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Human Protein Atlas lpcat1 protein
Lpcat1 Protein, supplied by Human Protein Atlas, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/lpcat1 protein/product/Human Protein Atlas
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lpcat1 protein - by Bioz Stars, 2026-06

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A Transcriptomic analysis of the TCGA database shows the mRNA expression levels of phosphatidylcholine-metabolizing enzymes, including LPCAT1, CEPT1, CHKA, PEMT, PCYT1A, LPCAT2, LPCAT3, and LPCAT4, in HNSCC ( n = 519) and normal tissues ( n = 44). B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database. C Representative immunohistochemical staining of LPCAT1 protein in normal oral mucosa and HNSCC from the Human Protein Atlas database. Scale bar, 100 μm in the upper image, 20 μm in the lower image. LPCAT1 mRNA ( D ) and protein ( E ) in normal tissues ( n = 6) and tumor tissues ( n = 12) were measured by qPCR and western blotting, respectively. F Representative immunohistochemical staining of normal tissues ( n = 13) and tumor tissues ( n = 13) with LPCAT1 antibody. Scale bar, 200 μm in the upper image, 50 μm in the lower image. Statistical significance determined by Mann–Whitney test ( B , F ) or Student’s t test ( A ) or Student’s t test with Welch’s correction ( D , E ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: Cell Death Discovery

Article Title: Lysophosphatidylcholine acyltransferase 1 promotes head and neck squamous cell carcinoma progression by enhancing COX17-dependent oxidative phosphorylation

doi: 10.1038/s41420-026-02994-3

Figure Lengend Snippet: A Transcriptomic analysis of the TCGA database shows the mRNA expression levels of phosphatidylcholine-metabolizing enzymes, including LPCAT1, CEPT1, CHKA, PEMT, PCYT1A, LPCAT2, LPCAT3, and LPCAT4, in HNSCC ( n = 519) and normal tissues ( n = 44). B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database. C Representative immunohistochemical staining of LPCAT1 protein in normal oral mucosa and HNSCC from the Human Protein Atlas database. Scale bar, 100 μm in the upper image, 20 μm in the lower image. LPCAT1 mRNA ( D ) and protein ( E ) in normal tissues ( n = 6) and tumor tissues ( n = 12) were measured by qPCR and western blotting, respectively. F Representative immunohistochemical staining of normal tissues ( n = 13) and tumor tissues ( n = 13) with LPCAT1 antibody. Scale bar, 200 μm in the upper image, 50 μm in the lower image. Statistical significance determined by Mann–Whitney test ( B , F ) or Student’s t test ( A ) or Student’s t test with Welch’s correction ( D , E ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, MANN-WHITNEY

A CCK-8 assay was used to detect the proliferation of FaDu and SCC15 cells with or without LPCAT1 knockdown. B Cell growth was measured by colony formation assays. C Live/Dead staining for cell death measurement. Representative images were shown. Green represents live cells, and red represents dead cells. The percentage of dead cells was calculated. Scale bar, 50 μm. D Wound-healing assays were used to detect cell migration. Scale bar, 200 μm. E Transwell invasion assays were used to evaluate cell invasive potential. Scale bar, 50 μm, n = 3 for ( A – E ). Statistical significance determined by Student’s t test ( B – E ) or two-way ANOVA ( A ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: Cell Death Discovery

Article Title: Lysophosphatidylcholine acyltransferase 1 promotes head and neck squamous cell carcinoma progression by enhancing COX17-dependent oxidative phosphorylation

doi: 10.1038/s41420-026-02994-3

Figure Lengend Snippet: A CCK-8 assay was used to detect the proliferation of FaDu and SCC15 cells with or without LPCAT1 knockdown. B Cell growth was measured by colony formation assays. C Live/Dead staining for cell death measurement. Representative images were shown. Green represents live cells, and red represents dead cells. The percentage of dead cells was calculated. Scale bar, 50 μm. D Wound-healing assays were used to detect cell migration. Scale bar, 200 μm. E Transwell invasion assays were used to evaluate cell invasive potential. Scale bar, 50 μm, n = 3 for ( A – E ). Statistical significance determined by Student’s t test ( B – E ) or two-way ANOVA ( A ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database.

Techniques: CCK-8 Assay, Knockdown, Staining, Migration

A Body weight was monitored every 5 days for 15 days after tumor implantation. B In vivo fluorescence imaging of FaDu-shLPCAT1 and FaDu-shNC xenografts at endpoint and quantification of fluorescence intensity from endpoint imaging. C Tumor volume was measured at the endpoint. Statistical analysis of tumor volume differences between the LPCAT1 knockdown and control groups. D Representative immunohistochemical staining of tumor tissues from FaDu-shLPCAT1 and FaDu-shNC xenografts with Ki-67 antibody. Scale bar, 50 μm. The percentages of Ki-67-positive cells were calculated. Statistical significance determined by Student’s t test ( C ), Student’s t test with Welch’s correction ( D ), Mann–Whitney test ( B ). * P < 0.05.

Journal: Cell Death Discovery

Article Title: Lysophosphatidylcholine acyltransferase 1 promotes head and neck squamous cell carcinoma progression by enhancing COX17-dependent oxidative phosphorylation

doi: 10.1038/s41420-026-02994-3

Figure Lengend Snippet: A Body weight was monitored every 5 days for 15 days after tumor implantation. B In vivo fluorescence imaging of FaDu-shLPCAT1 and FaDu-shNC xenografts at endpoint and quantification of fluorescence intensity from endpoint imaging. C Tumor volume was measured at the endpoint. Statistical analysis of tumor volume differences between the LPCAT1 knockdown and control groups. D Representative immunohistochemical staining of tumor tissues from FaDu-shLPCAT1 and FaDu-shNC xenografts with Ki-67 antibody. Scale bar, 50 μm. The percentages of Ki-67-positive cells were calculated. Statistical significance determined by Student’s t test ( C ), Student’s t test with Welch’s correction ( D ), Mann–Whitney test ( B ). * P < 0.05.

Article Snippet: B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database.

Techniques: Tumor Implantation, In Vivo, Fluorescence, Imaging, Knockdown, Control, Immunohistochemical staining, Staining, MANN-WHITNEY

A CCK-8 assay was used to monitor cell proliferation. B Volcano plot of differentially expressed genes (DEGs) in LPCAT1-knockdown (FaDu-siLPCAT1) and control (FaDu-siNC) cells, showing 622 downregulated and 371 upregulated genes (fold change ≥2, P adjust <0.05). C KEGG pathway enrichment analysis of differentially expressed genes ( P adjust <0.01). D Differential pathway activity between LPCAT1-knockdown and control groups ( P adjust <0.05). E Heatmaps displaying GSVA enrichment scores per sample for each module. F The mitochondrial membrane potential was detected by JC-1 staining. Images were acquired using fluorescence microscopy. Scale bar, 50 μm. G ATP production was measured by quantification assays following LPCAT1 knockdown or overexpression. Mitochondrial respiration was measured by Seahorse XFp analysis following LPCAT1 knockdown ( H ) or overexpression ( I ). Basal OCR, ATP-linked OCR, and maximal OCR were calculated. n = 3 for ( A – I ). Statistical significance determined by two-way ANOVA test ( A ), or one-way ANOVA with Dunnett’s multiple comparison test ( F ), or Student’s t test ( G – I ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Cell Death Discovery

Article Title: Lysophosphatidylcholine acyltransferase 1 promotes head and neck squamous cell carcinoma progression by enhancing COX17-dependent oxidative phosphorylation

doi: 10.1038/s41420-026-02994-3

Figure Lengend Snippet: A CCK-8 assay was used to monitor cell proliferation. B Volcano plot of differentially expressed genes (DEGs) in LPCAT1-knockdown (FaDu-siLPCAT1) and control (FaDu-siNC) cells, showing 622 downregulated and 371 upregulated genes (fold change ≥2, P adjust <0.05). C KEGG pathway enrichment analysis of differentially expressed genes ( P adjust <0.01). D Differential pathway activity between LPCAT1-knockdown and control groups ( P adjust <0.05). E Heatmaps displaying GSVA enrichment scores per sample for each module. F The mitochondrial membrane potential was detected by JC-1 staining. Images were acquired using fluorescence microscopy. Scale bar, 50 μm. G ATP production was measured by quantification assays following LPCAT1 knockdown or overexpression. Mitochondrial respiration was measured by Seahorse XFp analysis following LPCAT1 knockdown ( H ) or overexpression ( I ). Basal OCR, ATP-linked OCR, and maximal OCR were calculated. n = 3 for ( A – I ). Statistical significance determined by two-way ANOVA test ( A ), or one-way ANOVA with Dunnett’s multiple comparison test ( F ), or Student’s t test ( G – I ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database.

Techniques: CCK-8 Assay, Knockdown, Control, Activity Assay, Membrane, Staining, Fluorescence, Microscopy, Over Expression, Comparison

A Transcriptomic analysis highlighting COX17 as a top dysregulated gene within the oxidative phosphorylation pathway in LPCAT1-knockdown cells. B qPCR validation of top 10 genes mRNA levels. C COX17 protein was measured by western blotting after LPCAT1 knockdown or overexpression. Representative immunofluorescence staining of LPCAT1 knockdown ( D ) or overexpression ( E ) cells with mitotracker and COX17 antibody. Images were acquired using confocal microscopy. Scale bar, 20 μm. F Western blotting analysis of electron transport chain complex core subunits (I–V). G Microplate assay measurement of CcO activity after LPCAT1 knockdown or overexpression. Overexpress COX17 in LPCAT1-knockdown cells or silence COX17 in LPCAT1-overexpression cells. CcO activity ( H ) and ATP production ( I ) were detected, and a mitochondrial stress test ( J ) was performed. K The potential transcription factors of COX17 were predicted by Genecard, KnockTF, and hTFtarget. Representative immunofluorescence staining of LPCAT1 knockdown ( L ) or overexpression ( M ) cells with SP1 antibody. Scale, 20 μm. SP1 in the nucleus is indicated by white arrows. n = 3 for ( B – J , L , M ). Statistical significance determined by Student’s t test ( B , C , F , G ), or one-way ANOVA with Tukey’s multiple comparison test ( H – J ). * P < 0.05; ** P < 0.01, *** P < 0.001, # P < 0.05, ## P <0.01.

Journal: Cell Death Discovery

Article Title: Lysophosphatidylcholine acyltransferase 1 promotes head and neck squamous cell carcinoma progression by enhancing COX17-dependent oxidative phosphorylation

doi: 10.1038/s41420-026-02994-3

Figure Lengend Snippet: A Transcriptomic analysis highlighting COX17 as a top dysregulated gene within the oxidative phosphorylation pathway in LPCAT1-knockdown cells. B qPCR validation of top 10 genes mRNA levels. C COX17 protein was measured by western blotting after LPCAT1 knockdown or overexpression. Representative immunofluorescence staining of LPCAT1 knockdown ( D ) or overexpression ( E ) cells with mitotracker and COX17 antibody. Images were acquired using confocal microscopy. Scale bar, 20 μm. F Western blotting analysis of electron transport chain complex core subunits (I–V). G Microplate assay measurement of CcO activity after LPCAT1 knockdown or overexpression. Overexpress COX17 in LPCAT1-knockdown cells or silence COX17 in LPCAT1-overexpression cells. CcO activity ( H ) and ATP production ( I ) were detected, and a mitochondrial stress test ( J ) was performed. K The potential transcription factors of COX17 were predicted by Genecard, KnockTF, and hTFtarget. Representative immunofluorescence staining of LPCAT1 knockdown ( L ) or overexpression ( M ) cells with SP1 antibody. Scale, 20 μm. SP1 in the nucleus is indicated by white arrows. n = 3 for ( B – J , L , M ). Statistical significance determined by Student’s t test ( B , C , F , G ), or one-way ANOVA with Tukey’s multiple comparison test ( H – J ). * P < 0.05; ** P < 0.01, *** P < 0.001, # P < 0.05, ## P <0.01.

Article Snippet: B LPCAT1 protein expression in HNSCC ( n = 109) and normal tissues ( n = 70) from the Human Protein Atlas database.

Techniques: Phospho-proteomics, Knockdown, Biomarker Discovery, Western Blot, Over Expression, Immunofluorescence, Staining, Confocal Microscopy, Activity Assay, Comparison

Lpcat1 catalyzes histone H4 protein palmitoylation in vitro and in vivo. A, in vitro palmitoylation. Histone H4 protein palmitoylation reactions were conducted in the presence of Lpcat1, heat-inactivated (denatured) Lpcat1, and a related palmitoyltransferase, SPTLC2, using [14C]palmitoyl-CoA as a donor and recombinant histone H4 substrate. The lower panel shows histone H4 protein input controls. B, the relevant bands on nitrocellulose membranes were cut, and the radioactivity was counted using a scintillation counter. *, p = 0.0017, radioactivity of Lpcat1 versus dpm of heat-inactivated Lpcat1. C, histone H4 protein palmitoylation reactions were conducted in the presence of lung microsomes, heat-inactivated Lpcat1, and SPTLC2. *, p = 0.0002, radioactivity of microsome versus dpm of heat-inactivated microsome. D, in vivo palmitoylation. MLE cells were pulse-labeled with [3H]palmitoyl acid in the presence of 2 mm Ca2+ for 2 h. Cell lysates were immunoprecipitated (IP) with anti-H4 antibody or IgG to detect palmitoylated H4 followed by autoradiography. Cell lysates were analyzed by V5 immunoblotting as an input control in the lower panel. E, MLE cells were pulse-labeled with [3H]palmitoyl acid or [3H]oleic acid in the presence or absence of 2 mm Ca2+ for 2 h. Cell lysates were immunoprecipitated with H4 antibody, and the radioactivity of the precipitates was measured by scintillation counting. *, p = 0.017, radioactivity of H4 in palmitic acid group versus oleic acid group. F, HEK 293 cells were transfected with pcDNA3.1/Lpcat1 or a Lpcat1 catalytically inactive mutant (Lpcat1 H135A) for 24 h. Cells were pulse-labeled with [3H]palmitoyl acid with or without Ca2+ as above. Histone radioactivity was determined as described in E. The inset shows the protein expression levels of Lpcat1 wt and Lpcat1 H135A mutant and β-actin. *, p = 0.0005, radioactivity of wild type Lpcat1 versus dpm of H135A Lpcat1. The data represent three independent experiments. Error bars, S.E.

Journal: The Journal of Biological Chemistry

Article Title: Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (Lpcat1) Catalyzes Histone Protein O -Palmitoylation to Regulate mRNA Synthesis *

doi: 10.1074/jbc.M111.253385

Figure Lengend Snippet: Lpcat1 catalyzes histone H4 protein palmitoylation in vitro and in vivo. A, in vitro palmitoylation. Histone H4 protein palmitoylation reactions were conducted in the presence of Lpcat1, heat-inactivated (denatured) Lpcat1, and a related palmitoyltransferase, SPTLC2, using [14C]palmitoyl-CoA as a donor and recombinant histone H4 substrate. The lower panel shows histone H4 protein input controls. B, the relevant bands on nitrocellulose membranes were cut, and the radioactivity was counted using a scintillation counter. *, p = 0.0017, radioactivity of Lpcat1 versus dpm of heat-inactivated Lpcat1. C, histone H4 protein palmitoylation reactions were conducted in the presence of lung microsomes, heat-inactivated Lpcat1, and SPTLC2. *, p = 0.0002, radioactivity of microsome versus dpm of heat-inactivated microsome. D, in vivo palmitoylation. MLE cells were pulse-labeled with [3H]palmitoyl acid in the presence of 2 mm Ca2+ for 2 h. Cell lysates were immunoprecipitated (IP) with anti-H4 antibody or IgG to detect palmitoylated H4 followed by autoradiography. Cell lysates were analyzed by V5 immunoblotting as an input control in the lower panel. E, MLE cells were pulse-labeled with [3H]palmitoyl acid or [3H]oleic acid in the presence or absence of 2 mm Ca2+ for 2 h. Cell lysates were immunoprecipitated with H4 antibody, and the radioactivity of the precipitates was measured by scintillation counting. *, p = 0.017, radioactivity of H4 in palmitic acid group versus oleic acid group. F, HEK 293 cells were transfected with pcDNA3.1/Lpcat1 or a Lpcat1 catalytically inactive mutant (Lpcat1 H135A) for 24 h. Cells were pulse-labeled with [3H]palmitoyl acid with or without Ca2+ as above. Histone radioactivity was determined as described in E. The inset shows the protein expression levels of Lpcat1 wt and Lpcat1 H135A mutant and β-actin. *, p = 0.0005, radioactivity of wild type Lpcat1 versus dpm of H135A Lpcat1. The data represent three independent experiments. Error bars, S.E.

Article Snippet: Lpcat1 and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) recombinant proteins and Lpcat1 small hairpin RNA (shRNA) plasmid were from Origene (Rockville, MD).

Techniques: In Vitro, In Vivo, Recombinant, Radioactivity, Labeling, Immunoprecipitation, Autoradiography, Western Blot, Control, Transfection, Mutagenesis, Expressing

$(A) HEK293 cells transfected with LPCAT-HA or LPCAT-FLAG were costained with α-HA and α-CALR or α-FLAG and α-CALR. Note the localization of LPCAT-HA and LPCAT-FLAG proteins to the ER. Scale bars: 10 μm. (B) Hydropathy plot of mouse LPCAT1 protein as predicted from the TMpred algorithm. Scores greater than 500 (red line) are considered hydrophobic. A single TMD is predicted, encompassing amino acids 53–74. Bottom panel: Diagram depicts the position of the TMD of LPCAT1 protein relative to the acyltransferase (PlsC) domain. (C) Membrane fractionation assay on HEK293 cells expressing LPCAT1-HA. Pellet (P) and supernatant (S) fractions were isolated after incubation of the membranes in the listed additives and subjected to immunoblot analysis with α-HA antibody. Blots were stripped and reprobed with an antibody against endogenous CANX, an ER-resident transmembrane protein. (D) Trypsin protection assay. Microsomes isolated from HEK293 cells transiently expressing LPCAT-HA or LPCAT-FLAG were incubated with buffer only, trypsin, or trypsin plus Triton X-100 and subjected to immunoblot analysis. Note the approximate 5-kDa shift of the LPCAT-HA protein in the presence of trypsin (IB: HA and IB: LPCAT1) and failure to detect LPCAT-FLAG in the presence of trypsin (IB: FLAG), indicating a type II orientation. A model depicting LPCAT1 in a type II orientation within a microsome is shown on the right.$

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) HEK293 cells transfected with LPCAT-HA or LPCAT-FLAG were costained with α-HA and α-CALR or α-FLAG and α-CALR. Note the localization of LPCAT-HA and LPCAT-FLAG proteins to the ER. Scale bars: 10 μm. (B) Hydropathy plot of mouse LPCAT1 protein as predicted from the TMpred algorithm. Scores greater than 500 (red line) are considered hydrophobic. A single TMD is predicted, encompassing amino acids 53–74. Bottom panel: Diagram depicts the position of the TMD of LPCAT1 protein relative to the acyltransferase (PlsC) domain. (C) Membrane fractionation assay on HEK293 cells expressing LPCAT1-HA. Pellet (P) and supernatant (S) fractions were isolated after incubation of the membranes in the listed additives and subjected to immunoblot analysis with α-HA antibody. Blots were stripped and reprobed with an antibody against endogenous CANX, an ER-resident transmembrane protein. (D) Trypsin protection assay. Microsomes isolated from HEK293 cells transiently expressing LPCAT-HA or LPCAT-FLAG were incubated with buffer only, trypsin, or trypsin plus Triton X-100 and subjected to immunoblot analysis. Note the approximate 5-kDa shift of the LPCAT-HA protein in the presence of trypsin (IB: HA and IB: LPCAT1) and failure to detect LPCAT-FLAG in the presence of trypsin (IB: FLAG), indicating a type II orientation. A model depicting LPCAT1 in a type II orientation within a microsome is shown on the right.

Article Snippet: The cDNA encoding amino acids 413–534 of mouse LPCAT1 protein was amplified from LPCAT1/pcDNA3.1+ and cloned into the pET41a+ vector (Novagen) to create LPCAT1 413–534 /pET41a+.

Techniques: Transfection, Fractionation, Expressing, Isolation, Incubation, Western Blot

(A) Acyltransferase activity assays were conducted by incubating 150 μM lysoPC and 25 μM 1-14C-palmitoyl-CoA with 20 μg of lysates from cells transfected with empty vector (EV), wild-type LPCAT1 (LPCAT1 WT), or LPCAT1 with an alanine substitution for histidine at amino acid position 135 (LPCAT1 H135A). The complete abrogation of acyltransferase activity by the substitution of alanine for histidine identifies it as a critical residue. Data represent activities from 3 independent experiments with each group assayed in triplicate. *P < 0.001 versus EV. (B) Immunoblot analysis of whole cell lysates from A with α-HA and α-LPCAT1 antibodies. Note similar levels of expression of WT and H135A LPCAT1. (C) Subcellular localization of LPCAT WT and LPCAT H135A to ER in HEK293 cells. Merged images demonstrate colocalization (yellow) of HA epitope (red) and an ER marker, CALR (green). Scale bars: 10 μm. (D) Diagram depicting type II orientation of LPCAT1 in ER membrane with amino terminus (blue) in cytosol, a single-pass TMD (red) spanning the lipid bilayer, and carboxyl terminus (green) in ER lumen. Note the localization of His135 (H135) in ER lumen.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Acyltransferase activity assays were conducted by incubating 150 μM lysoPC and 25 μM 1-14C-palmitoyl-CoA with 20 μg of lysates from cells transfected with empty vector (EV), wild-type LPCAT1 (LPCAT1 WT), or LPCAT1 with an alanine substitution for histidine at amino acid position 135 (LPCAT1 H135A). The complete abrogation of acyltransferase activity by the substitution of alanine for histidine identifies it as a critical residue. Data represent activities from 3 independent experiments with each group assayed in triplicate. *P < 0.001 versus EV. (B) Immunoblot analysis of whole cell lysates from A with α-HA and α-LPCAT1 antibodies. Note similar levels of expression of WT and H135A LPCAT1. (C) Subcellular localization of LPCAT WT and LPCAT H135A to ER in HEK293 cells. Merged images demonstrate colocalization (yellow) of HA epitope (red) and an ER marker, CALR (green). Scale bars: 10 μm. (D) Diagram depicting type II orientation of LPCAT1 in ER membrane with amino terminus (blue) in cytosol, a single-pass TMD (red) spanning the lipid bilayer, and carboxyl terminus (green) in ER lumen. Note the localization of His135 (H135) in ER lumen.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Marker

(A) Immunohistochemistry of adult mouse lung with α-LPCAT1 antibody demonstrating restricted expression in alveolar type II cells. Note robust staining in alveolar type II cells (inset) and absence of signal in the proximal epithelium (arrowheads). Original magnification, ×40; inset magnification, ×100. (B) qPCR analysis of Lpcat1, Sftpc, Fasn, and Scd1 mRNA in primary mouse type II cells after treatment with LPCAT1 siRNA or a scrambled control siRNA (con siRNA) for 48 hours. Gene expression was normalized to Actb mRNA. Data represent 5 independent experiments with each group assayed in triplicate. *P < 0.01 versus HVJ-E only. (C) Immunoblot analysis of LPCAT1 from whole cell lysates described in B with α-LPCAT1 antibody at days 3 and 7 after siRNA administration. (D) Primary alveolar type II cells were cultured in the presence of siRNAs as outlined in B and labeled with [3H]palmitic acid (3H-PA). Palmitic acid incorporation into SatPC was measured and normalized to genomic DNA. Depletion of Lpcat1 mRNA significantly reduced [3H]palmitate incorporation into SatPC. Data represent 3 independent experiments; *P < 0.01 versus control siRNA or HVJ-E only. (E) MLE15 cells expressing empty vector or LPCAT-HA were labeled with [3H]palmitic acid. Palmitic acid incorporation into SatPC was significantly increased by expression of LPCAT1. Right: Increased expression of LPCAT1 in transfected cells. Data are pooled from 3 independent experiments, each performed in triplicate; *P = 0.006 versus EV.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Immunohistochemistry of adult mouse lung with α-LPCAT1 antibody demonstrating restricted expression in alveolar type II cells. Note robust staining in alveolar type II cells (inset) and absence of signal in the proximal epithelium (arrowheads). Original magnification, ×40; inset magnification, ×100. (B) qPCR analysis of Lpcat1, Sftpc, Fasn, and Scd1 mRNA in primary mouse type II cells after treatment with LPCAT1 siRNA or a scrambled control siRNA (con siRNA) for 48 hours. Gene expression was normalized to Actb mRNA. Data represent 5 independent experiments with each group assayed in triplicate. *P < 0.01 versus HVJ-E only. (C) Immunoblot analysis of LPCAT1 from whole cell lysates described in B with α-LPCAT1 antibody at days 3 and 7 after siRNA administration. (D) Primary alveolar type II cells were cultured in the presence of siRNAs as outlined in B and labeled with [3H]palmitic acid (3H-PA). Palmitic acid incorporation into SatPC was measured and normalized to genomic DNA. Depletion of Lpcat1 mRNA significantly reduced [3H]palmitate incorporation into SatPC. Data represent 3 independent experiments; *P < 0.01 versus control siRNA or HVJ-E only. (E) MLE15 cells expressing empty vector or LPCAT-HA were labeled with [3H]palmitic acid. Palmitic acid incorporation into SatPC was significantly increased by expression of LPCAT1. Right: Increased expression of LPCAT1 in transfected cells. Data are pooled from 3 independent experiments, each performed in triplicate; *P = 0.006 versus EV.

Techniques: Immunohistochemistry, Expressing, Staining, Western Blot, Cell Culture, Labeling, Plasmid Preparation, Transfection

(A) Top: Diagram of Lpcat1 locus. Boxes denote exons, and intervening lines denote introns. The β-geo selection cassette encoding a β-galactosidase/neomycin fusion protein was inserted in intron 9, approximately 400 bp 3′ of exon 9. f1, f2, and r2 denote primers used to confirm the presence of GT cassette in the Lpcat1 locus by qPCR analysis. Bottom: PCR analysis of the Lpcat1 locus in GT ES cells or parental ES cells (129J/Ola) using 2 separate primer sets. The presence of amplicons in GT ES cells with the absence of signal in parental cells demonstrates trapping of the Lpcat1 locus at exon 9. Vertical line indicates discontinuous lanes in the same gel. (B) qPCR analysis of GT ES cells. cDNA from GT ES cells (GT) and parental cells (WT) was subjected to qPCR analysis using 3 separate exon-spanning TaqMan primer/probe sets for Lpcat1 as depicted in the bottom panel: 2/3, 9/10, and 12/13. Data were normalized to Actb and represent 3 independent experiments, each performed in triplicate. Note the difference in signal between 2/3 probe and 9/10 probe in GT cells. †P < 0.05 versus 9/10 and 12/13 GT;*P < 0.05 versus 2/3 WT. RQ, relative quantification. (C) Multiplex PCR genotyping of genomic DNA from progeny of Lpcat1GT/+ intercrosses. Amplicon for WT allele is approximately 350 bp; GT, approximately 300 bp.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Top: Diagram of Lpcat1 locus. Boxes denote exons, and intervening lines denote introns. The β-geo selection cassette encoding a β-galactosidase/neomycin fusion protein was inserted in intron 9, approximately 400 bp 3′ of exon 9. f1, f2, and r2 denote primers used to confirm the presence of GT cassette in the Lpcat1 locus by qPCR analysis. Bottom: PCR analysis of the Lpcat1 locus in GT ES cells or parental ES cells (129J/Ola) using 2 separate primer sets. The presence of amplicons in GT ES cells with the absence of signal in parental cells demonstrates trapping of the Lpcat1 locus at exon 9. Vertical line indicates discontinuous lanes in the same gel. (B) qPCR analysis of GT ES cells. cDNA from GT ES cells (GT) and parental cells (WT) was subjected to qPCR analysis using 3 separate exon-spanning TaqMan primer/probe sets for Lpcat1 as depicted in the bottom panel: 2/3, 9/10, and 12/13. Data were normalized to Actb and represent 3 independent experiments, each performed in triplicate. Note the difference in signal between 2/3 probe and 9/10 probe in GT cells. †P < 0.05 versus 9/10 and 12/13 GT;*P < 0.05 versus 2/3 WT. RQ, relative quantification. (C) Multiplex PCR genotyping of genomic DNA from progeny of Lpcat1GT/+ intercrosses. Amplicon for WT allele is approximately 350 bp; GT, approximately 300 bp.

Techniques: Selection, Multiplex Assay, Amplification

(A) Measurement of lung tissue SatPC content in E18.5 mice demonstrates that Lpcat1GT/GT lungs contain significantly less SatPC. Data represent n = 5 per genotype and are normalized to tissue weight. *P < 0.01 versus Lpcat1+/+ and Lpcat1GT/+. (B) qPCR analysis of Abca3, Sftpb, and Sftpc from lung tissue of E18.5 mice shows that expression of these genes is unchanged. Data represent n = 5 for each genotype and are normalized to Actb. (C) Immunoblot analysis of lung homogenate from E18.5 mice using antisera directed against the C terminus of LPCAT1 (Mr ~60 kDa), pro-SFTPC (Mr ~21 kDa), the mature peptide of SFTPC (Mr ~4 kDa), or the mature peptide of SFTPB (Mr ~16 kDa, nonreduced). The blot for mature SFTPB was stripped and reprobed with α-ACTIN as a loading control. Note the lack of LPCAT1 immunoreactivity in Lpcat1GT/GT mice (arrow) and no change in pro-SFTPC, mature SFTPC, or mature SFTPB protein. (D) Immunohistochemical analysis of pro-SFTPC, mature SFTPB, LPCAT1, and ABCA3 in lung sections from Lpcat1GT/GT and Lpcat1+/+ mice confirms the results in C. Scale bars: 50 μm.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Measurement of lung tissue SatPC content in E18.5 mice demonstrates that Lpcat1GT/GT lungs contain significantly less SatPC. Data represent n = 5 per genotype and are normalized to tissue weight. *P < 0.01 versus Lpcat1+/+ and Lpcat1GT/+. (B) qPCR analysis of Abca3, Sftpb, and Sftpc from lung tissue of E18.5 mice shows that expression of these genes is unchanged. Data represent n = 5 for each genotype and are normalized to Actb. (C) Immunoblot analysis of lung homogenate from E18.5 mice using antisera directed against the C terminus of LPCAT1 (Mr ~60 kDa), pro-SFTPC (Mr ~21 kDa), the mature peptide of SFTPC (Mr ~4 kDa), or the mature peptide of SFTPB (Mr ~16 kDa, nonreduced). The blot for mature SFTPB was stripped and reprobed with α-ACTIN as a loading control. Note the lack of LPCAT1 immunoreactivity in Lpcat1GT/GT mice (arrow) and no change in pro-SFTPC, mature SFTPC, or mature SFTPB protein. (D) Immunohistochemical analysis of pro-SFTPC, mature SFTPB, LPCAT1, and ABCA3 in lung sections from Lpcat1GT/GT and Lpcat1+/+ mice confirms the results in C. Scale bars: 50 μm.

Techniques: Expressing, Western Blot, Immunohistochemical staining

(A) Cyanotic appearance of a newborn Lpcat1GT/GT mouse compared with wild-type littermate. (B) qPCR analysis of lung tissue from individual newborn mice using 2 primer/probe sets for Lpcat1. Data were normalized to Actb; each bar represents a single animal, and error bars represent error within technical replicates. Note the decreased amount of Lpcat1 in dead versus alive mice as detected with an exon 2/3 probe, suggesting variable mRNA stability. (C) Tissue SatPC content in newborn mice. Data represent n = 17 per genotype for Lpcat1GT/GT dead and alive, n = 9 for Lpcat1GT/+ and Lpcat1+/+. *P < 0.05 versus Lpcat1GT/GT alive; #P < 0.001 versus Lpcat1GT/GT dead and alive. (D) SatPC correlates with Lpcat1 mRNA in newborn mice. Lpcat1 mRNA was detected by qPCR using the exon2/3 primer/probe set and normalized to Actb. Data represent n = 9 for each genotype, and both assays were performed on lung tissue from the same animal. (E) Acyltransferase activity assays from lung tissue of newborn mice. Lpcat1GT/GT mice had significantly less acyltransferase activity than controls. Data represent n = 5 per genotype. *P < 0.05 versus Lpcat1+/+ mice. (F) SatPC content correlates with LPCAT1 activity in newborn mice. Data represent n = 5 for each genotype. (G) Tissue SatPC levels in E18.5 and P1 mice. Note the 3.3-fold increase in SatPC in Lpcat1+/+ mice from E18.5 to P1 versus 2.0-fold and 2.6-fold increases in Lpcat1GT/GT dead and alive mice, respectively. *P < 0.001 versus E18.5 of respective genotype; #P < 0.01 versus P1 Lpcat1GT/GT alive mice.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Cyanotic appearance of a newborn Lpcat1GT/GT mouse compared with wild-type littermate. (B) qPCR analysis of lung tissue from individual newborn mice using 2 primer/probe sets for Lpcat1. Data were normalized to Actb; each bar represents a single animal, and error bars represent error within technical replicates. Note the decreased amount of Lpcat1 in dead versus alive mice as detected with an exon 2/3 probe, suggesting variable mRNA stability. (C) Tissue SatPC content in newborn mice. Data represent n = 17 per genotype for Lpcat1GT/GT dead and alive, n = 9 for Lpcat1GT/+ and Lpcat1+/+. *P < 0.05 versus Lpcat1GT/GT alive; #P < 0.001 versus Lpcat1GT/GT dead and alive. (D) SatPC correlates with Lpcat1 mRNA in newborn mice. Lpcat1 mRNA was detected by qPCR using the exon2/3 primer/probe set and normalized to Actb. Data represent n = 9 for each genotype, and both assays were performed on lung tissue from the same animal. (E) Acyltransferase activity assays from lung tissue of newborn mice. Lpcat1GT/GT mice had significantly less acyltransferase activity than controls. Data represent n = 5 per genotype. *P < 0.05 versus Lpcat1+/+ mice. (F) SatPC content correlates with LPCAT1 activity in newborn mice. Data represent n = 5 for each genotype. (G) Tissue SatPC levels in E18.5 and P1 mice. Note the 3.3-fold increase in SatPC in Lpcat1+/+ mice from E18.5 to P1 versus 2.0-fold and 2.6-fold increases in Lpcat1GT/GT dead and alive mice, respectively. *P < 0.001 versus E18.5 of respective genotype; #P < 0.01 versus P1 Lpcat1GT/GT alive mice.

Techniques: Activity Assay

(A–F). H&E-stained lung tissue sections from newborn Lpcat1GT/GT mice that succumbed from RDS, showing areas of atelectasis and hemorrhaging (C, arrow) compared with less-affected areas in the same lung (C, arrowhead) and lungs of asymptomatic Lpcat1GT/GT (B) and wild-type littermates (A). (D–F) High-power magnifications of areas boxed in A–C. ABCA3 protein levels were assessed by immunostaining (G–I). Scale bars: 500 μm (A–C), 100 μm (D–F), 50 μm (G–I). (J) qPCR analysis of Abca3, Sftpb, and Sftpc from lung tissue of newborn mice. Data represent n = 5 for each genotype and were normalized to Actb. (K) Immunoblot analysis of lung homogenate from newborn mice using antisera directed against pro-SFTPC (Mr ~21 kDa), the mature peptide of SFTPC (Mr ~4 kDa), or the mature peptide of SFTPB (Mr ~16 kDa, nonreduced). The blot for mature SFTPB was stripped and reprobed with α-ACTIN as a loading control. (L–O) Representative transmission electron micrograph of lung tissue from a newborn Lpcat1GT/GT RDS mouse and a Lpcat1+/+ littermate control. Note the numerous lamellar bodies in alveolar type II cells (arrowheads, L and M) and abundant luminal surfactant and tubular myelin structures (arrows, N and O) in both genotypes. Scale bars: 2 μm (L–O).

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A–F). H&E-stained lung tissue sections from newborn Lpcat1GT/GT mice that succumbed from RDS, showing areas of atelectasis and hemorrhaging (C, arrow) compared with less-affected areas in the same lung (C, arrowhead) and lungs of asymptomatic Lpcat1GT/GT (B) and wild-type littermates (A). (D–F) High-power magnifications of areas boxed in A–C. ABCA3 protein levels were assessed by immunostaining (G–I). Scale bars: 500 μm (A–C), 100 μm (D–F), 50 μm (G–I). (J) qPCR analysis of Abca3, Sftpb, and Sftpc from lung tissue of newborn mice. Data represent n = 5 for each genotype and were normalized to Actb. (K) Immunoblot analysis of lung homogenate from newborn mice using antisera directed against pro-SFTPC (Mr ~21 kDa), the mature peptide of SFTPC (Mr ~4 kDa), or the mature peptide of SFTPB (Mr ~16 kDa, nonreduced). The blot for mature SFTPB was stripped and reprobed with α-ACTIN as a loading control. (L–O) Representative transmission electron micrograph of lung tissue from a newborn Lpcat1GT/GT RDS mouse and a Lpcat1+/+ littermate control. Note the numerous lamellar bodies in alveolar type II cells (arrowheads, L and M) and abundant luminal surfactant and tubular myelin structures (arrows, N and O) in both genotypes. Scale bars: 2 μm (L–O).

Techniques: Staining, Immunostaining, Western Blot, Transmission Assay

(A) Surface tension–lowering properties of lung surfactant isolated from newborn mice was measured on a captive bubble surfactometer. Minimum surface tensions of surfactant preparations isolated from Lpcat1GT/GT alive mice were significantly higher than those of Lpcat1GT/+ and Lpcat1+/+ mice. Data represent at least n = 5 per genotype. *P < 0.001 versus Lpcat1+/+ and Lpcat1GT/+. (B) Immunoblot analysis of phospholipid-associated mature SFTPB from an aliquot of samples used for captive bubble surfactometer analysis in A. Input was normalized to 40 nmol of total phospholipid. Graph represents densitometry of immunoblot. Levels of surfactant-associated, mature SFTPB protein were similar among all genotypes.

Journal: The Journal of Clinical Investigation

Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

doi: 10.1172/JCI38061

Figure Lengend Snippet: (A) Surface tension–lowering properties of lung surfactant isolated from newborn mice was measured on a captive bubble surfactometer. Minimum surface tensions of surfactant preparations isolated from Lpcat1GT/GT alive mice were significantly higher than those of Lpcat1GT/+ and Lpcat1+/+ mice. Data represent at least n = 5 per genotype. *P < 0.001 versus Lpcat1+/+ and Lpcat1GT/+. (B) Immunoblot analysis of phospholipid-associated mature SFTPB from an aliquot of samples used for captive bubble surfactometer analysis in A. Input was normalized to 40 nmol of total phospholipid. Graph represents densitometry of immunoblot. Levels of surfactant-associated, mature SFTPB protein were similar among all genotypes.

Techniques: Isolation, Western Blot

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