Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Detection of FASN protein in TG neurons during viral acute infection and latency by IHC. ( A ) TG tissues were collected from calves subjected to mock infection, acute infection (at 4 days post-infection), or latent infection (at 60 days post-infection). Thin sections of 10 µm were cut from formalin-fixed, paraffin-embedded TG tissues and subjected to FASN protein detection by IHC using FASN-specific polyclonal antibody (Proteintech, cat#10624-2-AP, 1:500). Biotinylated goat anti-rabbit IgG (Vector Laboratories) was used as the secondary antibody. ( B ) The percentage of FASN-positive neurons was calculated from 468 neurons of uninfected calves, 467 neurons from acutely infected calves, and 413 TG neurons from latency calves. Data shown are representative of two independent experiments. Scale bars = 100 µm.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Formalin-fixed Paraffin-Embedded, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: BoAHV-1 infection decreases the expression of FASN protein across various cell cultures. ( A ) MDBK cells that were confluent in 60 mm dishes were either mock infected or infected with BoAHV-1 at an MOI of 1. After 4, 8, 16, and 24 h of infection, cell lysates were prepared and analyzed by Western blotting using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) and virion-associated proteins (VMRD, cat# P170703-001, 1:5,000). ( C ) MDBK cells were either mock-infected or infected with BoAHV-1 at MOIs of 0.1, 1, and 10, respectively. At 24 hpi, cell lysates were prepared for Western blotting to assess the protein expression of FASN and virion-associated proteins. ( E ) Neuro-2A cells were either mock-infected or infected with BoAHV-1 at an MOI of 10. After 12, 24, 36, and 48 hours of infection, cell lysates were prepared and analyzed by Western blotting to detect FASN protein and virion-associated proteins. Tubulin was probed and served as a protein loading control and for subsequent quantitative analysis. ( B, D, and F ) Quantitative analysis of FASN band intensities was performed using the freeware software Image J. Changes in FASN protein levels after infection were calculated relative to mock-infected controls, which were set at 100%. ( G ) Neuro-2A cells in 24-well plates were either mock-infected or infected with BoAHV-1 at an MOI of 10. At 36 and 48 hpi, cell survival was assessed using the CCK-8 assay kit (Beyotime, Shanghai, China, cat# C0038), following the manufacturer’s instructions. ( H ) MDBK and Neuro-2A cells in six-well plates were either mock infected or infected with the virus (MOI of 1 for MDBK cells and MOI of 10 for Neuro-2A cells) for 24 h. Total RNA was then extracted from the cells for the detection of FASN mRNA using RT-qPCR. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed by standard t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Expressing, Western Blot, Control, Software, CCK-8 Assay, Virus, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN localization in BoAHV-1-infected MDBK cells using IFA assay. MDBK cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 4, 16, and 24 h, respectively. The cells were immunostained with antibodies against FASN (Green; Abclonal, cat# A21182 , 1:200) and the viral protein gD (Red; VMRD, cat# 1B8-F11, 1:1,000). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). Zoomed-in images framed in red highlight the typical co-localization of FASN with the viral protein gD. These images are representative of results from three independent experiments. Scale bars = 20 µm. ND, not done.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Cell Culture, Immunofluorescence, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN localization in BoAHV-1-infected Neuro-2A cells using IFA assay. Neuro-2A cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 48 and 72 h, respectively. They were immunostained with antibodies against the FASN protein (Red; Proteintech, cat# 10624-2-AP, 1:600) and the virion-associated protein (Green; VMRD, cat# P170703-001, 1:2,000). Nuclei were counterstained with DAPI (Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). These images are representative of results from three independent experiments. Scale bars = 20 µm.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Cell Culture, Immunofluorescence, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN protein in the Golgi apparatus in MDBK cells. ( A ) MDBK cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 24 h. They were immunostained with antibodies against the FASN protein (Green; Proteintech, cat# 10624-2-AP, 1:600) and GP73 (Red; Proteintech, cat# 66331-1-1g, 1:200). Nuclei were counterstained with DAPI (Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). These images are representative of results from three independent experiments. Scale bars = 20 µm. ( B ) MDBK cells in 100 mm dishes were either mock-infected or infected with BoAHV-1 (MOI = 1) for 24 h. The cells were then collected to isolate the Golgi fraction using a commercial kit (Beijing Biolabo Technology, cat# HR0247-50T), following the manufacturer’s protocol. The lysates were subjected to Western blot analysis using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) and GOLGA1 (Abclonal, cat# A14688, 1:1,000). GOLGA1 served as a marker for the Golgi apparatus and as a protein loading control. The data presented are representative of three independent experiments. ( C ) Quantitative analysis of FASN band intensities was performed using the freeware software Image J. Changes in FASN protein levels after infection were calculated relative to mock-infected controls, which were set at 100%. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed by a standard t -test (** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Cell Culture, Infection, Immunofluorescence, Confocal Microscopy, Western Blot, Marker, Control, Software

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in MDBK cells using transfection of siRNAs. ( A ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or individual siRNAs targeting FASN (150 pmol), referred to as siRNA1, siRNA2, siRNA3, and siRNA4, respectively. At 48 h after transfection, cell lysates were prepared and subjected to Western blot analysis to detect FASN protein levels using an antibody against FASN (Proteintech, cat# 10624-2-AP, 1:10,000). ( C and E ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or the indicated siRNAs (150 pmol). At 36 hpi, the cells were infected with BoAHV-1 at an MOI of 1. After 24 h of infection, the cells were collected and subjected to Western blot analysis using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) ( C ) and virus-associated proteins (VMRD, cat# P170703-001, 1:5,000) ( E ). ( B, D, and F ) The band intensities were quantified using the free software Image J. The intensity of each band was first normalized to that of the respective loading control Tubulin, and then normalized to that of the control transfected with scramble siRNA, which was arbitrarily set to 100%. ( G ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or the indicated siRNAs (150 pmol). At 36 h post-transfection, the cells were infected with the virus at an MOI of 1 for 24 h. Subsequently, total RNA was extracted from the cells for the detection of viral mRNA using RT-qPCR. Primers specific for bICP4, bICP27, viral DNA polymerase, and viral protein gC were used, respectively. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed using a Student’s t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Transfection, Western Blot, Virus, Software, Control, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in MDBK cells using a chemical inhibitor. ( A and C ) MDBK cells in six-well plates were infected with BoAHV-1 (MOI = 1) and treated either with DMSO control or with the FASN-specific inhibitor Cerulenin at the indicated concentrations. At 24 hpi, the cells were collected either to prepare cell lysates for Western blotting using an antibody against virus-associated proteins (VMRD, cat# P170703-001, 1:5,000) ( A ) or to extract DNA for subsequent qPCR analysis using gB-specific primers ( C ). ( B ) The intensity of bands for virus-associated proteins was quantified using the freeware software Image J. Changes in the levels of individual bands upon treatment with the inhibitor were calculated relative to those of the DMSO control, which was set at 100%. ( D ) Virus titers in the supernatants were determined and expressed as TCID 50 /mL. ( E ) MDBK cells in six-well plates were infected with the virus at an MOI of 1 for 24 h and treated with either DMSO control or 10 µM Cerulenin. Total RNA was then extracted from the cells for the detection of viral mRNA using RT-qPCR. Primers specific for bICP27, viral DNA polymerase, and viral protein gC were used, respectively. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed by a standard t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Control, Western Blot, Virus, Software, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in Neuro-2A cells using both siRNA and chemical inhibitor. ( A ) Neuro-2A cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or individual siRNAs targeting FASN (150 pmol), referred to as siRNA1, siRNA2, siRNA3, and siRNA4, respectively. At 48 h post-transfection, cell lysates were prepared and subjected to Western blot analysis to detect FASN protein levels using an antibody against FASN (Proteintech, cat# 10624-2-AP, 1:10,000). ( C ) Neuro-2A cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or siRNA1 (150 pmol). At 36 h post-transfection, the cells were infected with BoAHV-1 at an MOI of 10. After 24 h of infection, the cells were collected and subjected to Western blot analysis using a monoclonal antibody against viral protein gC (VMRD, cat#F2, 1:2,000). ( E ) Neuro-2A cells in six-well plates were infected with BoAHV-1 (MOI = 10) and treated either with DMSO control or with the FASN-specific inhibitor Cerulenin at the indicated concentrations. At 24 hpi, the cell lysates were prepared and subjected to Western blotting to detect viral protein gC. ( B, D, and F ) The band intensities were quantified using the free software Image J. The intensity of each band was first normalized to that of the respective loading control Tubulin, and then normalized to that of either DMSO-treated or scrambled siRNA-transfected control, which was arbitrarily set to 100%. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed using a Student’s t -test (ns, not significant; * P < 0.05; ** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Transfection, Western Blot, Control, Software

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the effects of FASN inhibitor, Cerulenin, on the accumulation of gD in the Golgi apparatus. ( A ) Diagram shows the treatment manner of virus-infected cells by both Cerulenin and Monensin. ( B ) MDBK cells of either mock-infected or virus-infected were treated either with vehicle control DMSO or with Cerulenin (10 µM) or Monensin (10 µM) for a duration of 4 h prior to the termination of infection. At 24 hpi, the cells were collected to purify the Golgi apparatus using a commercial purification kit (Beijing Biolabo Technology, cat# HR0247-50T). Subsequently, Western blot analysis was performed to detect the protein levels of gD in the Golgi fractions. GOLGA1, a marker of the Golgi apparatus, was used as a loading control and for subsequent quantitative analysis. ( C ) The band intensity was analyzed with the free software Image J. The intensity of each band was first normalized to that of the respective loading control GOLGA1, and then normalized to that of the control treated with DMSO, which was arbitrarily set to 1. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed with a Student’s t -test (* P < 0.05, ** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Virus, Infection, Control, Purification, Western Blot, Marker, Software

Journal: International Journal of Molecular Sciences

Article Title: Molecular Insights into the Inhibition of Lipid Accumulation in Hepatocytes by Unique Extracts of Ashwagandha

doi: 10.3390/ijms252212256

Figure Lengend Snippet: Effects of withanolides and Ashwagandha extracts on key regulators of lipid metabolism. ( A ) Western blot showing the effect of Withaferin A (Wi-A) and Withanone (Wi-N) on fatty acid synthase (FASN) protein level in Huh7 cells treated with PA. Quantitation from three independent experiments is shown below. ( B ) Western blot displaying the effect of Wi-A and Wi-N on peroxisome proliferator-activated receptor (PPAR)γ protein levels in palmitic acid (PA)-treated Huh7 cells. Quantitation from three independent experiments is shown below. ( C ) Western blot analysis showing the effect of Ashwagandha extracts on PPARγ protein levels in Huh7 cells treated with PA. Quantitation from three independent experiments is shown below. For the quantitation graphs, data were normalized against the PA group and plotted as fold differences (mean ± SD; n = 3). The statistical significance of the data (mean ± SD; n = 3; and * p < 0.05, ** p < 0.01, and *** p < 0.001) was calculated using an unpaired Student’s t -test.

Article Snippet: The membranes were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h. The blocked membranes were probed with the following target-protein-specific primary antibodies at 4 °C overnight: CARF (raised in our lab [ ]), SREBP-1c (sc-13551, Santa Cruz Biotechnology, Paso Robles, CA, USA), fatty acid synthase (FASN) (NBP1-84733, Novus biologicals, Littleton, CO, USA), PPARγ (PA50194884, Thermo Fisher Scientific, Waltham, MA, USA), and CD36 (ab252922, Abcam, Tokyo, Japan).

Techniques: Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Insights into the Inhibition of Lipid Accumulation in Hepatocytes by Unique Extracts of Ashwagandha

doi: 10.3390/ijms252212256

Figure Lengend Snippet: Effects of Ashwagandha extracts on the de novo lipogenesis pathway. ( A ) A schematic representation of the de novo lipogenesis pathway, key enzymes, and steps involved in fatty acid synthesis is shown. ( B ) Western blot showing the effects of Ashwagandha extracts on precursor and mature sterol regulatory element-binding protein-1c (SREBP-1c) expression levels in Huh7 cells treated with PA. β-actin was used as a loading control. Quantitation from three independent experiments is shown below. ( C ) Ashwagandha extracts decreased SREBP-1c mRNA and its target genes Acetyl-CoA Carboxylase 1 ( ACC1 ), fatty acid synthase ( FASN ) and stearoyl-CoA desaturase 1 ( SCD1 ) in PA-treated HepG2 cells. Data were normalized against the PA group for all panels and plotted as fold differences (mean ± SD; n = 3). The statistical significance of the data (mean ± SD; n = 3; and * p < 0.05, ** p < 0.01, and *** p < 0.001) was calculated using an unpaired Student’s t -test.

Article Snippet: The membranes were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h. The blocked membranes were probed with the following target-protein-specific primary antibodies at 4 °C overnight: CARF (raised in our lab [ ]), SREBP-1c (sc-13551, Santa Cruz Biotechnology, Paso Robles, CA, USA), fatty acid synthase (FASN) (NBP1-84733, Novus biologicals, Littleton, CO, USA), PPARγ (PA50194884, Thermo Fisher Scientific, Waltham, MA, USA), and CD36 (ab252922, Abcam, Tokyo, Japan).

Techniques: Western Blot, Binding Assay, Expressing, Control, Quantitation Assay

Journal: Nature Communications

Article Title: Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat

doi: 10.1038/s41467-023-39266-y

Figure Lengend Snippet: A Schematic representation of the catalytic cycle for de novo fatty acid synthesis in metazoans. B Domain organization and schematic of full length h FASN. Location of the engineered TEV cleavage site is shown by the black-red arrowhead. The expected cleavage fragments are shown at the bottom of the panel.

Article Snippet: A C-terminal 6 × His-tag containing vector harboring h FASN was purchased from Addgene (item # 107138) and primers for cloning were synthesized by Integrated DNA Technologies (idtdna.com).

Techniques:

Journal: Nature Communications

Article Title: Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat

doi: 10.1038/s41467-023-39266-y

Figure Lengend Snippet: A SDS-PAGE analysis using Coomassie staining of the TEV cleavage reaction is shown. Blue and red arrows are full length and cleaved h FASN, respectively. B Separation of cleavage product using size exclusion chromatography. A representative 2D class of traces of uncleaved hFASN in fraction 21 is shown. C SDS-PAGE analysis of the elution fractions (same staining as in ( A )). Representative 2D classes from particle images frozen in vitreous ice are shown from fraction 21 highlighted by *. D Elution profile of h FASN modifying region expressed and purified as an independent construct. SDS-PAGE analysis of elution fractions and 2D classes of fraction 3 are shown. Experiments from ( A ) and ( C ) were repeated once and D repeated two independent times.

Article Snippet: A C-terminal 6 × His-tag containing vector harboring h FASN was purchased from Addgene (item # 107138) and primers for cloning were synthesized by Integrated DNA Technologies (idtdna.com).

Techniques: SDS Page, Staining, Size-exclusion Chromatography, Purification, Construct

Journal: Nature Communications

Article Title: Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat

doi: 10.1038/s41467-023-39266-y

Figure Lengend Snippet: Slices of atomic model of h FASN fitted into the cryoEM density is shown for each domain. Sharpened map is used. Amino acid side chains within the pseudo domains were modeled as alanine when no corresponding densities were observed.

Article Snippet: A C-terminal 6 × His-tag containing vector harboring h FASN was purchased from Addgene (item # 107138) and primers for cloning were synthesized by Integrated DNA Technologies (idtdna.com).

Techniques:

Journal: Nature Communications

Article Title: Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat

doi: 10.1038/s41467-023-39266-y

Figure Lengend Snippet: A Inhibition of the KR activity of full-length TEV engineered h FASN (left) and modifying region expressed and purified as a truncated construct (right) by addition of Denifanstat. Assays are done in two biological replicates (i.e., two independent protein preparations). First 600 s are used to measure specific activity for both assays. Source data are provided as a file. B CryoEM model of ΨME:ΨKR:KR tridomain bound to NADPH (shown as sticks) and Denifanstat (shown as ball and stick). C Model to map density for the drug carved at 2 Å (see supplementary Fig. for uncarved map). D 2D interaction landscape for Denifanstat. Residues are colored based on domain color scheme in Fig. . KR residues in the predicted ACP interaction region are highlighted within boxes. 2D interaction network is generate using LigPlot .

Article Snippet: A C-terminal 6 × His-tag containing vector harboring h FASN was purchased from Addgene (item # 107138) and primers for cloning were synthesized by Integrated DNA Technologies (idtdna.com).

Techniques: Inhibition, Activity Assay, Purification, Construct

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: Combined effect of PD‐1 mAb and Fatostatin combination treatment is dependent on CD8 + T cells and SREBP1‐deficiency of cancer cells. (A) Overall survival of the PDAC patients following Sintilimab therapy stratified by median serum TC levels ( n = 43). (B‐F) C57BL/6 mice were implanted with 7 × 10 5 KPC cells and co‐treated with Fatastatin and PD‐1 mAb. (B) A schematic view of the treatment plan. (C) Kaplan‐Meier survival curves for each group (10 mice/group). (D) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (E‐F) Representative images and statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells, and IFN‐γ + CD8 + T cells) are shown as indicated by flow cytometry (E) and IHC (F). (G‐I) C57BL/6 mice were implanted with 7 ×10 5 KPC cells and received Fatostatin, PD‐1 mAb, CD8α mAb or IgG isotype control (IgG2a) treatment. (G) A schematic view of the treatment plan. (H) Tumor image and statistical analysis of orthotopic PDAC model (6 mice/group). (I) Oil Red O staining was treated with Fatostatin or vehicle control in C57BL/6 mice xenograft tumor samples. (J) KEGG enrichment analysis of proteomics in KPC tumor treated with Fatostatin or vehicle control. (K‐M) C57BL/6 mice were implanted with 7 ×10 5 shSREBP1 KPC or shNC cells and received PD‐1 mAb or IgG2a treatment. (K) Kaplan‐Meier survival curves for each group (10 mice/group). (L) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (M) Statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells) are shown as indicated by flow cytometry. (N) Tumor image and statistical analysis of C57BL/6 mice implanted with 7×10 5 SREBP1‐OE or Vector KPC cells and treated Fatostatin plus PD‐1 mAb. Results are presented as mean ± SEM. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. PD‐1, programmed cell death protein 1; mAb, monoclonal antibody; SREBP1, sterol regulatory element binding proteins1; KPC, KrasG12D/Trp53R172H/Pdx1‐Cre; GZMB, granzyme B; KEGG, Kyoto Encyclopedia of Genes and Genomes; shNC, shRNA negative control; IFN‐γ, interferon‐γ; IHC, immunohistochemistry; OE, overexpression; SEM, standard error of the mean.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Flow Cytometry, Control, Staining, Plasmid Preparation, Binding Assay, shRNA, Negative Control, Immunohistochemistry, Over Expression

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: SREBP1 binds directly to the PCSK9 promoter in PDAC. (A) Signal densities of CUT & Tag‐seq at PCSK9 gene loci in SW1990 cells, showing SREBF1 binding. (B) PCSK9 expression of KPC and PANC02 cells transfected with shSREBP1 or shNC, as determined by RNA‐seq analysis. (C‐D) RT‐PCR (C) and Western blotting (D) analysis of PCSK9 expression of KPC and PANC02 cell transfected with shSREBP1 or shNC. (E) ELISA assay analyzed the Secreted PCSK9 of PDAC cell lines transfected with shSREBP1 or shNC in the culture supernatant. (F‐G) qRT‐PCR (F) and Western blotting (G) analysis of PCSK9 expression of PDAC cell lines treated with or without Fatostatin at the indicated doses for 48 h. (H) SREBF1 binding to the PCSK9 promoter was determined by chromatin immunoprecipitation‐RT‐PCR in human PDAC cell lines (BxPC3 and SW1990). (I) Schematic representation of the PCSK9 promoter cloned into the pGL4.1 vector. Three predicted SREBF1 binding motifs are shown, and promoter constructs containing mutations in these 3 regions to cause SREBF1‐binding deficiency are generated. (J) Analysis of PCSK9 WT or mutant promoter activity in 293T cell lines transfected with SREBF1‐Flag. (K‐M) SREBP1 is positively correlated with PCSK9 in PDAC by multi‐color IHC (K), ordinary IHC (L) and TIMER database (M). (N) The correlation of SREBF1 and CD8 + T cell in PAAD from the TIMER database. (O) Kaplan‐Meier analysis of survival in PDAC according to the expression of PCSK9 in the group with decreased or enriched intratumoral CD8 + T cell infiltration. (P) Comparison of serum PCSK9 levels in patients with objective response versus non‐objective response to anti‐PD‐1 therapy ( n = 43). Results are presented as mean ± SEM, n = 3. And all results were repeated for three times. ns, not significant; P > 0.05, *P < 0.05, **P < 0.01, *** P < 0.001. CUT & Tag‐seq, cleavage under targets and tagmentation sequencing; SREBF1, sterol regulatory element binding factor 1; shNC, shRNA negative control; RNA‐seq, RNA sequencing; ELISA, enzyme‐linked immunosorbent assay; qRT‐PCR:quantitative reverse transcription polymerase chain reaction; PAAD, pancreatic adenocarcinoma; TIMER, tumor immune estimation resource; IHC, immunohistochemistry; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Binding Assay, Expressing, Transfection, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Clone Assay, Plasmid Preparation, Construct, Generated, Mutagenesis, Activity Assay, Comparison, Sequencing, shRNA, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry

Journal: Cancer Communications

Article Title: Lipid metabolism reprograming by SREBP1‐PCSK9 targeting sensitizes pancreatic cancer to immunochemotherapy

doi: 10.1002/cac2.70038

Figure Lengend Snippet: The combined effect of targeting PCSK9 and PD‐1 mAb combination treatment is dependent on CD8 + T cells. (A‐D) C57BL/6 mice were implanted with 7×10 5 shPCSK9 or shNC KPC cells received PD‐1 mAb or IgG2a control. (A) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (B) Kaplan‐Meier survival curves for each group (10 mice/group). (C‐D) Flow cytometry showing tumor‐infiltrating lymphocytes (CD8 + T cells, GZMB + CD8 + T cells) and tumor cell PD‐L1 expression. (E‐F) C57BL/6 mice were implanted with 7×10 5 shPCSK9 KPC or shNC cells received PD‐1 mAb or CD8α mAb treatment. (E) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (F) Flow cytometry and quantification of CD8 + splenocytes confirming immune cell depletion. (G‐H) C57BL/6 mice were implanted with 7×10 5 KPC cells and co‐treated with PCSK9 neutralizing antibodies (Evolocumab or Alirocumab) and PD‐1 mAb. (G) Tumor image and statistical analysis of orthotopic PDAC model (5 mice/group). (H) Kaplan‐Meier survival curves for each group (10 mice/group). (I‐L) The HuPBMC‐NCG PDX model was co‐treated with PCSK9 neutralizing antibodies and PD‐1 mAb. The schematic diagram shows the process of constructing the patient‐derived PDAC‐huPBMC mice model (Top) and treatment schedule (Bottom). (J‐K) Plots of tumor growth (J) and tumor weight (K) for each group (5 mice/group). (L) Representative images and statistical results of tumor‐infiltrating lymphocytes (CD8 + T cells, PD1 + CD8 + T cells, and Tim3 + CD8 + T cells) are shown as indicated by flow cytometry. (M) Kaplan‐Meier survival curves for tumor‐bearing GEMM mice co‐treated with PCSK9 neutralizing antibodies and PD‐1 mAb. Treatments began when the solid tumor was palpable. (N) Diagram illustrating SREBP1‐mediated PD‐L1 regulation. SREBP1 binds to the PD‐L1 promoter to suppress its transcription. Meanwhile, SREBP1 binds to the PCSK9 promoter to promote its transcription, then PCSK9‐mediated degradation of PD‐L1 in the lysosome. Fatostatin (red arrows) impairs the activation of SREBP‐1 by inhibiting the ER‐Golgi translocation of SREBPs, which increases the PD‐L1 expression. Results are presented as mean ± SEM. ns, not significant; P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. PD‐1, programmed death‐1 monoclonal antibody; mAb, monoclonal antibody; IgG2a, immunoglobulin G2a; PDAC, pancreatic ductal adenocarcinoma; shNC, shRNA negative control; PD‐L1, programmed death‐ligand 1; GZMB, granzyme B; HuPBMC, human peripheral blood mononuclear cells; PDX, patient‐derived xenograft; GEMM, genetically engineered mouse model; ER‐Golgi:Endoplasmic Reticulum – Golgi; SREBP1, sterol regulatory element binding protein 1; Tim3, T‐cell immunoglobulin and mucin‐domain containing‐3.

Article Snippet: A week post‐tumor implantation, the mice were divided into six groups: Vehicle + IgG2a (BE0089, BioXCell, West Lebanon, NH, USA) (150 μg per mouse; intraperitoneal injection; every 3 days), Vehicle + PD‐1 mAb (BE0273, BioXCell) (150 μg per mouse; intraperitoneal injection; every 3 days), PD‐1 mAb + TVB‐2640 (HY‐112829, MedChemExpress, New Brunswick, NJ, USA) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Lovastatin (HY‐N0504, MedChemExpress) (10 mg/kg; intragastric administration; every day), PD‐1 mAb + Simvastatin (HY‐17502, MedChemExpress) (50 mg/kg; intragastric administration; every day), PD‐1 mAb + Fatostatin (S8284, Selleck, Houston, TX, USA) (30 mg/kg; intragastric administration; every day).

Techniques: Control, Flow Cytometry, Expressing, Derivative Assay, Activation Assay, Translocation Assay, shRNA, Negative Control, Binding Assay

Journal: Indian Journal of Forensic Medicine & Toxicology

Article Title: Serum Fatty Acid Synthase Level in Patients with Prostate Cancer and Benign Prostatic Hyperplasia

doi: 10.37506/ijfmt.v14i1.118

Figure Lengend Snippet: Figure 1 Concentration FASN in PC, BPH and control groups.

Article Snippet: A total of 100μl serum was analyzed using a commercially available ELISA kit, FASN ELISA (cusabio biotech), according to manufacturer’s recommendations.

Techniques: Concentration Assay, Control