Journal: Immunity

Article Title: Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway

doi: 10.1016/j.immuni.2022.01.003

Figure Lengend Snippet:

Article Snippet: Peroxidase-AffiniPure Goat Anti-Rat IgG (H+L) , Jackson ImmunoResearch Labs , Cat#: 112-035-003; RRID: AB_2338128.

Techniques: Transduction, Virus, Recombinant, Generated, Transfection, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Western Blot, Plasmid Preparation, Software, Flow Cytometry, Imaging, Microscopy, Real-time Polymerase Chain Reaction

Journal: Science signaling

Article Title: Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C

doi: 10.1126/scisignal.aap7336

Figure Lengend Snippet: (A to C) RNA was isolated from splenocytes at the indicated times after zymosan treatment. The expression of Hmox1 (A), Flvcr (B), and Spic (C) was measured relative to 18S. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test. n = 4 to 7 mice per time point (Hmox1 and Flvcr); n = 3 to 8 mice per time point (Spic). (D) Representative Western blot and quantification of BACH1 abundance in the spleen at the indicated time points after zymosan treatment. β-Actin is included as a loading control and was used for calculating the relative abundance of BACH1 using ImageJ software. Data represent the mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, n = 3 mice per time point. (E and F) Splenocytes from mice transfused with CFSE-labeled erythrocytes were sorted into F4/80+CFSE− and F4/80+CFSE+ populations 3 hours after zymosan treatment. Expression of Spic and Gdf15 was measured relative to 18S in RNA isolated from each population. Data represent the mean ± SEM. Student’s t tests were performed on log scale–transformed data. n = 4 mice per group. (G) Quantification of the abundance of SIRPα on the surface of bone marrow–derived macrophages (BMDMs) relative to that on unstimulated BMDMs as measured by flow cytometry 1 hour after zymosan treatment with or without the Syk inhibitor piceatannol or the PI3K inhibitor LY294002. All replicates were normalized to 0 min (baseline). Data represent the mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test, n = 3 to 6 independent biological replicates. (H) F4/80+CFSE+ populations, representing phagocytosis of CFSE-labeled erythrocytes, were measured by flow cytometry 1 hour after the indicated treatments. Data represent the mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test, n = 3 to 6 independent biological replicates. (I) Quantification of Gdf15 expression relative to 18S in BMDMs 3 hours after the indicated treatments. Data represent the mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test, n = 3 to 6 independent biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.005.

Article Snippet: TaqMan probes: Bmp4 (Mm00432087_m1), Gdf15 (Mm00442228_m1), Epo (Mm01202755_m1), Spic (Mm00488428_m1), Flvcr (Mm01320423_m1), Hmox1 (Mm00516005_m1), Tnfa (Mm00443258_m1), and 18S (Hs99999901_s1).

Techniques: Isolation, Expressing, Comparison, Western Blot, Control, Software, Labeling, Transformation Assay, Derivative Assay, Flow Cytometry

Journal: BMC Microbiology

Article Title: The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry

doi: 10.1186/1471-2180-9-111

Figure Lengend Snippet: Ectopic expression of CD81 in HCV-resistant Huh-7 cells restores HCV permissivity . A , Huh-7 cells and R1 cell population infected with JFH-1 HCVcc were processed for double-label immunofluorescence for capsid protein (green) and nuclei (blue, Hoechst). B , Cells were infected with virus pseudotyped with HCV envelope proteins from 1a (HCVpp 1a) or 2a (HCVpp 2a) or VSV G envelope protein (VSVpp). C , Huh-7 cells and R1 individual cellular clones were infected with HCVcc expressing Renilla luciferase. In parallel, Huh-7 cells and some of the clones were infected with HCVpp 1a, HCVpp 2a or VSVpp ( D ). Results are presented as relative percentages to HCVcc (C) and HCVpp (D) infectivity on Huh-7 cells. HCVpp infections (D) were also normalized to VSVpp infections on Huh-7 cells. E , Surface biotinylated cell lysates were immunoprecipitated with anti-CD81 (5A6), anti-SR-BI (NB400-104H3) or anti-CLDN-1 (JAY.8) mAbs. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. F , Flow cytometry analysis of CD81 cell surface expression. Cells were stained using an anti-hCD81 (1.3.3.22, left panel) or an anti-mCD81 (MT81, right panel), and secondary antibodies conjugated with PE. Ctrl corresponds to Huh-7 cells stained only with secondary antibodies. Cell lines were infected with HCVcc ( G ) and in parallel with HCVpp ( H ) generated with envelope proteins from different genotypes or virus pseudotyped with feline endogenous virus RD114 glycoprotein (Rd114pp). Results are presented as relative percentages to HCVcc (G) and HCVpp (H) infectivity on Huh-7 cells. P < 0.05 as calculated by the Mann-Whitney's test; *, statistically not significant difference in HCVpp entry compared to entry into Huh-7 cells.

Article Snippet: After rinsing with the lysis buffer, complexes were eluted with non-reducing Laemmli buffer, resolved by SDS-PAGE and immunoblotted with peroxidase-conjugated Streptavidin (Vector).

Techniques: Expressing, Infection, Immunofluorescence, Clone Assay, Luciferase, Immunoprecipitation, Western Blot, Flow Cytometry, Staining, Generated, MANN-WHITNEY

Journal: BMC Microbiology

Article Title: The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry

doi: 10.1186/1471-2180-9-111

Figure Lengend Snippet: Recognition of TEM-associated CD81 in Huh-7w7/mCD81 cells . A , Flow cytometry analysis of Huh-7w7/mCD81 cells stained with the mAbs MT81 and MT81 w . Cells stained only with PE-conjugated secondary antibody were used as control (dotted line). B , Cell lines were surface biotinylated and lysed in the presence of Brij97 or Triton X-100 before immunoprecipitation with MT81, MT81 w and 5A6 mAbs. Immunoprecipitates were revealed by western blotting using peroxidase-conjugated streptavidin. C , Cell lines were surface biotinylated and lysed in buffer containing 1% Brij97 and divalent ions. Immunoprecipitations were then performed with 5A6, MT81, MT81 w , 8A12 (anti-EWI-2), TS151 (anti-CD151) or irrelevant (CTL) mAbs. Immunoprecipitates were revealed by western blotting using peroxidase-conjugated streptavidin. The molecular weights of the prestained molecular ladders are indicated in KDa. The asterisks indicate dimers of CD81.

Article Snippet: After rinsing with the lysis buffer, complexes were eluted with non-reducing Laemmli buffer, resolved by SDS-PAGE and immunoblotted with peroxidase-conjugated Streptavidin (Vector).

Techniques: Flow Cytometry, Staining, Immunoprecipitation, Western Blot

Journal: BMC Microbiology

Article Title: The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry

doi: 10.1186/1471-2180-9-111

Figure Lengend Snippet: Cholesterol depletion and ceramide enrichment do not reduce cell surface expression of other HCV entry factors . Huh-7w7/mCD81 cells were treated with 7.5 mM MβCD, with 0.5 unit Smase/ml or left untreated (NT) 30 min at 37°C. Cells were then surface biotinylated and lysed in buffer containing 1% Brij97 and divalent ions. Immunoprecipitations were performed with indicated mAbs. Immunoprecipitates were revealed by western blotting using peroxidase-conjugated streptavidin.

Article Snippet: After rinsing with the lysis buffer, complexes were eluted with non-reducing Laemmli buffer, resolved by SDS-PAGE and immunoblotted with peroxidase-conjugated Streptavidin (Vector).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: A PERK-FOXO1 AXIS LINKS DNA DAMAGE TO FIBROBLAST SURVIVAL IN DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS

doi: 10.64898/2026.02.17.706443

Figure Lengend Snippet: A) Primary dermal fibroblasts (FB) were used to determine the resting mitochondrial membrane potential (ΔΨmt) using TMRM of live FB from independent biological replicates of each group, then quantified via flow cytometry analysis in HC (n=4), dcSSc (n=6), and post-ASCT (n=5) (with 2 independent replicates/group). B) qRT-PCR was used to determine the relative expression of various ETC complexes ( ND4, complex I; SDHA , complex II; Cytochrome b (CytB, complex III), and Cyclo-oxygenase 7C (Cox7C, complex IV), N: HC=5, dcSSc=8 and post-ASCT=5, independent biological replicates). C) Total cellular ROS was determined by measuring the fluorescence intensity of CellROXgreen then quantified via flow cytometry from independent biological replicates of HC (n=6), dcSSc (n=6), and post-ASCT (n=5) FB (with two independent replicates/FB). D) Nascent mitochondrial DNA was quantified by quantifying D-loop levels using qRT-PCR (N: HC=5, dcSSc=6 and post-ASCT=5, independent biological replicates). All data were presented as the means ± SEM. Statistical significance was determined by one-way ANOVA. Using GraphPad Prism, statistical significance was determined when *p<0.05, **p<0.005 and ***p<0.0005.

Article Snippet: The Taqman probes (Thermo Fisher Scientific) used for qRT-PCR (with targets) included: Hs02596861_s1 (D-loop), Hs02596876_g1 (ND4), Hs02596867_s1 (CytB), Hs00231106_m1 (FOXO1), Hs02758991_g1 (GAPDH), Hs00188166_m1 (Succinate dehydrogenase or SDHA), Hs00167309_m1 (SOD2), Hs01595220_g1 (COX7C), Hs00176875_m1 (PDK4), Hs01561847_m1 (PDK1), Hs00176865_m1 (PDK2).

Techniques: Membrane, Flow Cytometry, Quantitative RT-PCR, Expressing, Fluorescence

Journal: Cell Reports

Article Title: Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition

doi: 10.1016/j.celrep.2021.109784

Figure Lengend Snippet:

Article Snippet: Goat Anti-Human IgG Antibody (Peroxidase) , Jackson ImmunoResearch , Cat#109-035-088.

Techniques: Virus, Variant Assay, Mutagenesis, Recombinant, Conjugation Assay, High Throughput Screening Assay, Flow Cytometry, Next-Generation Sequencing, Sequencing, Software, Plasmid Preparation, Binding Assay

Journal: PLoS Biology

Article Title: A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure

doi: 10.1371/journal.pbio.2004624

Figure Lengend Snippet: (A) K562 lines were generated expressing the dCas9-KRAB, Clover-mApple ATP FRET sensor, and 1 sgRNA against MRPL10 or non-targeting control guides (Cont1, Cont2), and ATP levels were assessed by flow cytometry. Compared with the ATP level in basal conditions, MRPL10 knockdown decreased ATP to a greater extent in respiratory conditions (box and whisker plots; line = median; box = 25th–75th percentile; whisker = 5th–95th percentile). In contrast, in glycolytic conditions, ATP levels dropped similarly in MRPL10 and control groups. N = 3,128–11,971 cells examined per group for FACS. *** p < 0.001 versus both corresponding control groups by two-way ANOVA with Tukey multiple comparisons test. (B) Knockdown of MRPL10 in K562 cells also decreased ATP levels by luciferase to a greater extent in respiratory conditions. In contrast, in glycolytic conditions, ATP levels again dropped similarly in MRPL10 and control groups. Data show mean ± SEM; N = 4 wells per group from 2 independent experiments for luciferase. ** p < 0.01 versus both corresponding control groups by one-way ANOVA with Dunnett multiple comparisons test. (C) K562 cells expressing dCas9-KRAB were stably transduced with a single sgRNA corresponding to the indicated gene, identified as reducing ATP in the primary FRET-based screen. Cells were selected for sgRNA transduction as in the primary screen, allowed to recover, and then acutely incubated in respiratory or glycolytic conditions as in the primary screen. ATP concentration was measured by luciferase (CellTiter Glo 2.0). Bars represent ATP levels in drug/substrate treatment (respiratory or glycolytic) relative to cells untreated for the same time period from the same pool of cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Error bars are mean ± SEM; N = 10–32 wells/group, compiled from 2–4 experiments per cell line. (D, E) OCR (aerobic respiration rate) and ECAR (a surrogate of glycolysis) in K562 lines were measured using a 96-well Seahorse Extracellular Flux Analyzer. Arrows show the additions of the mitochondrial uncoupler FCCP (1 μM), the ATP synthase inhibitor oligomycin (1 μM), or the mitochondrial complex I inhibitor rotenone (1 μM). Knocking down of COX11 , KPNB1 , PDSS1 , PDSS2 , or ATP5MPL significantly decreased maximal respiration (after FCCP). Stars in legend box indicate significant difference of maximal respiration after FCCP (panel D). Oligomycin and rotenone additions similarly decreased OCR in all lines (panel E), indicating similar fractions of basal oxygen consumption devoted to mitochondrial respiration (oligomycin) and nonmitochondrial consumption (rotenone). Data are compiled from 4 experiments; n = 6–18 wells per group. Error bars represent SEM. NS, p > 0.05; * p < 0.005; ** p < 0.01; *** p < 0.001; using one-way ANOVA with Dunnett multiple comparisons test. Further information about this figure can be found in . Cont, control; dCas9, dead CRISPR-associated protein 9; ECAR, extracellular acidification rate; FACS, fluorescence-activated cell sorting; FCCP, carbonyl cyanide- 4 -(trifluoromethoxy)phenylhydrazone; FRET, fluorescence resonance energy transfer; KRAB, Kruppel-associated box; NS, not significant; OCR, oxygen consumption rate; sgRNA, single guide RNA.

Article Snippet: qRT-PCR gene relative expression quantifications were performed using 7900HT Fast Real-Time PCR System (Applied Biosystem) and using FAM-MGB TaqMan Gene Expression Assays (ThermoFisher, assay ID: Hs01680112_mH—COX11; Hs01550960_g1—COX16; Hs01043634_m1—ATP5MPL; Hs00372008_m1—PDSS1; Hs01047689_m1—PDSS2; and Hs00204417_m1—NDUFA8), together with VIC-MGB human ACTB (β-actin) (ThermoFisher number 4326315E) as endogenous control. cDNAs and PCR reactions were prepared according to the protocol for Cells-to-C T kit (ThermoFisher number AM1728), using the standard reverse transcription cycle (37 °C for 6 minutes, inactivation at 95 °C for 5 minutes, hold at 4 °C), and qRT-PCR conditions (UDG Incubation: 50 °C for 2 minutes; enzyme activation: 95 °C for 10 minutes; PCR cycle: 95 °C for 15 seconds, 60 °C for 1 minute—repeat 40 cycles).

Techniques: Generated, Expressing, Control, Flow Cytometry, Knockdown, Whisker Assay, Luciferase, Stable Transfection, Transduction, Incubation, Concentration Assay, CRISPR, Fluorescence, FACS, Förster Resonance Energy Transfer

Journal: PLoS Biology

Article Title: A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure

doi: 10.1371/journal.pbio.2004624

Figure Lengend Snippet: (A) CoQ10 rescue of ATP in respiratory conditions was assessed with a mini-library of sgRNAs. Cells were pre-incubated with 50 μM CoQ10 for 5 days and then placed under respiratory conditions before sorting. Four genes showed statistically significant improvement in ATP levels (difference of untreated and CoQ10-treated ATP phenotype). Black bars indicate untreated ATP phenotypes, and gray bars show the ATP phenotype with CoQ10. PDSS2 , COQ2 , and PDSS1 have biosynthetic roles in CoQ10, and CoQ10 blocks the decrease in ATP from knocking down these genes. CoQ10 also significantly blocked the low-ATP phenotype of COX11 , which has no known CoQ10 biosynthetic role. Data from 2 experiments, 1–2 guides studied per gene, 1 million cells sorted per group. *** p < 0.001 versus non-targeting guides (with a phenotype of 0 and no change in response to CoQ10) by one-way ANOVA with Dunnett multiple comparisons test. (B) Plot of genes identified as decreasing ATP in the initial screen for rescue with CoQ10 supplementation. Genes that decreased ATP in the respiratory screen were reanalyzed with and without supplementation with CoQ10. The positive (rescue) portion of the graph is shown. x-Axis is fold-rescue by CoQ10 (phenotype in CoQ10 minus phenotype in vehicle). y-Axis is the -log 10 of the p -value ( t test on null hypothesis that the rescue was 0) based on the multiple sgRNAs tested for that gene. The figure consists of 3 repetitions of the subgenome library screen with CoQ10 and 2 without. Genes showing strong rescue with CoQ10 (either having a t test p -value of <0.05 and rescue larger than 1 SD from the average rescue of non-targeting guides, or a t test p -value of less than 0.1 and a rescue larger than 2 SDs from the average rescue of non-targeting guides) are labeled with red squares. CoQ10 biosynthetic genes are labelled with red text. Other genes that were investigated in other parts of this figure are labelled with hollow circles and blue text. (C) Rescue of low ATP by CoQ10 as measured by luciferase. K562 cells expressing dCas9-KRAB and a single sgRNA were placed in respiratory or glycolytic conditions as in the primary screen. ATP concentrations were measured by luciferase as in . Data show mean ± SEM; N = 32 wells per group compiled from 4 independent experiments. * p < 0.05; *** p < 0.001 between untreated and CoQ10-treated conditions by two-way ANOVA with Dunnett multiple comparisons test. (D) K562 cells expressing dCas9-KRAB and a single sgRNA were collected, and total CoQ10 and CoQ10H2 levels were determined by HPLC and mass spectrometry with and without supplementation of 50 μM exogenous CoQ10 in the cell culture medium. The same cells assayed for total CoQ10 were also examined for the fraction of total CoQ10 in the oxidized state with and without CoQ10 supplementation. Untreated CoQ10 total pool sizes were compared with the untreated non-targeting guide by one-way ANOVA with Dunnett multiple comparison correction. The effect of CoQ10 treatment on total pool size and CoQ10 pool oxidation was analyzed within each gene knockdown line by two-way ANOVA with Dunnett multiple comparison correction. Data show mean ± SEM; N = 7–9 replicates per group compiled from 3 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. Further information about this figure can be found in . CoQ10, coenzyme Q10; dCas9, dead CRISPR-associated protein 9; HPLC, high-performance liquid chromatography; KRAB, Kruppel-associated box; NS, not significant; sgRNA, single guide RNA.

Article Snippet: qRT-PCR gene relative expression quantifications were performed using 7900HT Fast Real-Time PCR System (Applied Biosystem) and using FAM-MGB TaqMan Gene Expression Assays (ThermoFisher, assay ID: Hs01680112_mH—COX11; Hs01550960_g1—COX16; Hs01043634_m1—ATP5MPL; Hs00372008_m1—PDSS1; Hs01047689_m1—PDSS2; and Hs00204417_m1—NDUFA8), together with VIC-MGB human ACTB (β-actin) (ThermoFisher number 4326315E) as endogenous control. cDNAs and PCR reactions were prepared according to the protocol for Cells-to-C T kit (ThermoFisher number AM1728), using the standard reverse transcription cycle (37 °C for 6 minutes, inactivation at 95 °C for 5 minutes, hold at 4 °C), and qRT-PCR conditions (UDG Incubation: 50 °C for 2 minutes; enzyme activation: 95 °C for 10 minutes; PCR cycle: 95 °C for 15 seconds, 60 °C for 1 minute—repeat 40 cycles).

Techniques: Incubation, Labeling, Luciferase, Expressing, Mass Spectrometry, Cell Culture, Comparison, Knockdown, CRISPR, High Performance Liquid Chromatography

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: HO-1 overexpression increases stemness in CRC regardless of p53 status. Cells were pCMV6- HMOX1 or mock transfected, collected at 72 and 96 hours after transfection, and used to characterize the percentage of ALDH1+ cells by flow cytometry in the total population (TP) of ( a ) HCT-116, ( b ) HCT-116 p53 null, and ( c ) HT-29 CRC cells. In other experiments, cells were collected at 96 h after pCMV6- HMOX1 or empty vector (mock) transfections and used to quantify, in the TP, ( d ) the percentage of CD133 high /CD44 high /CD326 high and ( e ) the percentage of CD44 high /CD326 high cells or ( f ) the percentage of CD44 high /CD326 high cells in CSCs subpopulations, obtained as described in the Materials and Methods section. Data represent the mean ± SD of two experiments performed in duplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Over Expression, Transfection, Flow Cytometry, Plasmid Preparation

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: HO-1 overexpression increases self-renewal ability of isolated CRCssubpopulations in cultured cells, regardless of p53 status. The number of spheres formed by subpopulations obtained from mock and pCMV6- HMOX1 ( a ) HCT-116 and ( b ) HCT-116 p53 null transiently transfected cells. Representative images of tumorospheres formed from different subpopulations of pCMV6- HMOX1 ( c ) HCT-116 and ( d ) HCT-116 p53 null transiently transfected cells. TP: total population; CSCs: cancer stem cells subpopulation; non-CSCs: non-cancer stem cells subpopulation. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Over Expression, Isolation, Cell Culture, Transfection

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: HO-1 and ECE-1 expression is higher in the CSCs vs. non-CSCs subpopulation in CRC regardless of p53 status. The CSCs and non-CSCs subpopulations from HCT-116, HT-116 p53 null, and HT-29 cells were obtained as described in the Materials and Methods section and used to measure mRNA expression of ( a ) HO-1 and ( b ) ECE-1. Data represent the relative expression of HO-1 and ECE-1 to the TP of HCT-116 and express the mean ± SD of two experiments performed in duplicate. **, p < 0.01; ***, p < 0.001. ( c ) Protein expression of HO-1 and ECE-1 in HCT-116, HT-116 p53 null, and HT-29 cells in the TP, CSCs, and non-CSCs subpopulations. β-actin was used as housekeeping. TP: total population; CSCs: cancer stem cells subpopulation; non-CSCs: non-cancer stem cells subpopulation. Box plots representing the relative mRNA expression of ( d ) HO-1 and ( e ) ECE-1 in CRC samples from patients considering the levels of CSCs markers and the status of p53. Data represent the median and the interquartile range of the genes analyzed. *, distant values more than 3 box lengths from 75th percentile; °, distant values more than 1.5 box lengths from 75th percentile.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Expressing

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: HO-1 overexpression induces ECE-1 expression and ET-1 production in CRC cells regardless of p53 status. Cells were transiently transfected with pCMV6- HMOX1 or an empty vector and collected 24–96 h later for ( a ) HO-1 and ECE-1 protein expression by western blotting or ET-1 measurements in the supernatants of ( b ) HCT-116, ( c ) HCT-116 p53 null, ( d ) and HT-29 cells. Data represent the median ± SD of two experiments in duplicate. *, p < 0.05; ***, p < 0.001. Correlation of ECE-1 and HO-1 expression in tumors from CRC patients considering ( e ) all cases, ( f ) p53 wild-type tumors, and ( g ) p53 mutated tumors.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: Blockade of endothelin-1 receptors eliminates the HO-1-induced stemness only in p53 wild-type CRC cells. Cells were pCMV6- HMOX1 or mock transfected, treated with 10 μM bosentan 24 h after, collected at 72 and 96 h after transfection, and used to characterize the percentage of ALDH1+ cells by flow cytometry in the TP of ( a ) HCT-116, ( b ) HCT-116 p53 null, and ( c ) HT-29 CRC cells. In other experiments, cells were collected at 96 h after pCMV6- HMOX1 or empty vector (mock) transfections and used to quantify, in the TP, ( d ) the percentage of CD133 high /CD44 high /CD326 high and ( e ) CD44 high /CD326 high cells. Data represent the mean ± SD of two experiments performed in duplicate. ***, p < 0.001.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Transfection, Flow Cytometry, Plasmid Preparation

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: Blockade of endothelin-1 receptors eliminates the HO-1-induced self-renewal ability in p53 wild-type CRC cells and enhances it in p53 null cells. The number of spheres formed by subpopulations obtained from mock and pCMV6- HMOX1 ( a ) HCT-116 and ( b ) HCT-116 p53 null transiently transfected cells. Representative images of tumorospheres formed from different subpopulations of pCMV6- HMOX1 ( c ) HCT-116 and ( d ) HCT-116 p53 null transiently transfected cells. ***, p < 0.001.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Transfection

Journal: Journal of Personalized Medicine

Article Title: Endothelin-1 as a Mediator of Heme Oxygenase-1-Induced Stemness in Colorectal Cancer: Influence of p53

doi: 10.3390/jpm11060509

Figure Lengend Snippet: Influence of HO-1 overexpression on therapy resistance in CRC. As described in the Materials and Methods section, cells were seeded in 96 well-plates, transfected with vector or pCMV6- HMOX1 plasmid, and treated with increasing doses of 5-FU (0–4 Μm) during 72 h in the presence or absence of 10 μM bosentan. The viability of cells was analyzed using the MTT assay in ( a ) HCT-116, ( b ) HCT-116 p53 null, and ( c ) HT-29 cells. Data represent the mean ± SD of two experiments performed in quadruplicate. *, p < 0.05; **, p < 0.01.

Article Snippet: Plasmid expression vector pCMV6 containing the human HMOX1 gene and the corresponding empty vector were purchased from Origene Technologies (Rockville, MA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, MTT Assay

Journal: Cellular and Molecular Immunology

Article Title: Tumor cells that resist neutrophil anticancer cytotoxicity acquire a prometastatic and innate immune escape phenotype

doi: 10.1038/s41423-025-01283-w

Figure Lengend Snippet: Bacterial stimulation of tumor cells induces PMN activation. A description of the supernatants can be found in Table . A Exemplary scheme of PMN stimulation: Upper part: Control PMNs (unstimulated) were treated with the supernatant of untreated FaDu cells (FaDu (F)); lower part: PMNs were treated with supernatant from bacteria-stimulated tumor cells (SAFs). Supernatants from 20 h stimulated or unstimulated PMNs were harvested, and the concentrations of MIF B and IL-8 C were determined via ELISA, n = 6. D PMNs were stimulated for 3 h, and ROS production was determined via 123Dihydrorhodamine and flow cytometry, n = 6. E PMNs were stimulated for 24 h, and the number of dead cells was determined via AnnexinV/7AAD staining and flow cytometry, n = 6. Supernatants from PMNs stimulated for 20 h under the indicated conditions were harvested, and the concentration F and activity G of NE were determined, n = 1–6. H NET formation was analyzed in PMNs stimulated for 30 min with different supernatants. The release of myeloperoxidase (MPO, green) and citrullinated histone 3 (CitH3, orange) was detected by immunofluorescence to confirm NET formation, n = 3. I , J PMNs were stimulated with the designated supernatants for 30 min and subsequently added to tumor cells on an E-plate. The proliferation of tumor cells was monitored with xCELLigence for 72 h, n = 3. Tumor tissues from ten SCCHN patients (also shown in Fig. ) were stained for CD66b (neutrophils), neutrophil elastase (NE) and matrix metalloproteinase-9 (MMP-9). Patients were grouped according to their nodal metastasis status (gray for cN0 and yellow for cN+ ); the frequencies of neutrophils expressing NE (CD66b + NE + ) K , MMP-9 (CD66b + MMP-9 + ) L , and both markers (CD66b + NE + MMP-9 + ) M in the tumor tissue were quantified. N Representative spatial plots showing the distribution of total cells (DAPI + , gray), neutrophils (CD66b + , pink), and neutrophils expressing NE (CD66b + NE + , orange), MMP-9 (CD66b + MMP-9 + , green), or both markers (CD66b + NE + MMP-9 + , red). O – T Frequencies (as a percentage of total DAPI + cells) of neutrophils expressing NE (CD66b + NE + ), MMP-9 (CD66b + MMP-9 + ), or both markers (CD66b + NE + MMP-9 + ) in SCCHN tissue are shown according to bacterial (16S + ) enrichment. For this analysis, regions showing high and low bacterial colonization were analyzed (41 high bacterial ROIs and 31 low bacterial ROIs). U Representative spatial plots showing the spatial distribution of bacterial RNA (16S + ), neutrophils (CD66b + , pink), neutrophils expressing NE (CD66b + NE + , orange), MMP-9 (CD66b + MMP-9 + , green), and both markers (CD66b + NE + MMP-9 + , red) in regions with high or low bacterial loads. B – E, H n indicates the number of independent PMN donors tested. F , G n indicates the number of independent technical replicates. I, J n indicates the number of independent experiments and PMN donors. K – M Each symbol represents one patient. O – T Each symbol represents one unique intratumoral region. All the statistical analyses were performed via unpaired t tests: * p < 0.05, ** p < 0.01, *** p < 0,001, **** p ≤ 0.0001. The data are displayed as the means ± SDs. Spatial plots were generated via HALO AI®

Article Snippet: To visualize NET formation, the slides were stained with unconjugated antibodies targeting myeloperoxidase (MPO; mouse anti-human MPO; Bio-Rad) and citrullinated histone H3 (CitH3; rabbit anti-human histone H3; Abcam) for 1 h at RT.

Techniques: Activation Assay, Control, Bacteria, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Concentration Assay, Activity Assay, Immunofluorescence, Expressing, Generated

Journal: Immunity

Article Title: Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway

doi: 10.1016/j.immuni.2022.01.003

Figure Lengend Snippet:

Article Snippet: Peroxidase-AffiniPure Goat anti-Mouse IgG (H+L) , Jackson ImmunoResearch Labs , Cat#: 115-035-003; RRID: AB_10015289.

Techniques: Transduction, Virus, Recombinant, Generated, Transfection, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Western Blot, Plasmid Preparation, Software, Flow Cytometry, Imaging, Microscopy, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in lung adenocarcinoma

doi: 10.1101/2020.04.09.030403

Figure Lengend Snippet: (A-C) Kaplan-Meier curves for OS and FP in NSCLC (A) LUAD (B) and LUSC patients (C). (D) COX6B2 mRNA expression (RNA-seq RSEM, log2(norm count +1)) from TCGA Lung Cancer dataset. Bars represent median (normal: n = 110; tumor: n = 1017). P values calculated by Mann-Whitney test. (E-F) Whole tissue homogenates of LUAD tumors (E) and LUAD cell line lysates (F) were immunoblotted with indicated antibodies. Molecular weight (MW) markers are indicated. (G) IHC staining of non-malignant testis (a positive control), non-malignant lung (adjacent normal) and LUAD tissues. Scores ranged from 0 to 3. Scale bar, 50 μm. Bars represent mean ± SEM. P values calculated by Mann-Whitney test. (H) Representative confocal images of endogenous COX6B2 in HCC515 cells. Tom20 is used as a mitochondrial marker. Images were shown as Z-stack maximum projection from 0.3 μm-thick-image. Scale bar, 10 μm.

Article Snippet: The TaqMan ® qPCR probes were as follows: COX6B2 (Hs00376070_m1), COX6B1 (Hs01086739_g1).

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Molecular Weight, Immunohistochemistry, Positive Control, Marker

Journal: bioRxiv

Article Title: Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in lung adenocarcinoma

doi: 10.1101/2020.04.09.030403

Figure Lengend Snippet: (A) Left: Whole cell lysates from HCC515 cells +/- COX6B2-V5 were immunoblotted with indicated antibodies. MW markers are indicated. Right: Quantitation of COX6B2 protein expression from blots on left. Bars represent mean ± SD (n = 4). (B) Mitochondrial DNA content. Bars represent mean + range (n = 2). (C) Quantification of total mitochondria using FACS analysis of Mitotracker green stained cells. Bars represent mean ± SD (n = 3). (D) Left: Oxygen consumption rate (OCR) as a function of time in indicated cell lines following exposure to electron transport chain complex inhibitors. Bars represent mean ± SEM (n = 6). Right: Mean and distribution of individual values for basal, ATP-linked, maximal and reserve OCRs. P values calculated by Student’s t -test. (E) Left: Whole cell lysates from H2122 cells +/- COX6B2-V5 were immunoblotted with indicated antibodies. MW markers are indicated. Right: Quantitation of COX6B2 protein expression. Bars represent mean ± SD (n = 3). (F) As in (D) (n = 8). (G) ATP content of indicated cell lines. Bars represent mean + SEM (n ≥ 6). HCC515 P value calculated by Student’s t -test and H2122 P value calculated by Mann-Whitney test. (H) NAD + /NADH measurement in indicated cell lines. Bars represent mean ± SEM (n ≥ 5). P value calculated by Student’s t -test. (I) Basal extracellular acidification rate (ECAR) are presented as mean ± SEM (n ≥ 6) from CTRL and COX6B2-V5 cells of HCC515 and H2122 cells. P value calculated by Student’s t -test. (J) Immunoblots of indicated whole cell lysates with specified antibodies. MW markers are indicated. (K) As in (D) using indicated cell lines. Bars represent mean ± SEM (n ≥ 9). Basal and ATP-link OCR P value calculated by Student’s t -test. Maximal OCR and reserve capacity P value calculated by Mann-Whitney test.

Article Snippet: The TaqMan ® qPCR probes were as follows: COX6B2 (Hs00376070_m1), COX6B1 (Hs01086739_g1).

Techniques: Quantitation Assay, Expressing, Staining, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in lung adenocarcinoma

doi: 10.1101/2020.04.09.030403

Figure Lengend Snippet: (A) Schematic of complex IV activity measurements in isolated mitochondria. Details are described in Material and Methods. antA, Antimycin A; Cyt c, Cytochrome c; ATP Syn, ATP synthase. (B) Complex IV activity in indicated cell lines using TMPD/ascorbate as a substrate. Bars represent mean + SEM (n ≥ 8). P value calculated by Student’s t -test. (C) As in (B) in HCC515 cell lines expressing CTRL and COX6B1-V5 cDNA. Bars represent mean + SEM (n = 8). P value calculated by Student’s t -test. (D) Indicated lysates from HCC515 cells were run on a BN-PAGE gel and stained with Colloidal Blue (left two panels) or immunoblotted with anti-COX6B2, anti-COX6B1 and anti-COXIV (right panels). Representative image of n ≥ 3. MW markers are indicated to identify different complexes . (E) Left: Distribution of COX6B1 and COX6B2 incorporated in complex IV monomers, dimers or supercomplexes as detected by BN-PAGE in (D). Right: Bars represent mean ± SEM (n ≥ 3) based on quantification of bands in (D). In monomeric complex IV, P value calculated by Mann-Whitney test whereas others calculated by Student’s t -test. (F) Left: Distribution of COX6B1/COX6B2 incorporated in monomeric complex IV, dimeric complex IV and supercomplexes based on BN-PAGE in COX6B2-V5 or COX6B1-V5 overexpressing cell lines (D). Right: Bars represent mean ± SEM (n ≥ 3) based on quantification of bands in (D). In monomeric complex IV, P value calculated by Mann-Whitney test whereas others calculated by Student’s t -test. (G) Left: Distribution of COXIV incorporated in monomeric complex IV, dimeric complex IV and supercomplexes by blots from (D). Right: Bars represent mean ± SEM (n ≥ 3) of COXIV based on quantification of band in (D). P value calculated by Student’s t -test. (H) Relative superoxide in indicated cell lines. Bars represent mean + SEM (n = 5). P value calculated by Student’s t -test.

Article Snippet: The TaqMan ® qPCR probes were as follows: COX6B2 (Hs00376070_m1), COX6B1 (Hs01086739_g1).

Techniques: Activity Assay, Isolation, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in lung adenocarcinoma

doi: 10.1101/2020.04.09.030403

Figure Lengend Snippet: (A) Percentage of cells positive for EdU incorporation in indicated samples. Values represent the mean ± SEM (n ≥ 9). P value calculated by Student’s t -test. (B) Population doubling rate in indicated samples incubated ± 1mM pyruvate for five days. Bars represent mean + SEM (n = 4). P value calculated by Student’s t -test. (C) Top: Representative images of soft agar assays for indicated cell lines. Arrows indicate formed colonies. Bottom: Graphs represent mean colony numbers in indicated cell lines. Bars represent mean ± SEM (n = 6). P value calculated by Student’s t -test. (D) EdU positive HCC515 cells at indicated times in hypoxia. Values are relative to cells incubated in normoxia. Bars represent mean ± SEM (n = 8). P value calculated by Student’s t -test. (E) Average mRNA expression of COX6B2 measured following 12 hours of hypoxia culture in HCC515 cells. Bars represent mean ± SEM (n = 6). P calculated by Student’s t -test. (F) Whole cell lysates from indicated cell lines were immunoblotted with indicated antibodies. Hypoxia exposure was for 12 hours. Representative immunoblots from n = 3. MW markers are indicated.

Article Snippet: The TaqMan ® qPCR probes were as follows: COX6B2 (Hs00376070_m1), COX6B1 (Hs01086739_g1).

Techniques: Incubation, Expressing, Western Blot

Journal: bioRxiv

Article Title: Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in lung adenocarcinoma

doi: 10.1101/2020.04.09.030403

Figure Lengend Snippet: (A-B) Left: Oxygen consumption rate (OCR) as a function of time in COX6B2 depleted HCC515 cells using an shRNA pool (A) or an siRNA pool (B) following exposure to electron transport chain complex inhibitors. Bars represent mean ± SEM (n ≥ 4). Right: Bars represent mean ± SEM 20 minutes following the addition of each drug (on left). P values calculated by Student’s t -test. (C) Relative NAD + /NADH measurement in indicated cell lines. Bars represent mean + SEM (n = 4). P value calculated by Student’s t -test. (D) Relative ATP content in indicated cell lines transfected with indicated individual siRNAs for 96 hours. Bars represent mean + SEM (n = 6), **** P < 0.0001; *** P < 0.005 calculated by Student’s t -test. (E) Left panels: Flow cytometry measurement following JC-1 labelling of indicated HCC515 cells. Right graph: Bars represent mean of the aggregate to monomer ratio (red/green) in indicated cell lines + SEM (n = 3). P value calculated by Student’s t -test. (F) Indicated cell lines were labelled with CM-H2DCFDA to measure H 2 O 2 as described in material and methods. Bars represent mean fold change in median fluorescent intensities ± SEM (n = 4). P value calculated by Student’s t -test.

Article Snippet: The TaqMan ® qPCR probes were as follows: COX6B2 (Hs00376070_m1), COX6B1 (Hs01086739_g1).

Techniques: shRNA, Transfection, Flow Cytometry