −/− testicular cells were determined and presented as mean ± SE (n = 3). D The mitochondrial membrane potential (MMP) of spermatozoa from wt and Prss55 −/− mice was determined using JC-1 probes (n = 4). JC-1 polymer/JC-1 monomer fluorescence ratios were calculated and shown as mean ± SE (n = 4). (*, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, n.s., no significant difference.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: In Vitro, Migration, Microscopy, Control, Membrane, Polymer, Fluorescence

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: PRSS55 is localized in mitochondria and facilitates mitochondrial energy metabolism in cultured cell lines. A Immunofluorescence staining shows co-localization of PRSS55 and mitochondria in NIH-3T3 cells (PRSS55, green; MitoTracker, red; DAPI, blue). pixel profile analysis revealed colocalization of green and red signals. Empty vector plasmid (Ctrl) was utilized as a control. B Immunofluorescence staining shows co-localization of PRSS55 and mitochondria in matured sperm (PRSS55, green; COXIV, red; DAPI, blue). Mouse and rabbit IgG staining (Ctrl) was used as a negative control. C Mitochondrial fraction from Prss55 transfected HEK293T cells was subject to immunoblotting analysis. Tubulin was used as a marker for cytoplasm, histone H2A for nuclear, and COXIV for mitochondria (Total, total cell lysates; Mito, isolated mitochondria). D ATP levels in HEK293T cells transfected with PRSS55-Myc expression vector were detected and shown as mean ± SE (n = 4). E NAD+ , NADH levels and NAD+ /NADH ratio in PRSS55-overexpressed HEK293T cells are shown as mean ± SE (n = 3). (*, P ≤ 0.05, **, P ≤ 0.01.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Cell Culture, Immunofluorescence, Staining, Plasmid Preparation, Control, Negative Control, Transfection, Western Blot, Marker, Isolation, Expressing

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Data-Independent Acquisition (DIA)-based quantitative proteomic analysis of wt and Prss55 −/− testicular layer 3 (TL3) cells and sperm. A Immunofluorescence staining of PRSS55 at the luminal side of the seminiferous tubules (PRSS55: red; PNA: green; DAPI: blue). B Work-flow displays the strategy of PRSS55-enrichment cells for both wild-type (wt) and Prss55 −/− mice. C Flow cytometry analysis of DNA ploid types of wt and Prss55 −/− mice T, L1, L2, and L3 testicular cells. D Immunoblotting analysis of PRSS55 levels in wild-type T, L1, L2, and L3 testicular cells. β-Actin was used as a loading control. E, F Volcano plot of TL3 ( E ) and sperm ( F ) proteomics showing significant (fold-change > 1.5, adjusted P ≤ 0.05, blue = down, red = up) differentially expressed proteins (DEPs). G , H Heatmap representation of TL3 DEPs ( G ) and sperm DEPs ( H )

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Data-independent acquisition, Immunofluorescence, Staining, Flow Cytometry, Western Blot, Control

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: DEPs between wt and Prss55 −/− TL3 cells and sperm are enriched in metabolic pathways. Top 5 significantly enriched GO and KEGG terms were selected to show potential functions of DEPs in TL3 cells A and sperm B and presented as bubble plots. Bubble size represents the number of DEPs. Bubble color represents the adjusted P. The x-axis shows the Z-score of proteins classified into each functional annotation. C Venn diagram exhibits the DEPs common in TL3 cells (red) and sperm (green). D The enriched canonical pathways identified in IPA by 153 DEPs common in testicular TL3 cells and sperm are indicated on the y-axis. On the x-axis, the enrichment score (- log 10 (P)) for each pathway is indicated by the bars. Color of each bar reflects its activation z-score upon IPA algorithm

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Functional Assay, Activation Assay

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Branched chain amino acids were accumulated in Prss55 −/− testes and sperm. A, B Heatmap representation ( A ) and orthogonal partial least squares discrimination analysis (OPLS-DA) score plot B of untargeted testicular metabolomic profiles between wt and Prss55 − / − . (wt, n = 6; Prss55 − / − , n = 8) C Volcano plot of untargeted testicular metabolomics showing significant abundant metabolites (fold-change > 1.2, FDR ≤ 0.1, green = down, red = up). D The enrichment of the untargeted Prss55 −/− differential metabolites in the KEGG pathways sorted by -log 10 (P). Color of each bar reflects its enrichment ratio. E, F Relative contents of BCAAs in testes ( E , n = 4) and sperm ( F , n = 3) detected by targeted metabolomic analysis. G, H Determination of BCAAs in testes ( G , wt = 3, Prss55 − /− = 4) and sperm ( H , wt = 3, Prss55 − /− = 5) by ELISA. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Interaction of PRSS55 with BCKDK and BCKDHA. A The V5-PRSS55-Flag fusion protein was immunoprecipitated from the testis lysates of 3 Prss55 KI/KI mice with an anti-V5 mAb magnetic beads and detected by anti-V5 antibody. Wt mice were used as negative controls. B The volcano plot shows 56 proteins identified with LC/MS in the V5-PRSS55 precipitates. BCKDK and DBT were pointed. C The PRSS55-EGFP fusion protein was immunoprecipitated from the HEK293T cell lysates. Endogenous BCKDK and DBT were probed with their specific antibodies, respectively. GAPDH was shown as a loading control. D, E Co-IP of PRSS55 ( D ) or BCKDK ( E ) tagged as indicated with anti-tag antibodies shows the existence of BCKDK or PRSS55 in the precipitates from the cell lysates of co-transfected HEK293T cells by immunoblot. F Immunoblotting analysis of endogenous BCKDHA protein levels upon overexpression of PRSS55 and BCKDK (left), and quantitative analysis of relative intensities of BCKDHA protein levels (right). G The PRSS55-EGFP fusion protein was immunoprecipitated from the HEK293T cell lysates. Endogenous BCKDHA was probed with specific antibody. H, I Co-IP of PRSS55 ( H ) or BCKDHA ( I ) tagged as indicated with anti-tag antibodies shows the existence of BCKDHA or PRSS55 in the precipitates from the cell lysates of co-transfected HEK293T cells by immunoblot. J Immunoblotting analysis of isolated mitochondria between wt and Prss55 −/− testes (left), and quantitative analysis of relative intensities of BCKDK, p-BCKDHA, and BCKDHA protein levels (right, n = 3). Bubble size and color represent the relative protein level. Tubulin was used as a marker for cytoplasm, Histone H2A for nuclei, and COXIV for mitochondria. (* P ≤ 0.05, ** P ≤ 0.01)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Immunoprecipitation, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Control, Co-Immunoprecipitation Assay, Transfection, Western Blot, Over Expression, Isolation, Marker

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Schematic illustration of PRSS55 participating in BCAA metabolism and energy homeostasis

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques:

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Representative intracellular calcium signal of AD293 cells transfected with hTRPC5 (1-764) evoked by 256 nM EA ( A ) or 14 mM extracellular calcium ( B, C ), and inhibited by different concentrations of HC-070 ( B ) or clemizole (CMZ) ( C ), measured by FLIPR calcium assay. Arrow 1 denotes the time point for application of buffer alone or buffer with inhibitors; arrow 2 denotes the time point for application of the activators. ( D ) Fluorescence size exclusion chromatography profile of full-length hTRPC5 and hTRPC5 (1-764) . ( E ) Representative size exclusion chromatography of hTRPC5 (1-764) purified in glycol-diosgenin (GDN) micelles. ( F ) SDS-PAGE gel of hTRPC5 (1-764) eluted from size exclusion chromatography. The position of TRPC5 is labeled. Fractions that were pooled for cryo-EM analysis are denoted by asterisks.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Transfection, Calcium Assay, Fluorescence, Size-exclusion Chromatography, Purification, SDS Page, Labeling, Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A, B ) Inhibitory effect of clemizole (CMZ) ( A ) and HC-070 ( B ) on the extracellular calcium-induced intracellular calcium increase of cells with wild-type hTRPC5 over-expression measured by FLIPR calcium assay (data are shown as means ± standard error, n = 3 independent experiments). ( C, D ) Cryo-EM density maps of CMZ-bound hTRPC5 shown in side view ( C ) and top view ( D ). Subunits A, B, C, and D are colored in purple, light blue, orange, and gray, respectively. Lipids are colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD, transmembrane domain; ICD, intracellular cytosolic domain. ( E ) Ribbon diagram of a single subunit with secondary structure elements represented in different colors. ARD, ankyrin repeats domain; LHD, linker-helix domain; CH1, C-terminal helix 1; CH2, C-terminal helix 2. Figure 1—source data 1. Inhibition of WT hTRPC5 by clemizole or by HC-070.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Over Expression, Calcium Assay, Cryo-EM Sample Prep, Inhibition

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of apo hTRPC5. ( C ) Flowchart for cryo-EM data processing of apo hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.9 Å) and corrected (purple line, 3 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of clemizole (CMZ)-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of apo hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 4.1 σ, gray mesh) with atomic models superimposed.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of CMZ-bound hTRPC5. ( C ) Flowchart for cryo-EM data processing of CMZ-bound hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.5 Å) and corrected (purple line, 2.7 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of CMZ-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of HC-070-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3 σ, gray mesh) with atomic model superimposed. ( E–F ) Cryo-EM density map of HC-070 shown in different views contoured at 3 and 7 σ, respectively. ( G–J ) Cryo-EM densities of HC-070-binding pocket in ( G ) HC-070-bound, ( H ) clemizole (CMZ)-bound, ( I ) apo, and ( J ) Pico145-bound hTRPC5 maps, contoured at 3.8 σ.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of CMZ-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3.8 σ, gray mesh) with atomic models superimposed. ( E ) Cryo-EM density of CMZ shown in different views (contoured at 3.7 σ). ( F ) Cryo-EM densities of CMZ-binding pocket in CMZ-bound hTRPC5 map (contoured at 4.6 σ). ( G, H ) Cryo-EM densities in HC-070-bound hTRPC5 and apo hTRPC5 maps corresponding to the CMZ-binding pocket contoured at the same σ as in ( F ).

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Comparison of experimental density map of diacylglycerol (DAG) (gray) (contoured at 3.2 σ) and simulated density map of lysophospholipid (yellow). Please note, the density for electron-dense phosphate head group is absent in the cryo-EM maps obtained experimentally. ( B–D ) Close-up views of lipid 1 ( B ), lipid 2 ( C ), and cholesteryl hemisuccinate (CHS) ( D ) binding sites. Subunits A and B are colored in purple and light blue, respectively. Residues that interact with lipids are shown as sticks. Insets show cryo-EM densities of these lipids, contoured at 2.3, 3.1, and 3.1 σ, respectively.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Bacterial strains and their sensitivities to phage EFLK1, vancomycin and combined treatment.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Isolation

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Representative depiction of vancomycin minimal inhibitory concentration (MIC) for E. faecalis V583 (VRE) by serial dilutions. The results are mean ± SD based on three independent biological replicates.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Concentration Assay

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Treatment of planktonic bacteria with 1.2 × 10 8 plaque forming units (PFU)/well phage EFLK1 (anti- E. faecalis phage) combined with 0.015 mg/mL vancomycin reduced VRE growth. Total growth was inhibited in the combination of 0.015 mg vancomycin/mL and 1.2 × 10 8 PFU/well phage EFLK1 (red). VRE control depicts untreated bacteria showing maximal growth (blue). A longer lag phase was observed after treatment with 0.015 mg/mL vancomycin (green) or phage EFLK1 (1.2 × 10 8 PFU/well) alone (purple). The combined treatment reduced the bacterial maximum optical density (OD) with better effect than that of the phages or vancomycin alone. The results are mean ± SD based on 3 independent biological replicates.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Bacteria, Control

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Viable counts of planktonic vancomycin-resistant E. faecalis following a combined treatment of phage EFLK1 and vancomycin. The colony forming units (CFU)/mL of VRE treated with 0.015 mg/mL vancomycin combined with phage EFLK1 1.2 × 10 8 PFU/well is presented. Bacteria were below the limit of detection after treating the cells by combining phage EFLK1 and vancomycin. Bacteria treated only with vancomycin showed survival scores like those of the untreated bacteria; cells treated with phage EFLK1 showed medium survival rates. Combining vancomycin and phage EFLK1 caused seven log reductions in CFU/mL. Light gray = vancomycin-treated bacteria, dark gray = phage EFLK1 treatment, black = phage EFLK1 + vancomycin. Statistically significant ( p < 0.01) compared to the untreated control. The results are mean ± SD based on three independent biological replicates.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Bacteria, Control

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: E. faecalis biofilm biomass following treatment with phage EFLK1 (1.2 × 10 8 PFU/well) and vancomycin (0.015 mg/mL). Treatment with combinations of phage EFLK1 and vancomycin significantly decreased bacterial biomass (87% reduction) as evaluated by crystal violet staining. The results are presented as percentages, normalized to the biofilm biomass controls. Light gray = vancomycin-treated bacteria, dark gray = phage EFLK1 treatment served as the control, black = phage EFLK1 + vancomycin treatment. Statistically significant ( p < 0.05) compared to the untreated control. The results are mean ± SD based on three independent biological replicates.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Staining, Bacteria, Control

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Combinations of phage EFLK1 and vancomycin target VRE. Scanning electron microscopy images (MAG: A – C : 20,000, D : 2000): ( A ) E. faecalis 72-h biofilm. ( B ) Biofilm exposed to vancomycin alone, showing no effect. ( C ) E. faecalis biofilm exposed to phage EFLK1 showing bacterial lysis, leaving mainly the extracellular matrix. ( D ) E. faecalis biofilm exposed to vancomycin and phage EFLK1 showing massive bacterial lysis, degradation and biofilm deformation, leaving almost no trace of biofilm.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Electron Microscopy, Lysis

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Alexa Fluor 647 wheat germ agglutinin (WGA) stains EFLK1 survivors . Confocal microscopy images (MAG: 60): ( A – B ) vancomycin resistant E. faecalis V583 show low fluorescence level, indicating no conjugation to N -acetylglucosamine. ( C – D ) EFLK1 survivors show high fluorescence levels followed the conjugation of Alexa Fluor 647 WGA to N -acetylglucosamine.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Confocal Microscopy, Fluorescence, Conjugation Assay

Journal: Viruses

Article Title: Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin

doi: 10.3390/v11100954

Figure Lengend Snippet: Fluorescence activated cell sorting (FACS) analysis: higher fluorescence levels are obtained followed phage treatment. Red = EFLK1 survivors, black = untreated control of E. faecalis V583. Statistically significant ( p < 0.01) compared to the untreated control. The results are mean ± SD based on six independent biological replicates.

Article Snippet: Vancomycin-resistant E. faecalis V583 (ATCC 700802, GenBank AE016830.1), clinically isolated from human blood [ , ], served as the test organism.

Techniques: Fluorescence, FACS, Control

Journal: Frontiers in Physiology

Article Title: A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics

doi: 10.3389/fphys.2022.772313

Figure Lengend Snippet: The first 20 nt of mito-ncR-805 is evolutionarily conserved in the mammals. (A) Alignment of mmu-mito-ncR-805 (accession number MI0005204 ) and hsa-mito-RNA-L-DL-1, which maps to the D-loop of the control region . Multiz alignment of hsa-mito-RNA-L-DL-1 with sequences from 100 vertebrates. The evolutionarily conserved region is shown in red. (B–H) Subcellular distribution of transfected mmu-CR805 (or control) in MLE12 cells. (B,C) Total, cytoplasmic (CP), nuclear pellet (NP), and nuclear extract (NE) fractions were prepared. NPs were treated with RNase I. (B) Purity of the fractions was assessed by the presence of the cytosolic protein, LDHA, mitochondrial protein SDHA, endoplasmic reticulum protein PDI, nuclear chromatin protein H3, and nucleoplasm protein nucleoplasmin via Western blot analysis. Ponceau S staining is shown as a loading control. (C) Small RNAs were extracted from generated fractions and analyzed for the expression levels of transcripts containing the conserved region of mito-ncR-805 by RT-qPCR. (D) MLE12 cells transfected with control or mmu-CR805 were grown on slides, fixed, and hybridized with a mito-ncR-805-specific probe. Images were acquired at 63 times magnification on a Leica DM IRE2 Leica SP8 laser confocal microscope. (E,F) Enlarged areas from Panel (D) are shown on the left. Graphs on the right show relative red channel fluorescence intensity through the line-scanned regions. (E) Line-scans drawn through cytoplasm (1 and 3) and structures positive for mmu-mito-ncR-805 and/or mmu-CR805 (2 and 4). (F) Line scans drawn through and around the perimeter of nuclei of cells transfected with non-targeting RNA (top image) and mmu-CR805 (bottom image). (G,H) MLE12 cells transfected with mmu-CR805 or non-targeting RNA oligos were lysed and analyzed (total fraction). Cytosolic fractions (Cyt) were obtained by separating the crude mitochondria, which were further purified using Percoll gradient and treated with RNase I to remove non-mitochondrial RNAs. The RNAs were isolated from the obtained fractions. (G) Purity of the fractions was evaluated by comparing the expression levels of mtDNA-encoded mRNA of Rnr2, nucleus-encoded and localized Sno55-RNA, and cytosolic GAPDH mRNA. Graph shows the difference between C T levels relative to the total lysates and with identical RNA inputs. (H) Expression levels of transcripts containing the mmu-CR805 sequence in different fractions as evaluated by RT-qPCR. Graph shows the difference in C T values between the control and mmu-CR805-transfected cells. (I) The MLE12 cells were transfected with FAM-labeled mmu-CR805, fixed, and stained for Tom20. Images were acquired at a magnification of 100 times and zoom of 3.37 times on a Leica TCS SP8 microscope with accelerated deconvolution (entire images are provided in ). Line scans were drawn through Tom20-labeled structures. Graphs show relative red and green channel fluorescence intensity through the line-scanned regions.

Article Snippet: The following program was used for random primers cDNA synthesis: 25°C for 10 min, followed by 37°C for 2 h, and 85°C for 5 min. TaqMan ® miRNA Looped Primer cDNA synthesis was carried out using the same kit, using the following program: 16°C for 30 min, followed by 42°C for 30 min, and 85°C for 5 min. Primers were from TaqMan assays (Life Technologies and Thermo Fisher Scientific): for the transcripts containing CR of mito-ncR-805 1–20 nt , cat. #4427975, the assay ID 002045 was used; for sno55 to normalize the expression levels of small RNAs, cat. #4427975, the assay ID 001228 was used; for TRIB3, Mm00454879-m1 was used; for RNR2, Mm04260181_s1 was used; and for GAPDH, Mm99999915_g1 (cytosolic marker and as a normalization of TRIB3 data) was used.

Techniques: Control, Transfection, Western Blot, Staining, Generated, Expressing, Quantitative RT-PCR, Microscopy, Fluorescence, Purification, Isolation, Sequencing, Labeling