Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells.

doi: 10.1159/000438569

Figure Lengend Snippet: Fig. 2. BHBA promotes de novo fatty acids synthesis. Cells were treated as described in Fig. 1. (A) Western blots of β-actin, SREBP1, FAS, ACC-α and Cidea. (B) Relative protein expression level of SREBP1 normalized to β-actin. (C) Relative protein expression level of Cidea normalized to β-actin. (D) Relati ve protein expression level of FAS normalized to β-actin. (E) Relative protein expression level of ACC-α normalized to β-actin. The data are shown as the mean ± SEM. *, stati stical significance (P < 0.05). **, sta tistical significance (P < 0.01). The symbol * and ** indicate statistical differences with the control group (no BHBA).

Article Snippet: Antibodies against Cidea (Abcam, Cambridge, UK) diluted at 1:500 in TBS-T, SREBP1 (Novus, USA) diluted at 1:1000 in TBS-T, FAS (CST, Danvers, USA) diluted at 1:500 in TBS-T, ACC (Abcam, Cambridge, UK) diluted at 1:1000 in TBS-T, β-actin (Santa Cruz, USA) diluted at 1:1000 in TBS-T. HRP-conjugated goat, anti-mouse or anti-rabbit secondary antibody (Boster, Wuhan, China) diluted at 1:5000 in TBS-T.

Techniques: Western Blot, Expressing, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells.

doi: 10.1159/000438569

Figure Lengend Snippet: Fig. 5. Immunofluore scence staining of SREBP1 in bovine mammary epit helial cells. Cells were tre ated as described in Fig. 3. Immunofluorescence staining of SREBP1 (a, d, g and j) in bovine mammary epithelial cells. Images (c, f, i and m) were mer ged with Hoechst 33258 staining images (b, e, h and k).

Article Snippet: Antibodies against Cidea (Abcam, Cambridge, UK) diluted at 1:500 in TBS-T, SREBP1 (Novus, USA) diluted at 1:1000 in TBS-T, FAS (CST, Danvers, USA) diluted at 1:500 in TBS-T, ACC (Abcam, Cambridge, UK) diluted at 1:1000 in TBS-T, β-actin (Santa Cruz, USA) diluted at 1:1000 in TBS-T. HRP-conjugated goat, anti-mouse or anti-rabbit secondary antibody (Boster, Wuhan, China) diluted at 1:5000 in TBS-T.

Techniques: Staining, Immunofluorescence

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 2. Hepatic endoplasmic reticulum (ER) stress and lipogenic state in dairy cows with fatty liver. (A) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), C/EBP-homologous protein (CHOP), and β-actin. (B) The ratios of p-PERK/PERK, p-IRE1α/IRE1α, GRP78, ATF6, and CHOP in healthy (n = 6) and fatty liver (n = 6) cows. (C) Relative mRNA expression levels of GRP78, ATF4, and sXBP1 in healthy (n = 6) and fatty liver (n = 6) cows. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of ACC1, SREBP-1c, PPARγ in healthy (n = 6) and fatty liver (n = 6) cows. (F) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1) in healthy (n = 6) and fatty liver (n = 6) cows. The data of the control were used to normalize other treatments. Comparisons among groups were calculated using unpaired t-tests and expressed as means ± SEM.

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Western Blot, Expressing, Binding Assay, Control

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 3. Effects of free fatty acid (FFA) treatment on endoplasmic reticulum (ER) stress induction and lipogenic gene expression in calf primary hepatocytes. For the time-course analysis experiment, calf primary hepatocytes were treated with FFA (1.2 mM) for 1, 3, 6, and 12 h, respectively. (A) Identification of cytokeratin-18 immunofluorescence. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER ki- nase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) Caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, and (vi) CHOP were quantified in calf primary hepatocytes following NEFA treatment at different time points. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes following NEFA treatment at different time points. (F) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Comparisons among groups were calculated using a one-way ANOVA with subsequent Bonferroni correction. All data are presented as means ± SEM *P < 0.05, **P < 0.01 compared with the 1 h group.

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Gene Expression, Immunofluorescence, Western Blot, Binding Assay

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 4. The effect of caveolin 1 overexpression on free fatty acid (FFA)-induced endoplasmic reticulum (ER) stress induction and lipogenic gene and protein expression in calf primary hepatocytes. Cells were transfected with pCMV-T7-MCS-3 × FLAG-WPRE-Neo plasmid (pc-DNA) or pCMV-CAV1(cattle)-3 × FLAG-Neo (pc-CAV1) overexpression plasmid by lipofectamine 2000 for 36 h followed by FFA for another 12 h. (A) Relative hepatocytic mRNA levels of caveolin 1. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) Caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (F) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (G) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1) in calf primary hepatocytes. (H) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Immunofluorescence

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 5. The effect of silencing caveolin 1 on free fatty acid (FFA)-induced endoplasmic reticulum (ER) stress induction and lipogenic gene and protein expression in calf primary hepatocytes. Cells were treated with caveolin 1 transient transfection (siCAV1) for 36 h, and incubated with 1.2 mM FFA for another 12 h. (A) Relative hepatocytic mRNA levels of caveolin 1. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP- homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (F) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (G) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory ele- ment binding protein 1 (SREBP1). (H) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Expressing, Transfection, Incubation, Western Blot, Binding Assay, Immunofluorescence

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 6. Effects of caveolin 1 overexpression, tunicamycin (TM), or both, on endoplasmic reticulum (ER) stress induction and lipogenic gene expression in calf primary hepatocytes. Hepatocytes were cultured in the presence or absence of pc-CAV1 for 24 h, cells were treated with 0.5 μg/ mL TM for 24 h. (A) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requir- ing protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (B) The ratios of (i) caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (C) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (D) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (E) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (F) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1). (G) Co-IP analysis to assay the interaction between Flag-caveolin 1 and UPR indicators in calf primary hepatocytes. Calf primary hepatocytes were transfected with pCMV- CAV1(cattle)-3 × FLAG-Neo (pc-CAV1) overexpression plasmid for 48 h. Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Over Expression, Gene Expression, Cell Culture, Western Blot, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: Primary RCC cells (“RCC1/RCC2/RCC3”) or A498 cells, bearing the lentiviral S1P expression construct (“OE-S1P”) or empty vector (“Vec”), were established, expression of listed genes in these cells and parental control cells (“Pare”) was shown ( A – C and G ). Cells were further cultured for applied time periods, CCK-8 OD ( H ), proliferation (by recording nuclear EdU ratio, D and I ), migration, and invasion (“Transwell” assay, E , F , and J ) were tested. RCC1 cells bearing SREBP1 shRNA (“sh-SREBP1”) or SREBP1-expressing construct (OE-SREBP1) were established, control cells were transduced with scrambled control shRNA plus empty vector (“Vec+shC”), expression of SREBP1 mRNA and protein was shown ( K ). Cells were further cultured for applied time periods, viable cell number ( L ), proliferation ( M ), and migration ( N ) were tested similarly. For each assay, n = 5. Data were expressed as the mean ± standard deviation (SD). * P < 0.05 vs. “Vec”/“Vec+shC” group. In this figure, experiments were repeated three times, and similar results were obtained each time.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Expressing, Construct, Plasmid Preparation, Control, Cell Culture, CCK-8 Assay, Migration, Transwell Assay, shRNA, Transduction, Standard Deviation

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: RCC1 xenograft-bearing SCID mice were subjected to i.v . injection of PF-429242 (“PF”, at 10 mg/kg body weight, daily, for 21days) or vehicle control (“Veh”), tumor volumes ( A ) and mice body weights ( D ) were recorded every seven days; The estimated daily tumor growth, in mm 3 per day, was calculated using the described formula ( B ). On Day-35 tumors of the two groups were separated through surgery and individually weighted ( C ). On Day-7 and Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed genes was shown ( E and F ). RCC1 xenograft-bearing SCID mice were subjected to intratumoral injection of SREBP1 shRNA lentivirus (“shSREBP1”), S1P shRNA lentivirus (“shS1P”) or scramble control shRNA lentivirus (“shC”), daily for five days, tumor volumes ( G ) and mice body weights ( H ) were recorded every seven days; At Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed proteins was shown ( I ). Data were expressed as the mean ± standard deviation (SD).* P < 0.05 vs. “Veh”/ “shC” group.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Injection, Control, Isolation, Lysis, Expressing, shRNA, Standard Deviation

Journal: Cell Death & Disease

Article Title: SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

doi: 10.1038/s41419-021-03999-9

Figure Lengend Snippet: Eight ( n = 8) pairs of human RCC tumor tissues (“T”) and surrounding normal renal tissues (“N”) were obtained, expression of listed genes was tested by RT-qPCR ( A - C ) and Western blotting ( D , E ) analyses, results were normalized and quantified. The TCGA cohort shows relative SREBP1 transcripts in 533 cases of ccRCC tissues (“Primary Tumor”) and 72 cases of normal renal tissues (“Normal”) ( F ). Kaplan–Meier Survival analyses of SREBP1 -low ( n = 398, in blue) and SREBP1 -high ( n = 133, in red) ccRCC patients were shown ( G ). Data were expressed as the mean ± standard deviation (SD). * P < 0.05 vs. “N” tissues.

Article Snippet: SREBP1 shRNA lentiviral particles (sc-44327-V, Santa Cruz Biotech, Santa Cruz, CA) or S1P shRNA lentiviral particles (sc-36496-V, Santa Cruz Biotech) were added to RCC cells for 48 h. Puromycin was added to the medium.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation