Journal: Scientific Reports

Article Title: USP10 promotes cell proliferation, migration, and invasion in NSCLC through deubiquitination and stabilization of EIF4G1

doi: 10.1038/s41598-024-74490-6

Figure Lengend Snippet: Regulation of EIG4G1 expression by USP10 . A Two shRNAs downregulated the expression of USP10 in NSCLC cell lines H1299, H157, and H23. EIF4G1 levels were detected using western blotting, with β-actin as the internal reference. B Overexpression of USP10 . Western blotting for the protein levels of EIF4G1, with β-actin as the internal reference. C Transfection of a USP10-overexpressing plasmid in USP10 knockdown H1299 cells; EIF4G1 protein levels were detected using western blotting, with β-actin as the internal reference. D Downregulation of USP10 expression. qRT-PCR detection of the mRNA levels of EIF4G1 , with GAPDH as the internal reference. E Overexpression of USP10 . qRT-PCR detection of the mRNA levels of EIF4G1 , with GAPDH as the internal reference. Mean ± SD, n = 3. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Original blots are presented in Supplementary Fig. 2.

Article Snippet: NSCLC cell lines H157 and H23 were obtained from iCellBioscience Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Over Expression, Transfection, Plasmid Preparation, Knockdown, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas

doi: 10.1172/JCI133081

Figure Lengend Snippet: (A) Volcano plot of the differential label-free proteomic analysis performed between PZOX+ and HTOX+ LUADs. (B) Proteins involved in glycolysis were repressed in PZOX+, whereas proteins involved in FAO were upregulated. The mean fold change ratio between OX+ PZ and OX+ HT is indicated. (C) Analysis of HADHA expression in 586 human LUAD samples (https://www.cbioportal.org/). Tumors with a positive HADHA z score higher than 1.2 (HADHA+) are indicated in red; tumors with HADHA z score less than 0.8 (HADHA–) are shown in blue. The heatmap gives the z score for the genes listed on the left. (D) Venn diagram showing the overlap between the TCGA LUAD OX+ tumors and the TCGA LUAD HADHA+ tumors identified in panel C. (E) HADHA absolute mRNA expression in HADHA+ and HADHA– LUAD tumors. (F) RNA-Seq data from the HADHA+ and the HADHA– LUADs were analyzed using DESeq2 to generate the list of genes that differed between the 2 groups (adjusted P < 0.005). This list was further analyzed using Metascape (metascape.org) to identify all statistically enriched GO/KEGG terms. The significant terms were then hierarchically clustered into a tree and converted into a network layout. Each term is represented by a circle node, where its size is proportional to the number of genes, and its color represents its cluster. Terms with a similarity score higher than 0.3 are linked by an edge (the thickness represents the similarity score) and visualized with Cytoscape (v3.1.2). One term from each cluster was selected as label. (G) Genes coexpressed with HADHA in TCGA lung tumors. The top genes with a Pearson coefficient higher than 0.35 are shown in the bar graph. Data are expressed as mean ± SEM.

Article Snippet: The 12 human lung cancer cells listed in Supplemental Table 1 , sheet 2 were obtained from ATCC (A549, H1975, H460, LLC, H1299, H596, H1770, H1437) and from D. Santamaria’s group (INSERM U1218, Bordeaux, France) (H23, H358, PC9, H2279, H2935, HCC4006, HCC364, and HCC1395).

Techniques: Expressing, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas

doi: 10.1172/JCI133081

Figure Lengend Snippet: (A) HADHA immunohistology staining in mouse heart (40× zoom). A strong HADHA cytosolic staining (brown) can be observed in the myofibers (nuclei were stained in blue). (B) This method was applied to study HADHA expression in paraffin-embedded sections of lung tumors stained with hematoxylin (blue), eosin (red), and with a monoclonal antibody recognizing HADHA (brown staining). Representative tumors with either high HADHA expression (HADHA+ LUAD; left panel) or low HADHA expression (HADHA– LUAD; right panel) are shown. (C) HADHA tissue expression in the tumor and the noncancer tissue was used to calculate the HADHA histology score in 32 tumor samples. Tumors with HADHA score greater than median absolute deviation (MAD) were denominated HADHA+ LUADs (shown in red) and tumors with HADHA score less than MAD were denoted HADHA– LUADs (shown in blue). (D) Distribution of the [18F]-FDG–PET scan SUVmax values. (E) HADHA expression was determined by Western blot on a panel of 12 human lung cancer cell lines. (F) HADHA expression normalized to actin levels (mean expression value at dashed line) was used to segregate the cell lines with high (HADHA+; red) or low HADHA expression (HADHA–; blue). Cells with a black symbol show no difference to the median value. (G) Mitochondrial respiration was measured in the 12 human lung cancer cell lines using the Seahorse extracellular flux analyzer. The mean (dashed line) was used to segregate the cell lines with high (red) or low (blue) respiration. Cells with a plain symbol correspond to the HADHA–/OX– group (plain blue) or to the HADHA+/OX+ group (plain red). Data are expressed as mean ± SEM. **P < 0.01.

Article Snippet: The 12 human lung cancer cells listed in Supplemental Table 1 , sheet 2 were obtained from ATCC (A549, H1975, H460, LLC, H1299, H596, H1770, H1437) and from D. Santamaria’s group (INSERM U1218, Bordeaux, France) (H23, H358, PC9, H2279, H2935, HCC4006, HCC364, and HCC1395).

Techniques: Staining, Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas

doi: 10.1172/JCI133081

Figure Lengend Snippet: (A) Reduction of OX+ LUAD spheroid growth using HADHA shRNA or 50 μM TMZ on A549 cells cultured in anchorage-independent conditions. (B) Effect of MTP inhibition on A549 OX+ LUAD spheroid diameter. (C) Representative (10×) fields of 2 separate areas of A549 OX+ tumors: H&E staining (left; pink) and anti-human marker HLA (right; blue) staining of A549 cells in excised orthotopic human A549-OX+ LUAD tumors in NSG mice. (D) Immunohistology staining of HADHA in excised orthotopic human A549-OX+ LUAD tumors in NSG mice. H&E staining 20× zoom. Anti-HADHA 40× zoom. (E) Representative evolution of the bioluminescence signal from day 1 to day 18 in 2 groups of NSG mice: (i) orthotopic model of A549 expressing luciferase, (ii) orthotopic model of A549 shHADHA expressing luciferase. (F) Relative tumor volume obtained from the luminescence signal in the groups of mice treated with shRNA scramble and shRNA HADHA (N = 20 animals per group). (G) Animal survival (Kaplan-Meier representation) in the 2 groups of mice treated with shRNA scramble and shRNA HADHA (N = 20 animals per group). Values represent mean ± SEM; N = 4–6 for the in vitro experiments; N = 8–20 for the in vivo experiments. One-way ANOVA with Dunnett’s correction was used to analyze the results of panels A and B. Two-sided unpaired Student’s t test was used to compare the groups of mice in panel F. Log-rank (Mantel-Cox) test was used to compare animal survival in panel G. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The 12 human lung cancer cells listed in Supplemental Table 1 , sheet 2 were obtained from ATCC (A549, H1975, H460, LLC, H1299, H596, H1770, H1437) and from D. Santamaria’s group (INSERM U1218, Bordeaux, France) (H23, H358, PC9, H2279, H2935, HCC4006, HCC364, and HCC1395).

Techniques: shRNA, Cell Culture, Inhibition, Staining, Marker, Expressing, Luciferase, In Vitro, In Vivo

Journal: The Journal of Clinical Investigation

Article Title: Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas

doi: 10.1172/JCI133081

Figure Lengend Snippet: (A) NAD+/NADH ratio in A549 (OX+) cells treated with 500 μM trimetazidine (TMZ) for 48 hours. (B) NADH-oxidizing complex I enzymatic activity on A549 cells treated with 500 μM TMZ for 48 hours, on A549 cells expressing a shRNA against HADHA, or on A549 cell homogenate treated with 100 μM palmitoyl-CoA. (C) Impact of HADHA downregulation using shRNA on complex I–HADHA physical association. HADHA detection was performed by Western blot. A negative control IgG was also used to verify the specificity of the capture. (D) Changes in complex I composition in A549 cells expressing the shRNA targeting HADHA or in cells treated with TMZ. Complex I subunit content was quantified using mass spectrometry. The data are expressed as a percentage of the corresponding control. (E) NDUFAF1 and NDUFAF4 subunit mRNA content in A549 cells treated with TMZ. (F) Differential proteomic analysis performed on A549 cells treated with TMZ versus untreated cells, or between A549 cells expressing an shRNA against HADHA and cells expressing a scramble shRNA. The proteome modifications were compared using the “comparative analysis module” of IPA (QIAGEN). Only proteins with changes greater than 15% and P < 0.05 were kept for this analysis. The IPA canonical pathways altered in the 2 conditions are shown as well as the –log P value (purple color scale). (G) The mechanism of action of TMZ in lung cancer cells. Three related effects are proposed: HADHA inhibition, dissociation from complex I, and reduced content of NDUFAF1 and NDUFAF4. (H) Transcription factors potentially activated or inhibited by the shHADHA or TMZ treatments in A549 cells, as predicted using the upstream analysis module of IPA. The activation z score is shown as a color code (orange = activated and blue = inhibited). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01.

Article Snippet: The 12 human lung cancer cells listed in Supplemental Table 1 , sheet 2 were obtained from ATCC (A549, H1975, H460, LLC, H1299, H596, H1770, H1437) and from D. Santamaria’s group (INSERM U1218, Bordeaux, France) (H23, H358, PC9, H2279, H2935, HCC4006, HCC364, and HCC1395).

Techniques: Activity Assay, Expressing, shRNA, Western Blot, Negative Control, Mass Spectrometry, Control, Inhibition, Activation Assay

Journal: PLOS ONE

Article Title: Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis

doi: 10.1371/journal.pone.0312563

Figure Lengend Snippet: PCAIs inhibit the viability of NCI-H23 cells. Cells were seeded and treated with the respective PCAIs for 48 h followed by determination of the cell viability as described in the methods section. The molecular structures of the PCAIs can be found in Tawfeeq et al. [ 29 ].

Article Snippet: NCI-H23 (CRL-5800) cells were cultured in complete RPMI 1640, (Genesee Scientific, San Diego, CA) supplemented with 100 U/mL streptomycin,100 μg/mL penicillin and 10% heat-inactivated fetal bovine serum (FBS) (Genesee Scientific, San Diego, CA) at 37°C in 5% CO 2 /95% humidified air.

Techniques: Control

Journal: Bioinformatics

Article Title: MMFPh: a maximal motif finder for phosphoproteomics datasets

doi: 10.1093/bioinformatics/bts195

Figure Lengend Snippet: Summary of MMFPh and GrMFPh results for the NCI-H23 dataset at two occurrence thresholds

Article Snippet: 3.2.4 NCI-H23 non-small cell lung cancer cells dataset Finally, we perform a case study analysis of a set of 14 769 phosphopeptides from NCI-H23 non-small cell lung cancer cells ( Kettenbach and Gerber, 2011 ).

Techniques: