Journal:

Article Title: Evaluation of Etest for Direct Antifungal Susceptibility Testing of Yeasts in Positive Blood Cultures

doi: 10.1128/JCM.39.4.1328-1333.2001

Figure Lengend Snippet: Effect of inoculum concentrations on the MICs determined by Etest

Article Snippet: If the manufacturer's recommended McFarland turbidity of 0.5 was used as a standard concentration for the Etest, all MICs obtained by the other inoculum densities were within 1 log 2 dilution. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Microorganism and McFarland turbidity MIC (μg/ml) of: Amphotericin B Flucytosine Fluconazole Itraconazole Ketoconazole C. krusei ATCC 6258 2 0.5 16 32 0.25 0.125 0.5 0.5 16 32 0.25 0.125 0.05 0.5 16 16 0.25 0.125 C. albicans ATCC 18804 2 0.125 0.062 0.25 0.016 0.008 0.5 0.125 0.031 0.125 0.016 0.008 0.05 0.125 0.016 0.25 0.016 0.008 C. glabrata ATCC 2001 2 0.25 0.016 8 4 0.5 0.5 0.25 0.016 8 4 0.5 0.05 0.125 0.016 8 2 0.25 C. tropicalis C2-1 2 0.125 0.016 0.5 0.032 0.008 0.5 0.125 0.016 0.5 0.016 0.008 0.05 0.125 0.016 0.25 0.016 0.008 Open in a separate window Effect of inoculum concentrations on the MICs determined by Etest

Techniques:

Journal:

Article Title: Evaluation of Etest for Direct Antifungal Susceptibility Testing of Yeasts in Positive Blood Cultures

doi: 10.1128/JCM.39.4.1328-1333.2001

Figure Lengend Snippet: Differences between the DET and MD methods among yeast species

Article Snippet: If the manufacturer's recommended McFarland turbidity of 0.5 was used as a standard concentration for the Etest, all MICs obtained by the other inoculum densities were within 1 log 2 dilution. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Microorganism and McFarland turbidity MIC (μg/ml) of: Amphotericin B Flucytosine Fluconazole Itraconazole Ketoconazole C. krusei ATCC 6258 2 0.5 16 32 0.25 0.125 0.5 0.5 16 32 0.25 0.125 0.05 0.5 16 16 0.25 0.125 C. albicans ATCC 18804 2 0.125 0.062 0.25 0.016 0.008 0.5 0.125 0.031 0.125 0.016 0.008 0.05 0.125 0.016 0.25 0.016 0.008 C. glabrata ATCC 2001 2 0.25 0.016 8 4 0.5 0.5 0.25 0.016 8 4 0.5 0.05 0.125 0.016 8 2 0.25 C. tropicalis C2-1 2 0.125 0.016 0.5 0.032 0.008 0.5 0.125 0.016 0.5 0.016 0.008 0.05 0.125 0.016 0.25 0.016 0.008 Open in a separate window Effect of inoculum concentrations on the MICs determined by Etest

Techniques:

Journal: JCI Insight

Article Title: Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model

doi: 10.1172/jci.insight.163576

Figure Lengend Snippet: ( A ) Sacsin IP in SH-SY5Y cells (FT, flow through). ( B ) NFL IP in SH-SY5Y cells (with IgG as control) and immunodecoration with anti-sacsin antibody (asterisk indicates the specific band). ( C and D ) Sacsin IP in SH-SY5Y cells (with IgG and SACS –/– , respectively, as control) and immunodecoration with anti–myosin Va (asterisk indicates the specific band )( C ) and anti-plectin ( D ) antibodies. ( E ) Plectin IP in WT cerebellum (with IgG as control) and immunodecoration with anti-sacsin antibody. ( F ) WB analysis showing levels of plectin and myosin Va in insoluble fractions of Sacs –/– and WT cerebellum (normalized to calnexin). ( G ) Representative images (60 × ) of cerebellar slices stained with calbindin (in green) and plectin (in red) showing a more intense plectin signal in Sacs –/– PC soma and proximal dendrites compared with WT controls. Scale bar: 20 μm.

Article Snippet: For sacsin IPs, anti-sacsin antibody (181190; Abcam) was used; for plectin IP, anti-plectin (ab32528; Abcam) antibody was used; for NFL IP, anti-NFL antibody (8A1, sc-20012; Santa Cruz Biotechnology Inc.) was used.

Techniques: Control, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer

doi: 10.1016/j.bbrep.2024.101706

Figure Lengend Snippet: CYP4F2/CYP4F11 play key roles in the progression of ER + BC induced by E2 through ERα. (A) Immunoblot analysis of CYP4F2 and CYP4F11-induced expression in MCF-7 cells treated with E2 for 6, 24, and 48 h through MAPK signaling. (B) 20-HETE levels were quantified in cell lysates collected from cells treated with HET0016, E2 or control. (C) ERα in MCF-7 cells was knocked down with siRNA. RT‒qPCR analysis of the levels of ERα was performed. (D) Immunoblot analysis of ERα, P-Erk expression in MCF-7 cells knocked down with siERα and treated with E2. (E) Transcript levels of AA metabolism-related genes were measured using RT‒qPCR on RNA prepared from MCF-7-vec and MCF-7-siERα cells. (F) ERα was overexpressed in MDA-MB-231 cells. RT‒qPCR analysis of the levels of ERα was performed. For the transfection assay, we used empty plasmid as negative control. (G) Immunoblot analysis of ERα, p -Erk, 5-LOX, COX2, CYP4F2, CYP4F11, EP2, and EP3 expression by overexpression of ERα in MDA-MB-231 cells in the presence or absence of E2. (H) MDA-MB-231 cell viability after treatment with HET0016 was tested by MTT assay. (I) Transcript levels of AA metabolism-related genes were measured using RT‒qPCR on RNA prepared from MDA-MB-231 and 231-pcERα cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Student's t -test.

Article Snippet: The antibodies we used were as follows: anti -CYP4F2 (Abcam # ab230709), anti -CYP4F11 (Proteintech # 20012-1-AP), anti -ERα (H184) (Santa Cruz# sc-7207), anti -BCL2 (CST), anti-Cl-caspase 3 (CST), anti -β-Actin (CST), anti-Cl-PARP (CST), anti -P-Erk (CST), 17-β-estradiol (Sigma–Aldrich), NS398 (Cayman Chemical), indomethacin (Sigma–Aldrich), estradiol cypionate (HY–B1100), HET0016 (ApexBio, C5344), and 20-HETE (Cayman, 90030).

Techniques: Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Negative Control, Over Expression, MTT Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer

doi: 10.1016/j.bbrep.2024.101706

Figure Lengend Snippet: CYP4F2/CYP4F11 accumulated in ER + BC. (A) Kaplan–Meier analysis of CYP4F2 and CYP4F11 expression for different molecular subtypes of ER + BC. (B) Immunohistochemical and HE analysis of CYP4F2 and CYP4F11 protein expression in ER + BC tissues. Representative IHC images in matched normal tissues and tumors from three patients are shown. (C) Serum levels of 20-HETE in ER + BC patients (n = 16) and healthy women (n = 10) were assessed (ELISA). (D) RT‒qPCR analysis of the transcript levels of CYP4F2 and CYP4F11 in ER + BC patients. *, P < 0.05; ***, P < 0.001; ns, not significant. Student's t -test for transcript levels; Mann‒Whitney test for 20-HETE quantification in clinical data.

Article Snippet: The antibodies we used were as follows: anti -CYP4F2 (Abcam # ab230709), anti -CYP4F11 (Proteintech # 20012-1-AP), anti -ERα (H184) (Santa Cruz# sc-7207), anti -BCL2 (CST), anti-Cl-caspase 3 (CST), anti -β-Actin (CST), anti-Cl-PARP (CST), anti -P-Erk (CST), 17-β-estradiol (Sigma–Aldrich), NS398 (Cayman Chemical), indomethacin (Sigma–Aldrich), estradiol cypionate (HY–B1100), HET0016 (ApexBio, C5344), and 20-HETE (Cayman, 90030).

Techniques: Expressing, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer

doi: 10.1016/j.bbrep.2024.101706

Figure Lengend Snippet: CYP4F2/CYP4F11 overexpression enhances ER + BC cell viability and antiapoptotic ability. (A and C) Western blotting and RT‒qPCR assays were performed to verify the transfection efficiency. (B) The metabolite 20-HETE levels were quantified in cell lysates collected from MCF-7 cells overexpressing CYP4F2 and CYP4F11 (ELISA). (D) MCF-7, MCF-7-CYP4F2, and MCF-7-CYP4F11 cells in the absence of FBS. Cell death was measured using a PI exclusion assay. For the stably overexpressed cell line construct, we used empty plasmid as negative control. (E) Proliferation of MCF-7 cells treated with 20-HETE and 5 μM indomethacin was measured by MTT assay. (F) Tumor growth of MCF7-vec, MCF-7-CYP4F2, and MCF-7-CYP4F11 cells implanted subcutaneously in athymic mice in the presence of growth factor-reduced Matrigel. The tumor formation rate was 80–90% after MCF-7 cells were transfected with the CYP4F2 or CYP4F11 plasmid. (G) H&E staining and IHC analysis of the expression of CYP4F2, CYP4F11, and Ki67 in tumors. (H and I) MCF-7, MCF-7-CYP4F2 and MCF-7-CYP4F11 cells were cultured in FBS-free medium and treated with 10 nM E2 and 100 μM H 2 O 2 for 60 h. Cells were stained with Annexin V-APC and 7-AAD, and apoptosis was determined (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student's t -test.

Article Snippet: The antibodies we used were as follows: anti -CYP4F2 (Abcam # ab230709), anti -CYP4F11 (Proteintech # 20012-1-AP), anti -ERα (H184) (Santa Cruz# sc-7207), anti -BCL2 (CST), anti-Cl-caspase 3 (CST), anti -β-Actin (CST), anti-Cl-PARP (CST), anti -P-Erk (CST), 17-β-estradiol (Sigma–Aldrich), NS398 (Cayman Chemical), indomethacin (Sigma–Aldrich), estradiol cypionate (HY–B1100), HET0016 (ApexBio, C5344), and 20-HETE (Cayman, 90030).

Techniques: Over Expression, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Exclusion Assay, Stable Transfection, Construct, Plasmid Preparation, Negative Control, MTT Assay, Staining, Expressing, Cell Culture

Journal: Biochemistry and Biophysics Reports

Article Title: Targeting estrogen mediated CYP4F2/CYP4F11-20-HETE metabolic disorder decelerates tumorigenesis in ER+ breast cancer

doi: 10.1016/j.bbrep.2024.101706

Figure Lengend Snippet: Inhibition of CYP4F2 or CYP4F11 attenuates E2 treated ER + BC cell viability. (A and B) RT‒qPCR was performed to measure CYP4F2 or CYP4F11 expression in MCF-7-shCYP4F2 and MCF-7-shCYP4F11 cells treated with or without E2. (C and D) Immunoblots of lysates from MCF-7 cells treated with E2 for 48 h and then CYP4F2 and CYP4F11 were knocked down with Bcl2, Cl-PARP, CYP4F2, and CYP4F11 antibodies. For the stably knock down cell line construct, we used scrambled shRNA as negative control. (E and F) 20-HETE levels were quantified in cell lysates collected from MCF-7-CYP4F2 and MCF-7-CYP4F11 cells transfected with CYP4F2/4F11 siRNA and control siRNA or treated with 50 μM HET0016. (G) MCF-7, MCF-7-shCYP4F2, and MCF-7-shCYP4F11 cell proliferation treated with or without E2 was measured by MTT assay for 72 h. (H) Immunoblot analysis of Cl-Caspase3, Cl-PARP, Bcl-2, Bad and ERα in MCF-7 cells treated with 50 μM and 100 μM HET0016 combined with or without E2 for 48 h. (I) MCF-7, MCF-7-shCYP4F2, and MCF-7-shCYP4F11 cells (7 × 10 5 ) were intravenously injected into female SCID mice. Baseline bioluminescent images were captured immediately after cell injection. Mice received imaging and final imaging at 2 and 24 h after cell injection, and bioluminescent images are shown. (J) Relative photon flux was calculated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Student's t -test.

Article Snippet: The antibodies we used were as follows: anti -CYP4F2 (Abcam # ab230709), anti -CYP4F11 (Proteintech # 20012-1-AP), anti -ERα (H184) (Santa Cruz# sc-7207), anti -BCL2 (CST), anti-Cl-caspase 3 (CST), anti -β-Actin (CST), anti-Cl-PARP (CST), anti -P-Erk (CST), 17-β-estradiol (Sigma–Aldrich), NS398 (Cayman Chemical), indomethacin (Sigma–Aldrich), estradiol cypionate (HY–B1100), HET0016 (ApexBio, C5344), and 20-HETE (Cayman, 90030).

Techniques: Inhibition, Expressing, Western Blot, Stable Transfection, Knockdown, Construct, shRNA, Negative Control, Transfection, Control, MTT Assay, Injection, Imaging