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: Molecular imaging

Article Title: Tryptophan PET Imaging of the Kynurenine Pathway in Patient-Derived Xenograft Models of Glioblastoma

doi: 10.1177/1536012116644881

Figure Lengend Snippet: Immunostaining for kynurenine pathway’s (KP) elements in patient and xenograft tumor tissues. Immunohistochemical staining for secondary antibody only control; the rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, and tryptophan 2,3-dioxygenase (TDO2); downstream enzymes kynureninase (KYNU) and kynurenine 3-monooxygenase (KMO); and l -type amino acid transporter 1 (LAT1), the membrane transporter that mediates the uptake of TRP and AMT. Left column: 13-058 patient tumor tissue and right column: mouse xenograft tumor tissue. Staining patterns are parallel between the 2 tumors with stronger signals found from IDO2 and TDO2 and the weakest signals found from IDO1 and KMO. The length of the scale bar is 100 µm. Insets: 4× greater magnification than the larger image.

Article Snippet: Primary antibodies used targeted the following proteins: IDO1 (0.1 mg/mL; cat.#NBP1-87702; Novus Biologicals, Littleton, Colorado), IDO2 (0.5 mg/mL; cat.#OAAB08672; Aviva Systems Biology, San Diego, California), TDO2 (0.3 mg/mL; cat# NBP2-13424; Novus Biologicals), KYNU (0.05 mg/mL; cat.#NBP1-56545; Novus Biologicals), KMO (concentration not provided by manufacturer; cat.#10698-1-AP; Proteintech, Chicago, Illinois), AHR (0.73 mg/mL; cat.#ab153744; Abcam, Cambridge, Massachusetts), and l -type amino acid transporter 1 (LAT1; 0.5 mg/mL; cat.#ab85226; Abcam).

Techniques: Immunostaining, Immunohistochemical staining, Staining, Control, Membrane

Journal: Molecular imaging

Article Title: Tryptophan PET Imaging of the Kynurenine Pathway in Patient-Derived Xenograft Models of Glioblastoma

doi: 10.1177/1536012116644881

Figure Lengend Snippet: Immunostaining for kynurenine pathway (KP) elements in patient-derived xenograft (PDX) tumor tissue. Immunohistochemical staining of left column: 13-062 PDX tumor tissue; middle column: 14-041 PDX tumor tissue; and right column: 14-066 PDX tumor tissue for secondary antibody only control; the rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, and tryptophan 2,3-dioxygenase (TDO2); downstream enzymes kynureninase (KYNU) and kynurenine 3-monooxygenase (KMO); and l -type amino acid transporter 1 (LAT1). Staining of KP enzymes is weakest in the 14-066 PDX, although a moderate signal for LAT1 is observed. The length of the scale bar is 100 µm. Insets: 4× greater magnification than the larger image.

Article Snippet: Primary antibodies used targeted the following proteins: IDO1 (0.1 mg/mL; cat.#NBP1-87702; Novus Biologicals, Littleton, Colorado), IDO2 (0.5 mg/mL; cat.#OAAB08672; Aviva Systems Biology, San Diego, California), TDO2 (0.3 mg/mL; cat# NBP2-13424; Novus Biologicals), KYNU (0.05 mg/mL; cat.#NBP1-56545; Novus Biologicals), KMO (concentration not provided by manufacturer; cat.#10698-1-AP; Proteintech, Chicago, Illinois), AHR (0.73 mg/mL; cat.#ab153744; Abcam, Cambridge, Massachusetts), and l -type amino acid transporter 1 (LAT1; 0.5 mg/mL; cat.#ab85226; Abcam).

Techniques: Immunostaining, Derivative Assay, Immunohistochemical staining, Staining, Control

Journal: Cells

Article Title: SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

doi: 10.3390/cells12060835

Figure Lengend Snippet: Figure 1. SCD5 is downregulated in VHL-deficient ccRCC tumors and cell lines. (a) Expression of SCD5 mRNA in TCGA ccRCC (n = 533) and normal kidney samples (n = 72). **** p < 0.0001. The analysis was performed using the UALCAN/TCGA platform. (b) SCD5 protein expression is reduced in ccRCC (n = 110) compared to normal kidney samples (n = 84) (CPTAC dataset). **** p < 0.0001. (c) Representative result of SCD5 expression using renal cancer (https://www.proteinatlas.org/ ENSG00000145284-SCD5/pathology/renal+cancer#img, accessed on 9 June 2021) and normal kidney samples (https://www.proteinatlas.org/ENSG00000145284-SCD5/tissue/kidney#img, accessed on 9 June 2021) from the Human Protein Atlas. (d) SCD5 protein levels are reduced in RCC4 and 786-O ccRCC cells compared to RPTEC. Cell lysates of RCC4, 786-O, and RPTEC cells were analyzed by Western blot using anti-SCD5 antibody. Protein levels of SCD5 were quantified using LabImage 1D software and normalized to β-actin protein levels. The right panel shows quantification of relative SCD5 protein levels from three independent experiments. Data are presented as mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2. (e) low SCD5 expression (n = 134) correlates with shorter overall survival (high SCD5 expression, n = 397). The analysis was performed using the UALCAN platform.

Article Snippet: Antibodies used in this study included antibody to SCD5 (Aviva systems biology, San Diego, CA, USA, SCD5 antibody-C-terminal region, ARP65045_P050, 1:1000 dilution), antibody to VHL (Cell Signaling, Leiden, The Netherlands, 1:1000 dilution), antibody to β-actin (Sigma, Darmstadt, Germany), antibody to γ-tubulin, and antibody to HIF2α from Abcam, Cambridge, UK.

Techniques: Expressing, Western Blot, Software

Journal: Cells

Article Title: SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

doi: 10.3390/cells12060835

Figure Lengend Snippet: Figure 2. SCD5 expression in ccRCC cells is VHL dependent. (a) mRNA levels of VHL in HeLa cells infected with inducible control-shRNA or VHL-specific shRNA assayed by RT-qPCR; HSPCB was used as the reference gene. n = four. Data are presented as mean ± SEM. *** p < 0.001 (t-test). (b) Representative blot showing VHL expression in HeLa cells infected with lenti-control shRNA or lenti-VHL shRNA. Full-length blots are presented in Supplementary Figure S2. (c) SCD5 mRNA levels are affected by VHL. qPCR analysis of HeLa cells expressing shRNA against control or VHL. Two different primer sets were used for SCD5 mRNA analysis; HSPCB was used as the reference gene. n = four for each experiment. Data are mean ± SEM. *** p < 0.001 (t-test). (d) Analysis of SCD5 mRNA levels in RCC4 cells and RCC4 cells expressing VHL. Cell lysates were prepared from VHL-deficient RCC4 cells and cell lines transduced with VHL (RCC4 + VHL); GAPDH was used as the reference. n = four for each experiment. Data are mean ± SEM. *** p < 0.001 (t-test). (e) Western blot demonstrating SCD5 expression in RCC4 cells (VHL deficient) and RCC4 cells transduced with VHL. Full-length blots are presented in Supplementary Figure S2. Right panel shows quantification of relative SCD5 levels from three independent experiments. * p < 0.05 (t-test).

Article Snippet: Antibodies used in this study included antibody to SCD5 (Aviva systems biology, San Diego, CA, USA, SCD5 antibody-C-terminal region, ARP65045_P050, 1:1000 dilution), antibody to VHL (Cell Signaling, Leiden, The Netherlands, 1:1000 dilution), antibody to β-actin (Sigma, Darmstadt, Germany), antibody to γ-tubulin, and antibody to HIF2α from Abcam, Cambridge, UK.

Techniques: Expressing, Infection, Control, shRNA, Quantitative RT-PCR, Transduction, Western Blot

Journal: Cells

Article Title: SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

doi: 10.3390/cells12060835

Figure Lengend Snippet: Figure 3. fat-7/SCD5 is downregulated in C. elegans vhl-1 mutants. (a) Volcano plot showing differentially expressed genes between vhl-1 mutants and wild-type. (b) fat-5 and fat-7 expression is downregulated in vhl-1 mutants. fat-5 and fat-7 mRNA expression in wild-type animals and vhl- 1(ok161) mutants was analyzed by RT-qPCR. Data are mean ± SEM of four independent experiments. t- test. *** p < 0.001. (c) vhl-1 mutants show reduced FAT-7::GFP expression. Representative fluorescence images of otherwise wild-type and vhl-1(ok161) animals expressing a fat-7::GFP translational reporter (nIs590). Scale bar indicates 100 µm. Right panel shows quantification of FAT-7::GFP in wild-type and vhl-1 mutants. Three independent experiments were performed. n > 50 for each condition. Mean ± SEM. **** p < 0.0001 (t-test). (d) Effect of hypoxia on FAT-7::GFP expression. Wild-type animals expressing FAT-7::GFP were exposed to low oxygen (0.5% in a hypoxia chamber) and compared to controls grown under standard conditions (21% oxygen). Quantification of FAT-7::GFP expression. n > 50 for each condition. Mean ± SEM. **** p < 0.0001 (t-test). (e) VHL-1 and HIF-1 antagonistically regulate fat-7 expression. Fluorescence micrographs of wild-type, hif-1(ia04), vhl-1(ok161), and hif- 1;vhl-1 mutants showing expression of FAT-7::GFP. Scale bar indicates 100 µm. Left panel shows quantification of FAT-7::GFP fluorescence intensity. Three independent experiments, n > 50 for each genotype. Mean ± SEM. **** p < 0.0001 (ANOVA). (f) Inactivation of hif-1 abrogates downregulation of fat-7 expression in vhl-1 mutants. RT-qPCR results showing fat-7 mRNA levels in wild-type, vhl-1(ok161), hif-1(i104), and hif-1;vhl-1 mutants. * p < 0.05, ** p < 0.01, **** p < 0.0001 (n = four for each genotype).

Article Snippet: Antibodies used in this study included antibody to SCD5 (Aviva systems biology, San Diego, CA, USA, SCD5 antibody-C-terminal region, ARP65045_P050, 1:1000 dilution), antibody to VHL (Cell Signaling, Leiden, The Netherlands, 1:1000 dilution), antibody to β-actin (Sigma, Darmstadt, Germany), antibody to γ-tubulin, and antibody to HIF2α from Abcam, Cambridge, UK.

Techniques: Expressing, Quantitative RT-PCR, Fluorescence

Journal: Cells

Article Title: SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

doi: 10.3390/cells12060835

Figure Lengend Snippet: Figure 4. SCD5 expression depends on HIF2α. (a) 786-O cells were incubated with the HIF2α inhibitor PT2385 (1 µmolar) for 72 h. RNA expression of SCD5, VEGF, and PAI-1 were assessed by RT-qPCR relative to HSPCB. n = three for each experiment. Data are represented as mean ± SEM. * p < 0.05 compared to untreated cells (t-test). (b) SCD5 protein levels are restored after treatment with the HIF2α inhibitor PT2385. The 786-O cells were incubated with the HIF2α inhibitor PT2385 (1 µmolar) or DMSO (control) for 72 h, and cell lysates were assessed by immunoblotting with anti- SCD5 and anti-HIF2α antibody. Quantification of three independent experiments. Data represent mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2. (c) Immunoblot of RCC4 cells that were treated with PT2385 (1 µM) or DMSO control. Quantification of relative SCD5 levels from three independent experiments. Data are presented as mean ± SEM. * p < 0.05 (t-test). Full-length blots are presented in Supplementary Figure S2. (d) SCD5 protein levels after hypoxia treatment of RPTEC cells. Quantification from three independent experiments. Data represent mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2.

Article Snippet: Antibodies used in this study included antibody to SCD5 (Aviva systems biology, San Diego, CA, USA, SCD5 antibody-C-terminal region, ARP65045_P050, 1:1000 dilution), antibody to VHL (Cell Signaling, Leiden, The Netherlands, 1:1000 dilution), antibody to β-actin (Sigma, Darmstadt, Germany), antibody to γ-tubulin, and antibody to HIF2α from Abcam, Cambridge, UK.

Techniques: Expressing, Incubation, RNA Expression, Quantitative RT-PCR, Control, Western Blot

Journal: Cells

Article Title: SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC.

doi: 10.3390/cells12060835

Figure Lengend Snippet: Figure 5. SCD5 affects cell proliferation. (a) RT-qPCR analysis of SCD5 and SCD in RCC4 cells infected with inducible control or SCD5-specific shRNA. Two different primer sets were used for SCD5 mRNA analysis. SCD5 shRNA does specifically affect SCD5, as SCD was not down regulated. HSPCB was used as the reference gene, n = three. Data are presented as mean ± SEM. * < 0.05 (t-test). (b) Western blot of shRNA-mediated knockdown of SCD5 in RCC4 cells. (c) mRNA levels of SCD5 in 786-O cells infected with control-shRNA or SCD5-specific shRNA, assayed by qPCR; β-actin was used as the reference gene. n = three. Data are presented as mean ± SEM. *** p < 0.001 (t-test). (d) Western blot of shRNA-mediated knockdown of SCD5 in 786-O. (e) Knockdown of SCD5 induces cell proliferation. Cell viability of RCC4 cells upon shRNA SCD5 knockdown was assayed compared to control. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 (t-test). (f) Cell viability of 786-O cells upon shRNA SCD5 knockdown was assayed compared to control. Data are presented as mean ± SEM of three independent experiments. ** p < 0.01 (t-test). (g) Overexpression of SCD5 in 293T and RCC4 cells reduces cell proliferation. Cell viability was assayed in 293T and RCC4 cells transfected with Flag-tagged SCD5, or transduced with Venus-tagged SCD5, as indicated. Data are presented as mean ± SEM of three independent experiments. ** p < 0.01 (t-test).

Article Snippet: Antibodies used in this study included antibody to SCD5 (Aviva systems biology, San Diego, CA, USA, SCD5 antibody-C-terminal region, ARP65045_P050, 1:1000 dilution), antibody to VHL (Cell Signaling, Leiden, The Netherlands, 1:1000 dilution), antibody to β-actin (Sigma, Darmstadt, Germany), antibody to γ-tubulin, and antibody to HIF2α from Abcam, Cambridge, UK.

Techniques: Quantitative RT-PCR, Infection, Control, shRNA, Western Blot, Knockdown, Over Expression, Transfection, Transduction

Journal: Scientific Reports

Article Title: Abrogation of atypical neurogenesis and vascular-derived EphA4 prevents repeated mild TBI-induced learning and memory impairments

doi: 10.1038/s41598-020-72380-1

Figure Lengend Snippet: Information of antibodies.

Article Snippet: Prox1 , ECM biosciences , CM4961 , 1:500.

Techniques:

Journal: Cell Death and Differentiation

Article Title: TFEB controls syncytiotrophoblast formation and hormone production in placenta

doi: 10.1038/s41418-024-01337-y

Figure Lengend Snippet: A Representative image of immunoblot analysis of TFEB expression in wild-type (WT) and CRISPR-Cas9 TFEB knock-out (KO#1 and KO#2) cells. GAPDH was used as a loading control. B ( Left) Representative immunofluorescence images of wild-type (WT) cells and TFEB knock-out (KO#1 and KO#2) cells upon Forskolin (FRSK) treatment immunostained for E-Cadherin (CDH1) and human chorion gonadotropin β subunit (CGB). Nuclei are counterstained blue using DAPI. Lack of CDH1 staining combined with DAPI fluorescence was used to denote clusters of fused cells (Fusion Index, see “Methods”). Scale bar 20 µm. ( Right ) High-content imaging-based quantification of CGB intensity and proportion of fused cells upon indicated treatments ( n = 4) shown as dot plots. Statistical analysis was performed by One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). C Gene-set enrichment Analysis was performed on genes ranked by their fold-change and significance (see Methods) in FRSK-treated BeWo cells upon TFEB KO against WT cells in both RNAseq (left) and LC–MS/MS (right). Upregulated genes from panel in Fig. (STB signature - BeWo) were used as geneset. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are reported. D Representative image of immunoblot analysis of TFEB, CYP19A1 and OVOL1 levels in human naive IPSC-derived trophoblast stem cells (TSC) and differentiated syncytiotrophoblast (STB) treated with siRNA targeting TFEB (siTFEB) and scramble sequences (SCR). H3 was used as a loading control. E Gene-set enrichment Analysis was performed on genes ranked by their fold-change and significance (see Methods) in syncytiotrophoblast (STB) treated with siRNA targeting TFEB (siTFEB) against human IPSC-derived trophoblast stem cells (TSC) in both RNAseq (left) and LC–MS/MS (right). Upregulated genes from panel in Fig. (STB signature - TSC-STB) were used as geneset. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are reported. F: Heatmap of Z-scored log 2- normalized expression values of selected differentially expressed genes in human naive IPSC-derived trophoblast stem cells (TSC) and differentiated syncytiotrophoblast (STB) treated with siRNA targeting TFEB (siTFEB) and scramble sequences (SCR). Genes displayed were grouped into categories indicated on the bottom.

Article Snippet: Primary antibodies (in TBST 0.1% BSA 3%) were incubated overnight at 4 °C (TFEB, Cell Signaling, # 4240 s; E-Cadherin, Cell Signaling, # 24E10; GAPDH, Santacruz, # sc32233; CYP19A1, Cell Signaling, # 14528; OVOL1, Aviva Systems Biology, #ARP38500T100).

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Control, Immunofluorescence, Staining, Fluorescence, Imaging, Liquid Chromatography with Mass Spectroscopy, Derivative Assay