Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A , B ) TAM isolated from ovarian cancer ascites were cultured in Glc low Gln low medium containing GM-CSF and IFNγ supplemented with Glc or lactic acid. Glycogen ( A ; n = 6–8) and relative mRNA expression of PCK2 ( B ; n = 5–13) were quantified in freshly purified TAM and after 3 days of culture. The boxplots display a median line, interquartile range (IQR) boxes, min to max whiskers. ( C ) Immunohistochemical analysis of CD163 expression in lung adenocarcinoma. On the same tissue section, glycogen was detected by PAS staining, after treatment or not with amylase; scale bar, 100 µm. ( D ) Lactic acid and glycogen quantification by FTIR spectroscopic imaging in lung adenocarcinoma. Left panel: unstained bright field image; right panel, glycogen and lactic acid maps; scale bar, 200 µm; linear correlation between lactic acid and glycogen contents in infiltrating lung adenocarcinoma was calculated (results are representative 1 out of 2 biologically independent experiments). ( E ) Day 3 TAM were treated or not with CP-91149 for 15 min before a 6 h stimulation with LPS. TNFα, VEGF and G-CSF were quantified by ELISA ( n = 3). Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by Welch’s ANOVA test followed by Dunnett’s multiple comparison post hoc test ( A ), or by two-tailed unpaired Welch t test ( B ) or by paired t test ( E ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001. .

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Isolation, Cell Culture, Expressing, Purification, Immunohistochemical staining, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A , B ) Day 5 M1 and M2 cells (generated in conventional medium or CM) were treated or not for 15 min with 50 µM CP-91149 before stimulation with 100 ng/mL LPS or with E. coli at a multiplicity of infection of 10. ( A ) Glycogen was quantified after 24 h activation ( n = 3). ( B ) Cytokines were quantified in the supernatants by ELISA after 24 h (IL-12p70) or 6 h (IL-10) stimulation ( n = 5). ( C ) GM-CSF-Mφ were switched on day 2 to Glc low Gln low medium supplemented or not with Gln, lactic acid or glycerol and treated or not with CP-91149 15 min before LPS stimulation. TNFα and IL-6 (2 h), or IL-12p70 (24 h) were quantified by ELISA in stimulated day 5 M1 cells culture supernatants ( n = 6–8). ( D ) M2 cells were pretreated with 50 µM CP-91149 or 10 µM cytochalasin B (CytoB) for 15 min before addition of 0.2 mg/mL pHrodo-conjugated E. coli BioParticles for 2 h at 37 °C. Fluorescence was determined by flow cytometry and expressed in MFI values ( n = 3). ( E ) G6PC1-3 mRNA expression was determined by RT-qPCR in monocytes and day 5 Mφ ( n = 6). Liver cells were used as a positive control. Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by paired t test ( A , B ) or by two-tailed unpaired Welch t test ( C , D ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Generated, Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Flow Cytometry, Expressing, Quantitative RT-PCR, Positive Control, Two Tailed Test

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A ) Day 5 M1 cells were cultured in CM or in Glc low medium and treated or not with CP-91149 or 6AN, 15 min before LPS stimulation. Cytokines were quantified by ELISA in the M1 cells culture supernatants after 2 h (TNFα and IL-6), 6 h (IL-1β) or 24 h (IL-12p70) activation with LPS ( n = 5–14). ( B , C ) Day 2 GM-CSF-Mφ were incubated either with siRNA targeting PYGL, PYGB, G6PD or a control siRNA. Cells were then stimulated with LPS at day 5, and cytokines were quantified by ELISA in M1 cell culture supernatants after 2 h (TNFα) or 6 h (IL-1β) ( B ) PYGL, PYGB and G6PD mRNA expression were determined by RT-qPCR in day 5 macrophages ( n = 4) ( C ). ( D ) Day 5 M2 cells were cultured in CM or in Glc low medium and treated or not with CP-91149, 15 min before LPS stimulation. Cytokines were quantified by ELISA in the M2 cells culture supernatants after 2 h (IL-6), 6 h (IL-10) activation with LPS ( n = 5–14). Phagocytosis was assessed by flow cytometry after 3 h LPS stimulation ( n = 6). ( E , F ) Lactate was quantified in 6 h LPS-stimulated M1 ( E ) and M2 ( F ) cells culture supernatants and oxygen consumption rate (OCR) of M1 and M2 cells was monitored after 2 h ( n = 4–6). Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by Welch’s ANOVA test followed by Dunnett’s multiple comparison post hoc test or two-tailed unpaired Welch t test for phagocytosis assay in ( D ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001. .

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Incubation, Control, Expressing, Quantitative RT-PCR, Flow Cytometry, Comparison, Two Tailed Test, Phagocytosis Assay

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: ( A ) Schematic of high-throughput screen (HTS) used to identify inhibitors of NMD. Mutant transcripts are represented by a smaller length in the cartoon for illustrative purposes only. All small molecules were tested at 10 μM. ( B ) Mutant RNA reads relative to wild-type reads for the top 8 hits from the HTS. The dotted line represents the minimum fraction required to be considered a hit (>5 standard deviations above dimethyl sulfoxide [DMSO] control). Full screen results are presented in . ( C ) Targeted RNA-sequencing results of isogenic RPTec knockout clones treated with the eight best hits from the HTS at 10 µM. The dotted line represents a relative RNA expression level of 1, equal to that of DMSO-treated wells. Data for ceritinib, which did not validate on any line, are presented only in . ( D ) TP53 western blot on RPE TP53 224, containing a homozygous TP53 mutation, using the four hit compounds that validated in RPTec isogenic lines at 10 μM. ( E ) Western blot analysis of full-length TP53α and isoform TP53β after treatment with two NMD inhibitor lead candidates at 10 μM. TP53β (expression known to be controlled by NMD) as well as mutant TP53 are prominently induced by LY3023414 whereas full length is not. Note that RPE TP53 223 is a heterozygous knockout clone with one near wild-type allele whereas RPTec TP53 588 contains a homozygous TP53 indel mutation. ( F ) Quantitative real-time PCR (qPCR) showing 10 μM LY3023414 treatment causes increased expression of the NMD controlled alternative transcript for TP53 , TP53β , in parent cell lines for RPE1 and RPTec. Significance determined by Student’s t -test. Unless indicated otherwise cells were exposed to test compound for 16 hr.

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: High Throughput Screening Assay, Mutagenesis, Control, RNA Sequencing, Knock-Out, Clone Assay, RNA Expression, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: ( A ) Mutant transcript recovery rates for genes containing heterozygous indel mutations based on RNA-sequencing results in cell lines treated with 5 µM LY3023414 for 16 hr. Strict inclusion criteria were used, such that only mutations with sufficient sequencing coverage are shown (see methods). Recovery is defined as at least two-fold increase over dimethyl sulfoxide (DMSO) treatment. ( B ) Targeted high coverage RNA-sequencing confirms recovery of mutant transcript levels in NCI-H358 and LS180 cancer cell lines treated with 5 µM LY3023414. RNF43 and DROSHA contain common heterozygous single-nucleotide polymorphisms (SNPs) and the mutant allele refers to the non-reference genome allele. Error bars indicate 95% confidence limits. ( C ) Western blot analyses of NCI-H358 cells showing mutant and wild-type protein levels in EXOC1 and ( D ) SPTAN1 with and without 5 µM LY3023414 treatment. The black arrow indicates the expected size of the mutant protein. The C-terminal SPTAN1 antibody is downstream of the out-of-frame indel mutation and is not expected to identify the mutant allele. ( E ) Fold change in the number of mutant RNA transcripts from deep-targeted RNA-sequencing of heterozygous mutated genes in NCI-H358 and LS180 xenografts treated by oral gavage with 60 mg/kg LY3023414 assayed 16 hr post-treatment. Student’s t -test for target genes are all p < 0.05, while the null hypothesis holds for RNF43 (common SNP).

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Mutagenesis, RNA Sequencing, Sequencing, Western Blot

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: Cells are treated with siRNAs targeting genes known to be inhibited by LY3023414. Error bars represent 95% confidence intervals.

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques:

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: ( A ) Fraction of mutant allele transcripts in genes with heterozygous indels previously established in this study as sensitive to NMD inhibition. Results show mutant levels after siRNA treatment targeting kinases inhibited by LY3023414. RNF43 and DROSHA are common heterozygous single-nucleotide polymorphisms (SNPs) (shaded gray) and serve as negative controls. ( B ) Fraction of mutant allele transcripts in genes with truncating mutations known to be sensitive to NMD inhibition after siRNA treatment with siUPF1 or non-targeting siRNA. Data from deep-targeted RNA-sequencing. ( C ) Structure of novel NMD inhibitor KVS0001. ( D ) Targeted RNA-sequencing on three genes with heterozygous, out-of-frame, indel mutations in LS180 cancer cells treated in a dose–response with KVS0001 or SMG1i-11. RNF43 serves as a control (common heterozygous SNP) and the mutant allele refers to the non-reference genome allele. ( E ) Western blot of EXOC1 protein in NCI-H358 cells treated with 5 µM novel inhibitor KVS0001, LY3023414, or SMG1i-11 for 24 hr. ( F ) Western blot of phosphorylated UPF1 on three cell lines treated with 5 µM KVS0001, SMG1i-11, or dimethyl sulfoxide (DMSO). Note that total UPF1 and p-UPF1 were run on different gels, loading controls correspond to indicated gel. ( G ) Fold change in the number of mutant allele transcripts measured by targeted RNA-seq in genes containing heterozygous out-of-frame indel mutations in NCI-H358 or ( H ) LS180 subcutaneous xenografts in bilateral flanks of nude mice. Mice were treated once with intraperitoneal (IP) injection of vehicle or 30 mg/kg KVS0001 and tumors harvested 16 hr post IP treatment. All genes shown contain heterozygous out-of-frame truncating mutations except RNF43 and DROSHA which serve as controls (contain heterozygous SNPs).

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Mutagenesis, Inhibition, RNA Sequencing, Control, Western Blot, Injection

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: Gene names are shown in the boxes above each slider plot, genes highlighted in gray are common single-nucleotide polymorphisms (SNPs) and serve as a negative control (not expected to change). In the case of the control SNPs, the mutant allele refers to the non-reference genome allele. TRIM21 did not show a large change in expression with either small molecule, while ANLN did not show a difference with LY3023414 but did respond to SMG1i-11. The remaining genes responded to nonsense-mediated decay (NMD) inhibition by both LY3023414 and SMG1i-11.

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Negative Control, Control, Mutagenesis, Expressing, Inhibition

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: ( A ) Western blot showing SPTAN1 expression after treatment with dimethyl sulfoxide (DMSO), 5 µM LY3023414, or 1 µM SMG1 inhibitor SMG1i-11 (lanes 1, 2, and 3, respectively, for each antibody). The arrow indicates the expected size of the mutant NMD-targeted protein. Antibody ab75755 (Abcam) binds C-terminal to the out-of-frame indel and does not show mutant protein as expected. Antibody A301-249 (Bethyl) is polyclonal and also did not bind mutant protein. Antibody ab11755 (Abcam) is located N-terminal to the indel and does display mutant protein expression. ( B ) Western blot showing EXOC1 expression after treatment with DMSO, 5 µM LY3023414, or 1 µM SMG1i-11 (lanes 1, 2, and 3, respectively). The arrow indicates the expected size of the mutant (NMD-targeted) protein.

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Western Blot, Expressing, Mutagenesis

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: DROSHA and RNF43 are common heterozygous single-nucleotide polymorphisms (SNPs) and serve as a negative control. In the case of the control SNPs, the mutant allele refers to the non-reference genome allele. Only the highest two concentrations were tested on LY3023414 which served as a positive control in this experiment. The dotted line indicates the mutant expression with dimethyl sulfoxide (DMSO) treatment and the solid line is a reference for equal expression of both the wild-type and mutant alleles.

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Negative Control, Control, Mutagenesis, Positive Control, Expressing

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet: LYO is LY3023414 and 5-FU is 5-fluorouracil. HEK293 parent cells are shown as a control (wild-type TP53 protein).

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Control

Journal: eLife

Article Title: Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

doi: 10.7554/eLife.95952

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , LY3023414 , Selleckchem , Cat #S8322 , .

Techniques: Western Blot, Flow Cytometry, Sequencing, Expressing, Plasmid Preparation, Luciferase, Transfection, Formulation, Saline, Gene Expression, Real-time Polymerase Chain Reaction, Protease Inhibitor, Recombinant, Lysis, Extraction, Drug discovery, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Knock-Out, CRISPR, Mutagenesis, Software

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IL-10 (human) ELISA set , Diaclone , 851 540 020.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry