Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae inhibited milk fat and protein synthesis in BMECs. (A–C) mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (D) DEGs heat maps. (E) Enrichment analysis of differentially expressed genes KEGG. (F) The protein levels of SREBP1 and β-casein were detected in BMECs. (G, H) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (I, J) mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (K) Typical images of BODIPY 493/503 staining in BMECs. * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae caused mitochondrial dysfunction in mammary gland and dyssynthesis of milk fat and protein. (A) Representative images of H&E staining. (B) The severity of mastitis was assessed by differences in histological score ( n = 6 cows per group) between the control and K. pneumoniae infection groups. (C–G) mRNA levels of TNF-α , IL-1β , IL-6, SREBP1 , and β-casein were detected by RT-qPCR method in mammary gland tissues (mean ± SEM, n = 6). (H) The protein levels of SREBP1, β-casein OPA1, MFN1, COX I, DRP1, and FIS1 were detected in mammary gland tissues. (I–O) Relative protein abundance of OPA1, MFN1, COX I, DRP1, FIS1, SREBP1, and β-casein were normalized to β-actin (mean ± SEM, n = 3). (P) Relative ATP levels (mean ± SEM, n = 6). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Staining, Control, Infection, Quantitative RT-PCR, Quantitative Proteomics

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: Mdivi-1 recovered K. pneumoniae -induced mitochondrial damage and dyssynthesis of milk fat and protein in BMECs. (A) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of OPA1, MFN1, COX I, DRP1, and FIS1. (B–F) Relative protein abundance of OPA1, MFN1, COX I, DRP1, and FIS1 were normalized to β-actin (mean ± SEM, n = 3). (G) Relative ATP levels (mean ± SEM, n = 3). (H) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of SREBP1 and β-casein. (I, J) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (K, L) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of SREBP1 and β-casein (mean ± SEM, n = 3). (M) Typical images of BODIPY 493/503 staining in BMECs. (N–P) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of TNF-α , IL-1β , and IL-6 (mean ± SEM, n = 3). (Q) Detection of the content of LDH (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: FNIP1 silencing alleviated K. pneumoniae -induced milk fat and protein dyssynthesis. (A) After FNIP1 silence, BMECs were infected with K. pneumoniae for 6 h to analyze the protein levels of SREBP1 and β-casein. (B, C) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (D, E) After FNIP1 silence, mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (F) Typical images of BODIPY 493/503 staining in BMECs. (G–I) After FNIP1 silence, mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Infection, Quantitative Proteomics, Quantitative RT-PCR, Staining

Journal: Toxins

Article Title: PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells

doi: 10.3390/toxins12040238

Figure Lengend Snippet: RT-qPCR validation of the differentially expressed genes (DEGs) obtained by RNA sequencing (RNA-Seq). ( A ) The genes involved in inflammation. ( B ) The genes involved in casein synthesis. Data represent the mean and standard deviation ( n = 6), and the asterisk indicates statistical difference (* p < 0.05) between the indicated columns, based on one-way analysis of variance and Duncan’s range test. IL-1β , interleukin-1β; IL-6 , interleukin-6; IL-8 , interleukin-8; TNF-α , tumor necrosis factor-α; CXCL1 , chemokine (C-X-C motif) ligand 1; CXCL6 , chemokine (C-X-C motif) ligand 6; CSN1S1 , αS1-casein; CSN2 , β-casein; CSN3 , κ-casein; RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction; CON, control group; PGN, peptidoglycan group; LTA, lipoteichoic acid group; MIX, PGN + LTA group.

Article Snippet: The primary antibodies included CSN1S1 (Bioss Antibodies, bs-10034R, Beijing, China), CSN2 (Bioss Antibodies, bs-10032R, Beijing, China), CSN3 (Bioss Antibodies, bs-10031R, Beijing, China), Histone H3 (17168-1-AP, Proteintech, Wuhan, China), Histone H4 (16047-1-AP, Proteintech, Wuhan, China), acetylated Histone H3 (Acetyl Lys9/14, Bioss Antibodies, bs-4316R, Beijing, China), acetylated Histone H4 (Acetyl K5, Bioss Antibodies, bs-10721R, Beijing, China), and GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Quantitative RT-PCR, RNA Sequencing Assay, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Toxins

Article Title: PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells

doi: 10.3390/toxins12040238

Figure Lengend Snippet: The protein expression of the three caseins (CSN1S1, CSN2, and CSN3). The total protein was isolated to examine casein expression by Western Blot analysis, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a housekeeping protein. Quantitation of blots is representative of three independent experiments. Data represent the mean and standard deviation ( n = 6), and the asterisk indicates statistical difference (* p < 0.05) between the indicated columns, based on one-way analysis of variance and Duncan’s range test. CSN1S1, αS1-casein; CSN2, β-casein; CSN3, κ-casein; CON, control group; PGN, peptidoglycan group; LTA, lipoteichoic acid group; MIX, PGN + LTA group.

Article Snippet: The primary antibodies included CSN1S1 (Bioss Antibodies, bs-10034R, Beijing, China), CSN2 (Bioss Antibodies, bs-10032R, Beijing, China), CSN3 (Bioss Antibodies, bs-10031R, Beijing, China), Histone H3 (17168-1-AP, Proteintech, Wuhan, China), Histone H4 (16047-1-AP, Proteintech, Wuhan, China), acetylated Histone H3 (Acetyl Lys9/14, Bioss Antibodies, bs-4316R, Beijing, China), acetylated Histone H4 (Acetyl K5, Bioss Antibodies, bs-10721R, Beijing, China), and GAPDH (60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Expressing, Isolation, Western Blot, Protein Quantitation, Standard Deviation

Journal: Cancer & Metabolism

Article Title: Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l -type amino acid transporter 1

doi: 10.1186/s40170-022-00295-8

Figure Lengend Snippet: Changes in the phosphorylation of substrates of suggested key kinases and their regulatory proteins by LAT1 inhibition. Protein extracted from cells treated with 30 μM JPH203 and control cells was analyzed by Western blot. (A and B) The decrease in phosphorylation associated with JPH203 treatment for indicated time: Thr-389 of p70 S6K ( A ) and Ser-1469 of TOP2A ( B ). The data are presented as a ratio of the signal intensity of phosphorylated protein to total protein ± SD ( n = 3). * p value < 0.05. ns = not significant (one sample t test) ( C ) Decreased phosphorylation of part of CK2 substrates by LAT1 inhibition with JPH203 for 24 h. Phosphorylation on the consensus CK2 substrate motif was detected by a specific antibody. D Decreased phosphorylation of Ser-209 of CK2β, a regulatory subunit of CK2, in cells treated with JPH203 for 24 h. E CK2 activity of BTC cells treated with JPH203. The extracted protein of BTC cells treated with 30 μM JPH203 for 24 h was subject to CK2 activity assay by ELISA using p53 N-terminal peptide and p53-pS46 antibody conjugated with horseradish peroxidase. F The decrease of CK2α co-immunoprecipitated with NOLC1. Protein extracted from KKU-055 and KKU-213 cells treated with JPH203 for 24 h was subject to immunoprecipitation using anti-NOLC1 antibody

Article Snippet: In Western blotting, the following antibodies were used at the indicated dilutions: p70 S6K–pT389 (1:1000, #9234), p70 S6K (1:1000, #9202), phospho–CK2 substrate (1:1000, #8738) from Cell Signaling Technology (Danvers, MA, USA); CK2β (1:500, 22418-1-AP), NOLC1 (1:1000, 11815-1-AP), and β-actin (1:5000, 66009-1-Ig) from Proteintech Group (Chicago, IL, USA); TOP2A-pS1469 (1:1000, PA5-64536) from Affinity Biosciences (Cincinnati, OH, USA); CK2β–pS209 (1:1000, 44-1090G) from Invitrogen (Waltham, MA, USA); TOP2A (1:200, sc-365916), CK2α (1:1000, sc-373894) from Santa Cruz Biotechnology (Dallas, TX, USA); goat anti-mouse IgG-HRP (1:5000, 115-035-062), goat anti-rabbit IgG-HRP (1:5000, 111-035-003) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Techniques: Phospho-proteomics, Inhibition, Control, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Cancer & Metabolism

Article Title: Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l -type amino acid transporter 1

doi: 10.1186/s40170-022-00295-8

Figure Lengend Snippet: Evaluation of the combined treatment of JPH203 and CX-4945. A Effect of the combination of JPH203 with CX-4945 on cell proliferation. BTC cell lines were treated with JPH203 or CX-4945 alone, or in combination. After 3 days of treatment, cell growth was assessed by WST assay. The absorbance at 450 nm of each sample was expressed as % of the control without JPH203 treatment. B Wound healing assay using KKU-100 treated with 30 μM JPH203 or 5μM CX-4945 alone, or in combination. C The number of migrating cells of KKU-055 and KKU-100 treated with 30 μM JPH203 or 5 μM CX-4945 alone, or in combination. The significance of the difference between the treatment with each inhibitor alone and the combination was determined by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent means ± SD ( n = 4 for WST assay, n = 3 for wound healing assay). *Tukey post-hoc p value < 0.05. D Decreased phosphorylation of part of CK2 substrates by LAT1 inhibition with 30 μM JPH203 and 1 μM CX-4945, or in combination for 24 h. Phosphorylation on the consensus CK2 substrate motif was detected by a specific antibody

Article Snippet: In Western blotting, the following antibodies were used at the indicated dilutions: p70 S6K–pT389 (1:1000, #9234), p70 S6K (1:1000, #9202), phospho–CK2 substrate (1:1000, #8738) from Cell Signaling Technology (Danvers, MA, USA); CK2β (1:500, 22418-1-AP), NOLC1 (1:1000, 11815-1-AP), and β-actin (1:5000, 66009-1-Ig) from Proteintech Group (Chicago, IL, USA); TOP2A-pS1469 (1:1000, PA5-64536) from Affinity Biosciences (Cincinnati, OH, USA); CK2β–pS209 (1:1000, 44-1090G) from Invitrogen (Waltham, MA, USA); TOP2A (1:200, sc-365916), CK2α (1:1000, sc-373894) from Santa Cruz Biotechnology (Dallas, TX, USA); goat anti-mouse IgG-HRP (1:5000, 115-035-062), goat anti-rabbit IgG-HRP (1:5000, 111-035-003) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Techniques: WST Assay, Control, Wound Healing Assay, Comparison, Phospho-proteomics, Inhibition

Journal: Nutrients

Article Title: Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat

doi: 10.3390/nu14234948

Figure Lengend Snippet: Milk Protein β-casein in Mice Mammary Epithelial Cultures (MEC) . Immunostained images of F-actin (green) as a marker of epithelial cells and β-casein (red) as a marker of milk and merged image. Blue represents the nuclei stained with DAPI. Scale bars are 20 µm.

Article Snippet: The primary antibodies anti-FAS (SC-20140) and anti-SREBP (SC-8984) were from Santa Cruz Biotechnology, Inc (Dallas, TX), the anti-β-casein antibody from Abbexa (abx430060, Sugar Land, TX, USA), and the anti-adipophilin antibody from LSBio (LS-C146977-100, Seattle, WA, USA).

Techniques: Marker, Staining

Journal: Nutrients

Article Title: Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat

doi: 10.3390/nu14234948

Figure Lengend Snippet: Milk Protein β-casein and Milk Lipid in Mammary Epithelial Cell (MEC) Cultures. Panel 1 shows immunostained MECs images for DAPI, F-actin/BODIPY, adipophilin, with merged and enlarged images. Panels 2 and 3 show immunostained MECs images for DAPI, F-actin/BODIPY, β-casein, with merged and enlarged images. Scale bar 10 µm.

Article Snippet: The primary antibodies anti-FAS (SC-20140) and anti-SREBP (SC-8984) were from Santa Cruz Biotechnology, Inc (Dallas, TX), the anti-β-casein antibody from Abbexa (abx430060, Sugar Land, TX, USA), and the anti-adipophilin antibody from LSBio (LS-C146977-100, Seattle, WA, USA).

Techniques:

Journal: Nutrients

Article Title: Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat

doi: 10.3390/nu14234948

Figure Lengend Snippet: Secreted Milk Protein β-casein in the Apical Medium . Immunoblot of β-casein in MEC “milk” (mouse 25 kDa), human milk as a positive control (23 kDa), and lactogenic medium (no band detected).

Article Snippet: The primary antibodies anti-FAS (SC-20140) and anti-SREBP (SC-8984) were from Santa Cruz Biotechnology, Inc (Dallas, TX), the anti-β-casein antibody from Abbexa (abx430060, Sugar Land, TX, USA), and the anti-adipophilin antibody from LSBio (LS-C146977-100, Seattle, WA, USA).

Techniques: Western Blot, Positive Control

Journal: Nutrients

Article Title: Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat

doi: 10.3390/nu14234948

Figure Lengend Snippet: Effects of Palmitate on Milk Protein β-casein and Lipid Synthesis in Mammary Epithelial Cells . MECs were treated with varying doses of palmitic acid added to the lactation medium in the basolateral chamber for 48 h. Cellular MECs were analyzed for ( a ) protein expression (representative immunoblot shown) and ( b ) mRNA expression of milk lipid regulators. Each treatment was performed in quadruplicate, and differences between treated and untreated MECs were compared using ANOVA with Dunnett’s post-hoc test. Values are fold changes (Mean ± SE); * p < 0.001 treated vs. untreated MECs.

Article Snippet: The primary antibodies anti-FAS (SC-20140) and anti-SREBP (SC-8984) were from Santa Cruz Biotechnology, Inc (Dallas, TX), the anti-β-casein antibody from Abbexa (abx430060, Sugar Land, TX, USA), and the anti-adipophilin antibody from LSBio (LS-C146977-100, Seattle, WA, USA).

Techniques: Expressing, Western Blot

Journal: Nutrients

Article Title: Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat

doi: 10.3390/nu14234948

Figure Lengend Snippet: Effects of Palmitate on Secreted Milk Protein β-casein and Milk Lipid . MECs were treated with varying doses of palmitic acid added to the lactation medium in the basolateral chamber for 48 h. The apical medium was analyzed for ( a ) protein expression of β-casein (representative immunoblot shown) and triglyceride concentration. ( b ) mRNA expression of lipid enzymes. Each treatment was performed in quadruplicate, and differences between treated and untreated MECs were compared using ANOVA with Dunnett’s post-hoc test. Values are fold changes (Mean ± SE); * p < 0.001 treated vs. untreated MECs.

Article Snippet: The primary antibodies anti-FAS (SC-20140) and anti-SREBP (SC-8984) were from Santa Cruz Biotechnology, Inc (Dallas, TX), the anti-β-casein antibody from Abbexa (abx430060, Sugar Land, TX, USA), and the anti-adipophilin antibody from LSBio (LS-C146977-100, Seattle, WA, USA).

Techniques: Expressing, Western Blot, Concentration Assay