human, wt galc complementary dna sc120078  (OriGene)

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Generation and validation of MO3.13/ GALC -KO cells. A , human GALC gene sequences ( red ) adjacent to PAM sites ( bolded ) are targeted for Cas9-mediated cleavage by the sgRNA vectors. A downstream nonsense mutation ( asterisk ) is introduced upon successful targeted deletion (highlighted yellow ). B , confirmation of targeted deletion of the 85 bp region in the KO cell line by PCR amplification, compared to control WT cells. C , Western blot analysis of GALC and GAPDH proteins in native MO3.13 cells (WT), GALC-KO cells (KO), and GALC-overexpressing cells (OE). D , GALC activity and ( E ) psychosine levels in WT and KO cells. Statistical significance is determined using an unpaired t test (two-tailed, 3–4 independent experimental replicates, 95% confidence interval, ∗∗ p < 0.01, ∗∗∗ p < 0.001). PAM, protospacer adjacent motif.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Biomarker Discovery, Mutagenesis, Amplification, Control, Western Blot, Activity Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Differential proteome of MO3.13 native cells (WT) and MO3.13/ GALC -KO (KO) cells. A , proteomics workflow. Proteins extracted from WT and KO cells were separated by SDS-PAGE. After in-gel digestion, peptides were labeled with tandem mass tags (TMTs) and high-pH reversed-phase fractioned. Peptides were identified and quantified by liquid chromatography mass spectrometry (LC/MS) using synchronous precursor selection (SPS) and MS3 method, and the differential expression profile between WT and KO cells was elucidated. B , volcano plot. A total of 2417 proteins were identified and quantified. Three hundred sixty-six proteins were identified as differentially expressed in KO versus WT cells (two-tailed t test; adjusted p value (Benjamini–Hochberg) < 0.05). A p value of 0.05 (-log 10 0.05 = 1.301) is indicated by a gray line . Differentially expressed proteins with a 1.25-fold change (KO/WT: log2 < −0.32 or > 0.32) are highlighted (upregulated in red ; downregulated in blue ). C , differentially expressed proteins. Scaled abundances of the 366 differentially expressed proteins are represented in a heat map in descending order based on average log2-fold change. The color intensity scale is arbitrary. D , differently expressed lysosomal proteins and proteins in the ceramide synthesis pathway. Percent change (KO versus WT), p values, and adjusted p values are listed. Scaled abundances of the target proteins are shown in a heat map in descending order based on average log2-fold change. The color intensity scale is arbitrary.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: SDS Page, Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Selection, Quantitative Proteomics, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Dramatic reduction of GALC activity in 26 clinically relevant MMVs. Residual GALC activity in transiently expressed cell models was measured in cell lysate and presented as relative percent of WT-GALC. MMVs are ranked in descending order based on GALC activity. Of the MMVs eliciting significant reductions compared to WT-GALC, seven variants had activity that remained relatively high (20–70% of WT, blue bars and I562T); six variants had low level activity (2–7% of WT, orange bars ); and 20 variants had little to no activity (0–2% of WT, red bars ). MMVs on the p.I562T background are annotated with (T). Data are presented as mean ± standard deviation from four independent experiments. Individual MMV (n = 4) was compared to WT (n = 24) by an unpaired t test (two-tailed, 95% confidence interval, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, # - no significant difference). MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Standard Deviation, Two Tailed Test, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: GALC activity in MMV cell models is highly correlated with the age of clinical onset in KD patients carrying the same GALC MMV genotypes. A , GALC activity levels in MMV cell models and the age of symptom onset in KD patients with either compound heterozygous MM-null mutation or homozygous MM-MM genotypes, based on reported cases from literature. B and C , correlation analysis between GALC activity and the age of symptom onset in KD patients with ( B ) heterozygous MM-null mutation genotypes (Pearson r = 0.94, p < 0.0001, n = 12) or ( C ) homozygous MM-MM genotypes (Pearson r = 0.98, p < 0.0001, n = 7). KD, Krabbe disease; MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Lys-GALC levels are highly correlated with GALC activity. A – D , Western blot results of lys-GALC and GAPDH in the lysate of GALC MMV expressing cells. Panel ( C and D ) shows MMVs in the absence (−) and presence (+) of the p.I562T polymorphic background. E , Lys-GALC levels (normalized to GAPDH levels) are highly correlated with GALC activity in the GALC MMV cell models (Pearson r = 0.93, p < 0.0001, n = 37). F , expanded plot of the red line area shown in ( E ). MMVs with lys-GALC levels greater than 1% are labeled in ( E ) and ( F ). MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Western Blot, Expressing, Labeling, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: GALC secretion is more impaired by MMVs located on the β-sandwich and lectin protein domains but does not correlate to the age of symptom onset in KD patients. A , Sec-GALC levels are plotted against the structural location of GALC MMVs, categorized by their position on the TIM barrel ( red ), β-sandwich ( green ), and lectin-binding ( blue ) domains. MMVs (11/12) significantly reduced sec-GALC levels compared with WT-GALC, specifically on the β-sandwich and lectin domains in our cell models. (Unpaired t test, two-tailed, four independent experimental replicates, 95% confidence interval; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). B and C , analysis reveals no significant correlation between sec-GALC levels and the age of symptom onset in KD patients with ( B ) heterozygous MM-null mutation genotypes or ( C ) homozygous MM-MM genotypes. KD, Krabbe disease; MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Binding Assay, Two Tailed Test, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Location and distribution of KD-related MMVs on the human GALC protein. The schematic diagram shows the distribution of clinically relevant MMVs on the human GALC protein. Human Genome Variation Society (HGVS) nomenclature is applied. The main structural domains of GALC are indicated: signal peptide (SP) (1–42 a.a.), TIM barrel domain (57–353 a.a.), β-sandwich domain (354–468 a.a.), and lectin-binding domain (488–685 a.a). Key residues involved in catalytic function and substrate-binding are labeled in red . Polymorphic variants are labeled in blue . Active site variants are in bolded font . KD, Krabbe disease; MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Binding Assay, Labeling, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Accumulation of pre-GALC protein in GALC MMV cell models. A , intracellular pre-GALC and GAPDH proteins were detected by Western blot. The bottom two blots show MMVs in the absence (−) and presence (+) of the p.I562T polymorphic background. The same GAPDH blots were used to normalize both pre-GALC ( current panel ) and lys-GALC protein levels ( , A – D ), as pre-GALC and lys-GALC were detected on the same blot but at different molecular weights. B , pre-GALC levels (normalized to GAPDH) detected by Western blot are significantly correlated with pre-GALC levels measured by sandwich ELISA in the MMV cell models (Pearson r = 0.71, p < 0.0001, n = 35). C , pre-GALC levels measured by ELISA plotted against the structural location of the MMVs, categorized by their position on the TIM barrel ( red ), β-sandwich ( green ) and lectin-binding ( blue ) domains. Ten out of 22 MMVs significantly increase pre-GALC levels compared with WT-GALC in the MMV cell models. (Unpaired t test, two-tailed, three independent experimental replicates, 95% confidence interval; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Western Blot, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: p.I562T background reduced GALC activity, sec-GALC levels and pre-GALC levels in the MMV cell models. A , GALC activity, ( B ) sec-GALC levels and ( C ) pre-GALC levels from MMV expressing cells containing the p.I562T variant and seven other p.I562T co-variants ( red bars ) are compared side-by-side with those from WT cells or cells expressing the corresponding single variants ( blue bars ), respectively. (Unpaired t test, two tails, three to four independent experimental replicates, 95% confidence interval; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). D , the percentage reductions of GALC activity are highly correlated with the percentage reductions in sec-GALC levels in MMV expressing cells containing the p.I562T and seven other p.I562T co-variants (Pearson r = 0.92, p < 0.001, n = 8). E , reductions in sec-GALC levels (%) correlated with the reductions in intracellular pre-GALC levels (%) in five p.I562T covariants (Pearson r = 0.88, p < 0.05, n = 5). MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Expressing, Variant Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: GALC activity correlates with sec-GALC levels in the MMV cell models. Correlations among GALC activity, sec-GALC levels, and pre-GALC protein levels in the GALC MMV expressing cells were analyzed using the Pearson correlation method. A , GALC activity is significantly correlated with sec-GALC levels in the cell models (Pearson r = 0.5, p < 0.01, n = 37). No significant correlations are found between ( B ) GALC activity and pre-GALC levels, and between ( C ) sec-GALC and pre-GALC levels. MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Cellular psychosine levels correlate with GALC activity in MMV cell models, but do not correlate with the onset of clinical symptoms in KD patients. A , expression studies were conducted in MO3.13/ GALC -KO cells for WT, p.I562T, and 12 clinically relevant GALC MMVs. Quantification of intracellular psychosine, GALC activity, and sec-GALC levels are summarized in . Psychosine levels significantly correlated with GALC activity (Pearson r = −0.63, p < 0.01, n = 15). B , consistent with our transient expression study ( , ), GALC activity was also highly correlated with the age of symptom-onset in KD patients with either homozygous MM-MM (Pearson r = 0.99, p < 0.0001, n = 7) or compound heterozygous MM-null mutation genotypes (Pearson r = 0.92, p < 0.0001, n = 12). C and D , however, psychosine levels were not significantly correlated with the age of symptom-onset, in either ( C ) compound heterozygous or ( D ) homozygous MMV genotypes. KD, Krabbe disease; MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Dramatic reduction in GALC secretion in 50% of the low-activity GALC MMVs. A , summary table listing GALC activity, sec-GALC levels, and intracellular pre-GALC levels for 26 low-activity GALC MMVs. Values are presented as percentages relative to WT-GALC levels. The color spectrum from green to red indicates values from high to low within each group. “ns” denotes no significant difference from WT-GALC. B , distribution of sec-GALC levels among the 26 low-activity GALC-MMVs. The red , blue , and gray sections of the pie chart represent MMVs with sec-GALC levels of 0% to 4%, 10% to 80%, and 100% of WT levels, respectively. MMV, missense mutation variant.

Article Snippet: Full-length, human, WT GALC complementary DNA (SC120078, NM_000153 ) was originally purchased from Origene.

Techniques: Activity Assay, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Generation and validation of MO3.13/ GALC -KO cells. A , human GALC gene sequences ( red ) adjacent to PAM sites ( bolded ) are targeted for Cas9-mediated cleavage by the sgRNA vectors. A downstream nonsense mutation ( asterisk ) is introduced upon successful targeted deletion (highlighted yellow ). B , confirmation of targeted deletion of the 85 bp region in the KO cell line by PCR amplification, compared to control WT cells. C , Western blot analysis of GALC and GAPDH proteins in native MO3.13 cells (WT), GALC-KO cells (KO), and GALC-overexpressing cells (OE). D , GALC activity and ( E ) psychosine levels in WT and KO cells. Statistical significance is determined using an unpaired t test (two-tailed, 3–4 independent experimental replicates, 95% confidence interval, ∗∗ p < 0.01, ∗∗∗ p < 0.001). PAM, protospacer adjacent motif.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Biomarker Discovery, Mutagenesis, Amplification, Control, Western Blot, Activity Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Location and distribution of KD-related MMVs on the human GALC protein. The schematic diagram shows the distribution of clinically relevant MMVs on the human GALC protein. Human Genome Variation Society (HGVS) nomenclature is applied. The main structural domains of GALC are indicated: signal peptide (SP) (1–42 a.a.), TIM barrel domain (57–353 a.a.), β-sandwich domain (354–468 a.a.), and lectin-binding domain (488–685 a.a). Key residues involved in catalytic function and substrate-binding are labeled in red . Polymorphic variants are labeled in blue . Active site variants are in bolded font . KD, Krabbe disease; MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Binding Assay, Labeling, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Accumulation of pre-GALC protein in GALC MMV cell models. A , intracellular pre-GALC and GAPDH proteins were detected by Western blot. The bottom two blots show MMVs in the absence (−) and presence (+) of the p.I562T polymorphic background. The same GAPDH blots were used to normalize both pre-GALC ( current panel ) and lys-GALC protein levels ( , A – D ), as pre-GALC and lys-GALC were detected on the same blot but at different molecular weights. B , pre-GALC levels (normalized to GAPDH) detected by Western blot are significantly correlated with pre-GALC levels measured by sandwich ELISA in the MMV cell models (Pearson r = 0.71, p < 0.0001, n = 35). C , pre-GALC levels measured by ELISA plotted against the structural location of the MMVs, categorized by their position on the TIM barrel ( red ), β-sandwich ( green ) and lectin-binding ( blue ) domains. Ten out of 22 MMVs significantly increase pre-GALC levels compared with WT-GALC in the MMV cell models. (Unpaired t test, two-tailed, three independent experimental replicates, 95% confidence interval; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Western Blot, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: GALC activity correlates with sec-GALC levels in the MMV cell models. Correlations among GALC activity, sec-GALC levels, and pre-GALC protein levels in the GALC MMV expressing cells were analyzed using the Pearson correlation method. A , GALC activity is significantly correlated with sec-GALC levels in the cell models (Pearson r = 0.5, p < 0.01, n = 37). No significant correlations are found between ( B ) GALC activity and pre-GALC levels, and between ( C ) sec-GALC and pre-GALC levels. MMV, missense mutation variant.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) DN32.D3 cells were co-cultured with 5.5 × 10 2 , 1.66 × 10 3 , 5.0 × 10 3 and 1.5 × 10 4 parental or CD1d-transduced MC38, LLC1, B16F10, 2B4, EL4, DN32.D3, HEK293T, HeLa, A549, MDA-MB-231, and PANC-1 cells for 16 h and analyzed for CD69 expression. (B) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of C57BL/6J mice ( n = 8). Tumor volume was measured every 3–4 days. (C) DN32.D3 cells were co-cultured with the indicated cell number of WT or Ugcg −/− Ugt8a −/− CD1d-transduced B16F10 cells for 16 h and analyzed as in A (left). Concentrations of IL-2 in the supernatants were measured (right). (D) CD1d-transduced B16F10 cells were cultured in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days. DN32.D3 cells were then co-cultured with those B16F10 cells for 16 h and analyzed as in A. (E) DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells that were cultured in RPMI 1640 supplemented with 0.6% FCS and 9.4% animal component-free cell culture supplement for 7 days as in D in the absence or presence of α-GalCer (t18:0/26:0) (KRN7000) for 16 h and analyzed as in A. (F) Lipids extracted from serum were separated into seven fractions by open column chromatography and analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) followed by staining with copper acetate reagent. Commercial β-GlcCer was used as a reference (right lanes). Open and closed arrowheads denote the origin and solvent front, respectively. (G) CD1d −/− or CD1d-transduced DN32.D3 cells were stimulated with each fraction separated from serum lipids in F for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (H) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids with or without hydrolysis treatment for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. (I) CD1d-transduced DN32.D3 cells were stimulated with the C:M = 19:1 fraction of serum lipids treated with Gba (left) or Galc (right) for 16 h and analyzed as in A. α-GalCer (t18:0/26:0) was used as a positive control. Data are presented as mean ± SD (A–E and G–I) and are representative of three independent experiments (A–I). Statistical significance was determined by Student’s t test. *, P < 0.05. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Cell Culture, Expressing, Injection, Column Chromatography, High Performance Thin Layer Chromatography, Staining, Solvent, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

doi: 10.1084/jem.20240728

Figure Lengend Snippet: Serum contains antigens for iNKT cells. (A) Surface expression of CD1d on CD1d-transduced cell lines. Filled histogram, anti-mouse CD1d antibody; open histogram, isotype control antibody. (B) WT or TCRα −/− DN32.D3 cells were co-cultured with CD1d-transduced B16F10 cells for 16 h and analyzed for CD69 expression. (C) CD1d −/− or CD1d-transduced B16F10 cells were seeded onto 24-well plates. Growth curves were generated using cell counting by flow cytometer every 24 h. (D) 5 × 10 5 CD1d −/− or CD1d-transduced B16F10 cells were injected subcutaneously into the right flank of Jα18-deficient mice ( n = 7). Tumor volume was measured every 3–4 days. (E) The crude lipids extracted from WT, Ugcg −/− , Ugt8a −/− , and Ugcg −/− Ugt8a −/− B16F10 cells were analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol) and stained with copper acetate reagent. (F) Lipid extracts from B16F10 cells (5 × 10 6 ) were separated into 84 fractions in a 96-well plate by LC-FRC system and evaporated. DN32.D3 cells were stimulated in the 96-well plate for 16 h and analyzed for CD69 expression. Fractionation was performed in triplicate. (G) The C:M = 19:1 fraction of serum lipids before and after hydrolysis treatment was analyzed by HPTLC as in E. (H) Commercial α- and β-GalCer (d18:1/16:0) (left) and α- and β-GalCer (d18:1/24:1) (right) were treated with Galc and analyzed by HPTLC as in E. (I) The C:M = 19:1 fraction of serum lipids and commercial β-GlcCer or β-GalCer were treated with Gba (left) or Galc (right) and analyzed by HPTLC as in E. (J) Screening of columns to separate three diastereomers of synthesized HexCer (d18:1/16:0). MRM chromatograms of SFC/MRM analysis using the columns in are shown. The MRM transition was set to 700.57 > 264.27 (precursor ions selected as [M+H] + ). The SFC analysis conditions for 1-AA, 2-PC, BEH 2-EP, BEH, DEA, Diol, P4VP (PEEK), and PTZ (PEEK) (left) were as follows: column temperature, 50°C; mobile phase A, supercritical carbon dioxide; mobile phase B, M:W (95:5, vol/vol) with 0.1% (wt/vol) ammonium acetate; flow rate of mobile phase, 1.0 ml min −1 ; flow rate of make-up pump, 0.1 ml min −1 ; back-pressure regulator, 10 MPa. The gradient conditions were as follows: 1% B, 0–1 min; 1–75% B, 1–24 min; 75% B, 24–26 min; and 1% B, 26–30 min. The SFC analytical conditions for other columns (center and right) were as described above with modification as follows: column temperature, 40°C; gradient conditions, 1% B, 0–1 min; 1–50% B, 1–17 min; 50% B, 17–26 min; and 1% B, 26–30 min. The MRM operating conditions were identical to those of the SFC/MRM analysis method. The colored shadows indicate the peaks coincident with the RT of synthesized α-GalCer (red), α-GlcCer (blue), β-GlcCer (green), and β-GalCer (yellow), respectively. Open and close arrowheads denote the origin and solvent front, respectively (E and G–I). Data are presented as mean ± SD (B–D and F) and are representative of three independent experiments (B–E and G–J). Statistical significance was determined by Student’s t test (C and D). NS, not significant. Source data are available for this figure: .

Article Snippet: N-stearoyl-D-erythro-sphingosine was from Katayama Chemical Industries Co. Recombinant human glucosylceramidase and recombinant human Galc were from R&D Systems.

Techniques: Expressing, Control, Cell Culture, Generated, Cell Counting, Flow Cytometry, Injection, High Performance Thin Layer Chromatography, Staining, Fractionation, Synthesized, Modification, Solvent