Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay

Journal: Cancer Communications

Article Title: Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1‐mediated MAPK signaling

doi: 10.1002/cac2.12187

Figure Lengend Snippet: SQLE accelerated the proliferation of CRC cells in vitro . A‐B. SQLE protein levels in HT29 (A) or RKO (B) cells after knockdown of SQLE (SQLE KD) were accessed by Western blotting analysis. shcontrol was served as control. C. Cell viabilities of HT29 cells after knockdown of SQLE (SQLE KD) were accessed by CCK8 assay. shcontrol was served as control. D‐E. Colony formation of HT29 cells after knockdown of SQLE (SQLE KD). shcontrol was served as control. F. Cell viabilities of RKO cells after knockdown of SQLE (SQLE KD) were accessed by CCK8 assay. shcontrol was served as control. G‐H. Colony formation of RKO cells after knockdown of SQLE (SQLE KD). shcontrol was served as control. I‐J. SQLE protein levels in HT29 (I) or RKO (J) cells after overexpression of SQLE (SQLE OE) were accessed by Western blotting analysis. Vector was served as control. K. Cell viabilities of HT29 cells after overexpression of SQLE (SQLE OE) were accessed by CCK8 assay. Vector was served as control. L‐M. Colony formation of HT29 cells after overexpression of SQLE (SQLE OE). Vector was served as control. N. Cell viabilities of RKO cells after overexpression of SQLE (SQLE OE) were accessed by CCK8 assay. Vector was served as control. O‐P. Colony formation of RKO cells after overexpression of SQLE (SQLE OE). Vector was served as control. Q‐R. Cell cycle analysis (G 0 /G 1 , S, G 2 /M phase) of HT29 cells after knockdown of SQLE (SQLE KD). shcontrol was served as control. S‐T. Cell cycle analysis (G 0 /G 1 , S, G 2 /M phase) of HT29 cells after overexpression of SQLE (SQLE OE). Vector was served as control. U. SQLE, NCEH1, LIPA, CYP24A1 and STAG2 protein level in shcontrol and SQLE KD HT29 cells by Western blotting analysis. β‐actin was used as loading control. Mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Article Snippet: Membranes were overnight incubated with primary antibodies against SQLE (1:1000 dilution; Cat#12544‐1‐AP; Proteintech, Rosemont, IL, USA), NCEH1 (neutral cholesterol ester hydrolase 1; 1:500 dilution; Cat#A17741; Abclonal, Wuhan, Hubei, China), LIPA (lipase A; 1:500 dilution; Cat#12956‐1‐AP; Proteintech, Rosemont, IL, USA), CYP24A1 (cytochrome P450 family 24 subfamily A member 1; 1:500 dilution; Cat#21582‐1‐AP; Proteintech, Rosemont, IL, USA), ATP2B1 (ATPase plasma membrane Ca2+ transporting 1; 1:1000 dilution; Cat#A18688; Abclonal, Wuhan, Hubei, China), STAG2 (stromal antigen 2; 1:1000 dilution; Cat#19837‐1‐AP; Proteintech, Rosemont, IL, USA), CACNG4 (1:1000 dilution; Cat#A14118; Abclonal, Wuhan, Hubei, China), ERK1/2 (1:1000 dilution; Cat#9102; Cell Signaling Technolgy), P‐ERK1/2 (1:1000 dilution; Cat#9101; Cell Signaling Technolgy, Danvers, Massachusetts, USA), Tubulin (1:5000 dilution; Cat#E7; DSHB, lowa city, IA, USA) and β‐actin (1:5000 dilution; Cat#AC026; Abclonal, Wuhan, Hubei, China) after being blocked at 4°C.

Techniques: In Vitro, Knockdown, Western Blot, Control, CCK-8 Assay, Over Expression, Plasmid Preparation, Cell Cycle Assay

Journal: Cancer Communications

Article Title: Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1‐mediated MAPK signaling

doi: 10.1002/cac2.12187

Figure Lengend Snippet: Knockdown of SQLE reduced the levels of CYP24A1 and calcitriol. A. The volcano plots of RNA‐seq analysis between shcontrol and SQLE KD HT29 cells. 1516 genes were up‐regulated, while 1376 genes were down‐regulated. SQLE, CYP24A1, CACNG4, STAG2, ATP2B1, NCEH1, LIPA were marked. B. The schematic diagram of Vitamin D3 synthesis. Down‐regulated genes are marked in green; up‐regulated genes are marked in red. C. The relative mRNA expressions of SQLE, CYP24A1, LIPA and NCEH1 in shcontrol and SQLE KD HT29 cells. D. Intracellular cholesterol level of shcontrol and SQLE KD HT29 cells by cholesterol measurement. E. CYP24A1 expression in 8 paired CRC samples by real‐time PCR analysis. F. The correlation between SQLE and CYP24A1 in 8 paired CRC samples by linear regression analysis. G. The volcano plots of untargeted metabolomics analysis between shcontrol and SQLE KD HT29 cells, 22(S)‐Hydroxycholesterol, 7‐Ketocholesterol and calcitriol were marked. H‐I. Intracellular Ca 2+ concentration of shcontrol and SQLE KD HT29 cells. J‐K. Intracellular Ca 2+ concentration of shcontrol and SQLE KD HT29 cells with calcitriol treatment (0.5 μmol/L). DMSO was used as control. Fluo‐4 probe and Hoechst represented calcium signal and nucleus, respectively. Scale bars, 50 μm. Mean ± SEM. ***, P < 0.001; ns, no significance. KD, knockdown

Article Snippet: Membranes were overnight incubated with primary antibodies against SQLE (1:1000 dilution; Cat#12544‐1‐AP; Proteintech, Rosemont, IL, USA), NCEH1 (neutral cholesterol ester hydrolase 1; 1:500 dilution; Cat#A17741; Abclonal, Wuhan, Hubei, China), LIPA (lipase A; 1:500 dilution; Cat#12956‐1‐AP; Proteintech, Rosemont, IL, USA), CYP24A1 (cytochrome P450 family 24 subfamily A member 1; 1:500 dilution; Cat#21582‐1‐AP; Proteintech, Rosemont, IL, USA), ATP2B1 (ATPase plasma membrane Ca2+ transporting 1; 1:1000 dilution; Cat#A18688; Abclonal, Wuhan, Hubei, China), STAG2 (stromal antigen 2; 1:1000 dilution; Cat#19837‐1‐AP; Proteintech, Rosemont, IL, USA), CACNG4 (1:1000 dilution; Cat#A14118; Abclonal, Wuhan, Hubei, China), ERK1/2 (1:1000 dilution; Cat#9102; Cell Signaling Technolgy), P‐ERK1/2 (1:1000 dilution; Cat#9101; Cell Signaling Technolgy, Danvers, Massachusetts, USA), Tubulin (1:5000 dilution; Cat#E7; DSHB, lowa city, IA, USA) and β‐actin (1:5000 dilution; Cat#AC026; Abclonal, Wuhan, Hubei, China) after being blocked at 4°C.

Techniques: Knockdown, RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Control

Journal: Cancer Communications

Article Title: Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1‐mediated MAPK signaling

doi: 10.1002/cac2.12187

Figure Lengend Snippet: Calcitriol and CYP24A1 restored the growth of SQLE KD HT29 cells through MAPK signaling. A. Cell viabilities of shcontrol and SQLE KD HT29 cells treated with calcitriol at 0.5, 1, 2.5, 5 μmol/L. DMSO was used as control. B. Cell viabilities of shcontrol and SQLE KD HT29 cells treated with calcitriol at 0.5, 1, 5, 10, 25, 50, 100, 250, 500 nmol/L for 96 h. DMSO was used as control. C. CYP24A1 was over‐expressed in shcontrol and SQLE KD HT29 cells. Cell viabilities of these cells were measured by CCK8. D‐E. CYP24A1 was over‐expressed in SQLE KD HT29 cells. Colony formation of these cells was measured. F. CYP24A1 was knocked down in shcontrol and SQLE KD HT29 cells. Cell viabilities of these cells were measured by CCK8. NC: negative control. G. GSEA set of MAPK signaling pathway by knockdown of SQLE in HT29 cells. H. The protein expression levels of SQLE, CACNG4, ERK1/2 and phosphorylation of ERK1/2 in shcontrol and SQLE KD HT29 cells were accessed by Western blotting. β‐actin was used as a loading control. I‐J. The protein expression levels of SQLE, CACNG4, CYP24A1, ERK1/2 and phosphorylation of ERK1/2 in shcontrol and SQLE KD HT29 xenograft tumors were accessed by Western blotting. Tubulin was used as a loading control. K. The protein expression levels of SQLE, CACNG4, CYP24A1, ERK1/2 and phosphorylation of ERK1/2 in SQLE KD HT29 cells treated with calcitriol (0.5 μmol/L) were accessed by Western blotting. DMSO was used as control. L‐M. The protein expression levels of SQLE, CACNG4, CYP24A1, ERK1/2 and phosphorylation of ERK1/2 in the cells used in C and F were accessed by Western blotting. N. Schematic diagram showing molecular mechanism of SQLE promoting CRC proliferation. SQLE increases the levels of calcitriol and CYP24A1.Then MAPK signaling is induced to accelerate CRC tumor growth. Mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. KD, knockdown

Article Snippet: Membranes were overnight incubated with primary antibodies against SQLE (1:1000 dilution; Cat#12544‐1‐AP; Proteintech, Rosemont, IL, USA), NCEH1 (neutral cholesterol ester hydrolase 1; 1:500 dilution; Cat#A17741; Abclonal, Wuhan, Hubei, China), LIPA (lipase A; 1:500 dilution; Cat#12956‐1‐AP; Proteintech, Rosemont, IL, USA), CYP24A1 (cytochrome P450 family 24 subfamily A member 1; 1:500 dilution; Cat#21582‐1‐AP; Proteintech, Rosemont, IL, USA), ATP2B1 (ATPase plasma membrane Ca2+ transporting 1; 1:1000 dilution; Cat#A18688; Abclonal, Wuhan, Hubei, China), STAG2 (stromal antigen 2; 1:1000 dilution; Cat#19837‐1‐AP; Proteintech, Rosemont, IL, USA), CACNG4 (1:1000 dilution; Cat#A14118; Abclonal, Wuhan, Hubei, China), ERK1/2 (1:1000 dilution; Cat#9102; Cell Signaling Technolgy), P‐ERK1/2 (1:1000 dilution; Cat#9101; Cell Signaling Technolgy, Danvers, Massachusetts, USA), Tubulin (1:5000 dilution; Cat#E7; DSHB, lowa city, IA, USA) and β‐actin (1:5000 dilution; Cat#AC026; Abclonal, Wuhan, Hubei, China) after being blocked at 4°C.

Techniques: Control, Negative Control, Knockdown, Expressing, Phospho-proteomics, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Urinary vitamin D-binding protein as a marker of ovarian reserve

doi: 10.1186/s12958-021-00762-9

Figure Lengend Snippet: Western blot analyses of VDBP from DOR, PCOS and NC urine samples

Article Snippet: Levels of VDBP in the urine samples were determined in all 140 validation cohort samples with commercially available human VDBP ELISA kits (R&D Systems, Mineapolis, MN, USA), according to the manufacturer’s protocols.

Techniques: Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Urinary vitamin D-binding protein as a marker of ovarian reserve

doi: 10.1186/s12958-021-00762-9

Figure Lengend Snippet: VDBP levels in the urine of patients in the DOR, PCOS and NC groups. Data are expressed as mean ± SD.* P < 0.05

Article Snippet: Levels of VDBP in the urine samples were determined in all 140 validation cohort samples with commercially available human VDBP ELISA kits (R&D Systems, Mineapolis, MN, USA), according to the manufacturer’s protocols.

Techniques:

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Urinary vitamin D-binding protein as a marker of ovarian reserve

doi: 10.1186/s12958-021-00762-9

Figure Lengend Snippet: Scatter plot of linear relations between urinary VDBP level and serum AMH level

Article Snippet: Levels of VDBP in the urine samples were determined in all 140 validation cohort samples with commercially available human VDBP ELISA kits (R&D Systems, Mineapolis, MN, USA), according to the manufacturer’s protocols.

Techniques:

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Urinary vitamin D-binding protein as a marker of ovarian reserve

doi: 10.1186/s12958-021-00762-9

Figure Lengend Snippet: The receiver operating characteristic (ROC) analysis of urinary VDBP in the diagnosis of DOR ( A ) and PCOS ( B )

Article Snippet: Levels of VDBP in the urine samples were determined in all 140 validation cohort samples with commercially available human VDBP ELISA kits (R&D Systems, Mineapolis, MN, USA), according to the manufacturer’s protocols.

Techniques: Biomarker Discovery