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: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: Analysis of ATT-mediated calcium regulation via RNA-seq. (A) The analysis of the KEGG pathway for ATT-treated cells (10 μM). (B) Heatmap showing the relative expression of differentially expressed genes in HCT-116 cells with or without ATT treatment (10 μM). (C) Volcano plots of DEGs in ATT vs. control HCT-116 cells. |Fold change| > 2 and P < 0.05 were shown in red (upregulated) or blue (downregulated). (D, E) Cellular calcium level of CRC cells in HCT-116 and HT-29 cells following ATT treatment (0, 5, 10, 20 μM) for 24 h. CRC cells were treated with ATT (0, 5, 10, 20 μM) for 24 h. The influence of ATT on the RNA (F) and protein levels (G, H) of the differentially expressed protein CYP24A1 were assessed. (I) The immunofluorescence analysis of CYP24A1 expression in CRC cells with ATT treatment (10 μM) for 24 h. Scale bar: 50 μm. Data are expressed as mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA followed by Dunnett's post hoc test. P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: RNA Sequencing, Expressing, Control, Immunofluorescence, Labeling

Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: The crucial role of LSH in ATT-induced ferroptosis in HCT-116. HCT-116 cells were pre-treated with siLSH (A) or Flag-LSH (B) for 24 h and then treated with or without ATT (10 μM) for 24 h. Cytotoxicity of ATT in cells was tested. Flow cytometry was performed to evaluate oxidation-dependent DCF fluorescence (C) , C11-BODIPY-detected lipid peroxidation (D) , and intracellular Ca 2+ levels (E) in CRC cells with LSH knockdown after ATT treatment. HCT-116 cells were pre-treated with Flag-LSH for 24 h and then treated with or without ATT (10 μM) for 24 h. Flow cytometry was used to analyze the effects of ATT on intracellular oxidative activity (DCFH-DA) (F) , lipid peroxidation (C11-BODIPY) (G) , and calcium levels (H) in CRC cells with LSH overexpression. Effects of ATT on target protein LSH and downstream protein CYP24A1 upon LSH knockdown (I) and LSH overexpression (J) . Data are expressed as mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test. P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: Flow Cytometry, Fluorescence, Knockdown, Activity Assay, Over Expression, Labeling

Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: Identification of LSH-interacting proteins perturbed by ATT. HCT-116 cells were treated with ATT (10 μM) for 24 h. (A) LC-MS/MS analysis of LSH-immunoprecipitated proteins in HCT-116 cells (left). Representative peptide counts and molecular weights of indicated proteins are shown (right). (B) Co-immunoprecipitation (Co-IP) analysis of the interaction between LSH and EWSR1 in CRC cells. (C) Immunofluorescence co-localization analysis of EWSR1 and LSH in CRC cells. Scale bar: 5 μm. (D) HCT-116 cells were pre-treated with Flag-LSH for 24 h and then treated with or without ATT (10 μM) for 24 h. Co-IP analysis of interactions between Flag-tagged wild-type LSH, mutant, and EWSR1. (E) HCT-116 cells were treated with ATT (0, 5, 10, 20 μM) for 24 h. Protein levels of EWSR1 in CRC cells were determined. (F) HCT-116 cells were treated with siEWSR1 for 48 h. Effect of EWSR1 knockdown on HCT-116 cell viability. Influence of EWSR1 knockdown on lipid peroxidation (C11-BODIPY) (G) and intracellular calcium levels (H). Statistical significance was assessed using one-way ANOVA followed by Dunnett's post hoc test. Impact of EWSR1 knockdown on (I) RNA and (J) protein expression of LSH and CYP24A1. Statistical significance was assessed using two-way ANOVA followed by Dunnett's post hoc test. (K) HCT-116 cells were transfected with siEWSR1 for 48 h, and a dual-luciferase assay was performed to examine its effect on the promoter activity of CYP24A1 mediated by exogenous LSH. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test. Data are expressed as mean ± SD (n = 3). P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Mutagenesis, Knockdown, Expressing, Transfection, Luciferase, Activity Assay, Labeling

Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: The effect of ATT on CRC progression in vivo . (A) Representative images of tumor samples from each group (n = 6). (B) Measurement of tumor volume and (C) weight (n = 6). (D) HE staining of tumor tissues (Scale bar: 100 μm) and IHC detection of Ki-67, CYP24A1, SCD, and LSH expression in tumor tissues (Scale bar: 50 μm). (E) Protein expression levels of CYP24A1, SCD, and LSH in tumor tissues were determined. (F) Impact of ATT on the interaction of LSH and EWSR1 in vivo was assayed by Co-IP assay. Statistical significance was assessed using one-way ANOVA followed by Dunnett's post hoc test. P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: In Vivo, Staining, Expressing, Co-Immunoprecipitation Assay, Labeling

Journal: Scientific Reports

Article Title: DNA methylation and Transcriptome Changes Associated with Cisplatin Resistance in Ovarian Cancer

doi: 10.1038/s41598-017-01624-4

Figure Lengend Snippet: CpG methylome and transcriptome differences between cisplatin sensitive and resistant ovarian cancer lines. DNA methylomes of the cells sensitive or resistant to cisplatin were profiled with Reduced Representation Bisulfite Sequencing and transcriptomes with mRNA-sequencing. In ( a ) are the CpG sites with coverage ≥ 10 and minimum methylation difference of 20% (qval ≤ 0.05) in M019i cells, (see also Supplementary Table ), ( b ) the distribution of differentially methylated cites in genomic regions, ( c ) distance of the differentially methylated sited from the closest transcription start sites, ( d ) the transcriptome differences (minimum absolute FC = 1.5, FDR ≤ 0.05) between cisplatin sensitive and resistant ovarian cancer cells (M019i), ( e–h ) qRT-PCR validation of AKR1C1 , CYP4F11 , CYP24A1 , MIR205HG , and SLC6A14 differences in M019i and OC001 cells (y-axis: relative expression level).

Article Snippet: Rabbit polyclonal anti-human CYP24A1 (Atlas Antibodies, HPA022261), mouse monoclonal anti-human GAPDH (Abcam, Ab9482), and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako) were used to evaluate protein expression.

Techniques: Methylation Sequencing, Sequencing, Methylation, Quantitative RT-PCR, Biomarker Discovery, Expressing

Journal: Scientific Reports

Article Title: DNA methylation and Transcriptome Changes Associated with Cisplatin Resistance in Ovarian Cancer

doi: 10.1038/s41598-017-01624-4

Figure Lengend Snippet: Identification of genes potentially associated with the drug resistance in ovarian cancer cells through integrative analysis. ( a ) Normalized relative gene expression counts and clustering of the genes differentially expressed by the cisplatin sensitive and resistant cells and regulated in response to 7 μM cisplatin treatment in resistant cells (M019i). ( b , c ) Normalized gene expression counts (RPKM) and statistics for CYP24A1 and IL6 genes in M019i cells. ( d – f ) Expression of KLF4 , CYP24A1 and IL6 was validated with qRT-PCR in two platinum sensitive cell lines M019i and OC002 and their resistant variants (y-axis: relative expression level). ( g ) CYP24A1 protein expression and ( h ) IL6 protein concentrations in cell culture supernatants of the same cell lines as in ( e and f ), respectively).

Article Snippet: Rabbit polyclonal anti-human CYP24A1 (Atlas Antibodies, HPA022261), mouse monoclonal anti-human GAPDH (Abcam, Ab9482), and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako) were used to evaluate protein expression.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Cell Culture

Journal: Scientific Reports

Article Title: DNA methylation and Transcriptome Changes Associated with Cisplatin Resistance in Ovarian Cancer

doi: 10.1038/s41598-017-01624-4

Figure Lengend Snippet: The potential drug resistance driver genes.

Article Snippet: Rabbit polyclonal anti-human CYP24A1 (Atlas Antibodies, HPA022261), mouse monoclonal anti-human GAPDH (Abcam, Ab9482), and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako) were used to evaluate protein expression.

Techniques: Migration, Marker, Expressing, Transformation Assay, Mutagenesis

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

doi: 10.1016/j.jsbmb.2024.106497

Figure Lengend Snippet: Fig. 6. Effect of PDIA3 silencing and 1,25-dihydroxyvitamin D3 treatment on CYP24A1 mRNA levels (a), CYP24A1 protein levels (b), PDIA3 protein levels (c) and CYP27B1 mRNA levels (d) in PC3 cells. PC3 cells were treated with 10 nM siRNA against PDIA3 (‘siPDIA3’) or control siRNA (‘siCtrl’) for 72 h, prior to treatment with 10 nM 1,25-dihydroxyvitamin D3 (‘1,25(OH)2-D3’) or vehicle control (‘EtOH’) for 24 h, as described in Materials and methods. (a, d) All mRNA levels were assessed with real-time PCR and analysed based on the 2-ΔΔCt method. Fold differences are shown relative to ‘siCtrl EtOH’. (b) CYP24A1 protein levels in whole-cell lysates were examined by Western blotting as described in Materials and methods. (c) Protein levels of PDIA3 were examined in parallel, in order to ensure silencing efficiency. Band intensities were normalised against total protein stain and fold differences are shown relative to ‘siCtrl EtOH’. Representative pictures are shown. Data were collected from 4 independent experiments. Error bars represent SEM. p ⩽ 0.05 (*), p ⩽ 0.01 (**).

Article Snippet: The membranes were subsequently blocked in Intercept® (TBS) Blocking Buffer for 2 h in room temperature under gentle agitation, before being incubated overnight at 4 ◦C with primary antibodies against CYP24A1 (anti-CYP24A1, 1:500, NBP1–85495, Novus Biologicals); PDIA3 (anti-PDIA3, 1:500, VMA00477, Bio-Rad Laboratories Inc.); VDR (antiVDR/NR1I1/vitamin D receptor, 1:1000, PP-H4537–00, R&D Systems); anti-c-Jun (anti-Jun, 1:1000, 610326, BD Biosciences); anti-c-Myc (recombinant anti-c-Myc [Y69], 1:1000, ab32072, Abcam).

Techniques: Control, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

doi: 10.1016/j.jsbmb.2024.106497

Figure Lengend Snippet: Fig. 7. Effect of PDIA3 silencing and 1,25-dihydroxyvitamin D3 treatment on CYP24A1 mRNA levels in U2OS cells. U2OS cells were treated with 10 nM siRNA against PDIA3 (‘siPDIA3’) or control siRNA (‘siCtrl’) for 72 h, prior to treatment with 10 nM 1,25-dihydroxyvitamin D3 (‘1,25(OH)2-D3’) or vehicle control (‘EtOH’) for 24 h, as described in Materials and methods. The mRNA levels were assessed with real-time PCR and analysed based on the 2-ΔΔCt

Article Snippet: The membranes were subsequently blocked in Intercept® (TBS) Blocking Buffer for 2 h in room temperature under gentle agitation, before being incubated overnight at 4 ◦C with primary antibodies against CYP24A1 (anti-CYP24A1, 1:500, NBP1–85495, Novus Biologicals); PDIA3 (anti-PDIA3, 1:500, VMA00477, Bio-Rad Laboratories Inc.); VDR (antiVDR/NR1I1/vitamin D receptor, 1:1000, PP-H4537–00, R&D Systems); anti-c-Jun (anti-Jun, 1:1000, 610326, BD Biosciences); anti-c-Myc (recombinant anti-c-Myc [Y69], 1:1000, ab32072, Abcam).

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: Oncology reports

Article Title: CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

doi: 10.3892/or.2016.5072

Figure Lengend Snippet: Figure 1. Establishment of CYP24A1-suppressed cell lines. (A) HPLC analysis to examine the metabolic activity of 1α,25(OH)2D3. MDA-MB‑231 cells were transfected with CYP24A1-specific siRNAs, and vitamin D metabolic activities were examined by HPLC at 48 h post-transfection. No metabolites were detected when cells were transfected with CYP24A1-specific siRNAs in 2 independent experiments. (B) Expression of CYP24A1 in MDA-MB‑231 cells transfected with different CYP24A1-specific shRNAs. (C) Metabolic activity of 1α,25(OH)2D3 in each transfectant. The summation of peak radioactivity from all metabolites as shown in (A) was analyzed by HPLC and expressed as the relative metabolic activity. Note the various suppression levels of vitamin D metabolic activity, which closely correlated with the CYP24A1 expression levels.

Article Snippet: Cells were also transfected with CYP24A1-specific small-interfering RNAs (siRNAs; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a plasmid containing the full-length CYP24A1 cDNA (OriGene, Rockville, MD, USA).

Techniques: Activity Assay, Transfection, Expressing, Radioactivity

Journal: Oncology reports

Article Title: CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

doi: 10.3892/or.2016.5072

Figure Lengend Snippet: Figure 2. CYP24A1 expression affects a wide array of signaling pathways involved in carcinogenesis. (A and B) Venn diagram representing the number of shared and cell-type specific changes caused by CYP24A1 suppression in MCF7 and MDA-MB‑231 cells (A). The numbers indicate genes whose expression increased or decreased by an average ≥200% (n=2), and the overlapping circles indicate the number of dysregulated genes observed in both cell lines. Genes were manually grouped according to function, and all genes that showed altered expression levels in both MCF7 and MDA-MB‑231 cells are displayed graphically, representing the mean expression values observed in both cell types (B). (C) Verification of the results of microarray analysis by RT-PCR in MDA‑MB‑231 cells, and quantitative analysis of gene-expression changes. *P<0.05 for control cells. (D) Verification of gene-expression alterations of PDLIM2 in MDA-MB‑231 cells transfected with various constructs, as indicated.

Article Snippet: Cells were also transfected with CYP24A1-specific small-interfering RNAs (siRNAs; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a plasmid containing the full-length CYP24A1 cDNA (OriGene, Rockville, MD, USA).

Techniques: Expressing, Protein-Protein interactions, Microarray, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control, Transfection, Construct

Journal: Oncology reports

Article Title: CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

doi: 10.3892/or.2016.5072

Figure Lengend Snippet: Figure 3. Suppression of CYP24A1 expression inhibits cell growth and promotes apoptosis. MDA-MB‑231 cells were exposed to 100 nM 1α,25(OH)2D3 or vehicle control in cells transfected with CYP24A1-specific or negative-control shRNA. (A) Cell proliferation. (B) Ki-67 labeling of cells. (C) Apoptosis was examined with a 24-h exposure to 50 µM H2O2. (D) Anoikis was induced after adding MDA-MB‑231 cells to agarose-coated dishes to avoid cell attachment. P<0.05 for control cells vs. cells treated with 1α,25(OH)2D3 (#). *P<0.05 for control cells vs. cells transfected with CYP24A1-specific shRNA.

Article Snippet: Cells were also transfected with CYP24A1-specific small-interfering RNAs (siRNAs; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a plasmid containing the full-length CYP24A1 cDNA (OriGene, Rockville, MD, USA).

Techniques: Expressing, Control, Transfection, Negative Control, shRNA, Labeling, Cell Attachment Assay

Journal: Oncology reports

Article Title: CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

doi: 10.3892/or.2016.5072

Figure Lengend Snippet: Figure 4. Suppression of CYP24A1 expression inhibits anchorage-independent growth. MDA-MB‑231 cells were exposed to 100 nM 1α,25(OH)2D3 or vehicle control following transfection with CYP24A1-specific or negative-control shRNA. (A and B) CYP24A1 suppression decreased colony formation in soft agar. Quantification of colony numbers (A) and representative morphology of the colonies (B). P<0.05 for control cells vs. cells treated with 1α,25(OH)2D3 (#). *P<0.05 for control cells vs. cells transfected with CYP24A1-specific shRNA.

Article Snippet: Cells were also transfected with CYP24A1-specific small-interfering RNAs (siRNAs; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a plasmid containing the full-length CYP24A1 cDNA (OriGene, Rockville, MD, USA).

Techniques: Expressing, Control, Transfection, Negative Control, shRNA

Journal: Oncology reports

Article Title: CYP24A1-induced vitamin D insufficiency promotes breast cancer growth.

doi: 10.3892/or.2016.5072

Figure Lengend Snippet: Figure 5. Suppression of CYP24A1 expression inhibits tumorigenicity in vivo. (A) MDA-MB‑231 cells transfected with CYP24A1-specific shRNAs grew significantly slower than did control cells. (B) Representative tumors developed in mice 18 days post-injection (upper panel) and their weights upon dissection (lower panel). (C) Histology of tumors (tm) developed from transplanted cells. Note the invasive growth (arrowheads) into surrounding fibrous tissue (fb) in tumors derived from control cells (upper panel). The tumors derived from cells with CYP24A1 suppression showed massive cell necrosis (nc) in the tumor centers (lower panel), while a limited number of tumor cells were viable only locating around the microvasculature (*). The middle panels represent magni- fications of the rectangular areas in the upper panels. (D) Apoptosis, Ki-67 labeling, and CD34-positive microvessels were quantitated in tumors developed in mice. *P<0.05 for control cells vs. cells transfected with CYP24A1-specific shRNA.

Article Snippet: Cells were also transfected with CYP24A1-specific small-interfering RNAs (siRNAs; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a plasmid containing the full-length CYP24A1 cDNA (OriGene, Rockville, MD, USA).

Techniques: Expressing, In Vivo, Transfection, Control, Injection, Dissection, Derivative Assay, Labeling, shRNA

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

doi: 10.1074/jbc.RA119.011869

Figure Lengend Snippet: CYP24A1 is a cell cycle–regulated protein involved in cell proliferation independent of its catabolic activity. A, CYP24A1 expression data from TCGA matched paired lung tissue RNA-Seq cohort (n = 58) of lung adenocarcinoma patients showing higher expressions of both Iso1 and Iso2 in the tumor tissue compared with normal lung. B, immunoblotting using specified antibodies showing overexpression of CYP24A1 Iso1 construct tagged with DDK and Iso2 construct tagged with V5 on different days (D1–D4) post irradiation in SK-Lu1 and Hcc827 cells. C and D, cell counting (left panels) and MTS assay (right panels) in SK-Lu1 (in C) and Hcc827 (in D) cells showing increased proliferation upon overexpression of both Iso1 and Iso2 in SK-Lu1 cells, however, similar studies in Hcc827 cells shows growth delay. E, HeLa cells were synchronized using double thymidine block and released. E and F, cells and cell lysates were collected at the indicated times and were subjected to either FACS (in E) or immunoblotting (in F). G and H, SK-Lu1 cells stably expressing CYP24A1-Iso2-V5 protein were synchronized and released and cell lysates were subjected to immunoblotting using indicated antibodies and assessed for cell cycle distribution (in H).

Article Snippet: We have also used pCMV6-CYP24A1 (Myc-DDK tagged) isoform 2 from OriGene Technologies (cat. no. RC225738).

Techniques: Activity Assay, Expressing, RNA Sequencing Assay, Western Blot, Over Expression, Construct, Irradiation, Cell Counting, MTS Assay, Blocking Assay, Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

doi: 10.1074/jbc.RA119.011869

Figure Lengend Snippet: CYP24A1 contains D-boxes, critical for APC-CDH1 regulation. A, schematics showing the location and sequence of two D-boxes (D#1 and D#2) and the catalytic cysteine (Cys-462) in CYP24A1. Both the D-boxes found to be conserved in vertebrates. B, phylogenetic analysis of CYP family members showing presence of D-boxes specifically in CYP24A1-related members (in red). C, HEK293 cells were transfected with indicated plasmids overexpressing either WT or D#1 mutant. 24 h post transfection, cell lysates were subjected to immunoprecipitation using anti-Myc agarose beads (for CYP24A1 pulldown) and immunoblotted with indicated antibodies. D, similar immunoprecipitation (using anti-V5 agarose beads) followed by immunoblotting experiments were performed as above. E, in HEK293 cells CYP24A1-WT protein was expressed in the presence of increasing (incline) concentrations of CDH1 and 24 h post-transfection cell lysates were immunoblotted with indicated antibodies. F, in vivo ubiquitination assay using CDC20 (as substrate) and CDH1 (as E3) in the presence and absence of either WT or D#2 mutant (mt) of CYP24A1. CYP24A1-WT reduced CDH1-mediated CDC20 polyubiquitination. G, in vitro ubiquitination assay using recombinant CYP24A1-His protein showing CDH1-mediated inhibition of cyclin B1 polyubiquitination. H and I, protein half-life of CYP24A1 (either WT or D#2) as determined by treating cells with cycloheximide (50 μg/ml). Data plotted were the mean ± S.E. from three independent experiments.

Article Snippet: We have also used pCMV6-CYP24A1 (Myc-DDK tagged) isoform 2 from OriGene Technologies (cat. no. RC225738).

Techniques: Sequencing, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, In Vivo, Ubiquitin Assay, In Vitro, Recombinant, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

doi: 10.1074/jbc.RA119.011869

Figure Lengend Snippet: Overexpression of WT CYP24A1-Iso2 enhances cell proliferation, whereas D#2 box mutant prevents mitotic exit and leads to cell death. A, SK-Lu1 cells stably transfected either with vector control or CYP24A1 (WT and D#2 mutant) were synchronized with double thymidine block and released. FACS data showing percentage of cells at different phases of cell cycle at different time points. B, FACS data showing increased sub-G1 (possibly mitotic catastrophe) percentage in D#2 overexpressing cells, which may be correlated with mitotic arrest (FACS data in panel A). C, cell lysates isolated from CYP24A1-WT and D#2 mutants were subjected to immunoblotting using indicated antibodies. D, schematic model showing CDH1 hyperactivation in D#2 mutant overexpressing cells leading to reduced cyclin B and CDC20 levels and increased p21 accumulation resulting in delay in mitotic exit causing mitotic catastrophe.

Article Snippet: We have also used pCMV6-CYP24A1 (Myc-DDK tagged) isoform 2 from OriGene Technologies (cat. no. RC225738).

Techniques: Over Expression, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Control, Blocking Assay, Isolation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

doi: 10.1074/jbc.RA119.011869

Figure Lengend Snippet: Alteration of CYP24A1 levels differentially impacts the clonogenic survival of lung cancer cells based on mutant KRAS dependence. A and B, H441 cells overexpressing CYP24A1-Iso2-V5 were treated either with PD98059 (20 μm), simvastatin (20 μm), or in combination showing significant down-regulation of CYP24A1 in combination treatment, which was correlated with the clonogenic survival as shown in (B). C, CYP24A1-Iso2-V5 overexpressing H441 cells were treated with a combination of simvastatin and PD98059 compounds as above for 20 h followed by a treatment with MG132 (2 μm for the last 4 h), where indicated. Cell lysates were then subjected to immunoblotting using indicated antibodies. D and E, mouse pancreatic adenocarcinoma cell lines (9805 and 4292) with doxycycline-inducible mutant KRASG12D expressing cassette (iKras*) were treated either with vehicle or with different concentrations (10 and 100 nm) of calcitriol. Cell numbers were counted on different days post calcitriol treatment as indicated (in D) and clonogenic survival was assayed (in E) as described in methods section. F, immunoblotting of cell lysates isolated from the above two mouse iKras* cell lines treated with calcitriol showing induction of CYP24A1. G and H, effect of calcitriol on cell proliferation and clonogenic survival in WT KRAS containing Hcc827 cells showing growth inhibitor effects. I, CYP24A1 induction was noted in Hcc827 cells upon calcitriol treatment in a dose- and time-dependent manner.

Article Snippet: We have also used pCMV6-CYP24A1 (Myc-DDK tagged) isoform 2 from OriGene Technologies (cat. no. RC225738).

Techniques: Mutagenesis, Western Blot, Expressing, Isolation

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity

doi: 10.1074/jbc.RA119.011869

Figure Lengend Snippet: Calcitriol treatment up-regulates CYP24A1 levels and increases tumor burden in Lsl-KrasG12D mouse lung tumor model. A, follow up of tumor progression using noninvasive bioluminescence -fold change in LSL-KrasG12D/+; LSL-Rosa26-Luc/+ tumor-bearing mice after 3 and 7 weeks post calcitriol (0.625 mg/kg, i.p., every other day) compared with vehicle-treated mice (n = 4 mice per group). B, similar groups of mice were imaged using micro CT, either prior to (left panels) or 2 weeks post calcitriol treatments. Increase in tumor volume and multiplicity were visible in calcitriol-treated group. C, representative H&E-stained sections at 4× (scale bar, 500 μm) and 10× (scale bar, 200 μm) magnifications of LSL-KrasG12D mice lungs treated with vehicle (top) and calcitriol (bottom). D, representative immunoblot showing CYP24A1 up-regulation in calcitriol-treated transgenic mice compared with vehicle control.

Article Snippet: We have also used pCMV6-CYP24A1 (Myc-DDK tagged) isoform 2 from OriGene Technologies (cat. no. RC225738).

Techniques: Micro-CT, Staining, Western Blot, Transgenic Assay, Control