Journal: Cells

Article Title: House Dust Mite Nebulization Drives Alarmin and Complement Activation in a Murine Tracheal Air–Liquid Interface Culture System

doi: 10.3390/cells14201598

Figure Lengend Snippet: Time-dependent complement activation and anaphylatoxin receptor expression in ALI cultures exposed to HDM. ( a ) C3 and C3a concentrations in cell lysates and the SN of unstimulated (steady-state) ALI cultures and 24, 48 and 72 h after HDM exposure (steady-state, 24, 48 and 72 h: n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001. ( b ) HDM and HI-HDM mediated cleavage of hC3 into hC3a ( n = 3 independent experiments). Data are shown as mean ± SEM. Data were analyzed using an unpaired T -test. *** p < 0.001. ( c ) C5 and C5a concentrations in cell lysates and the SN of steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). ( d ) Comparison of C3 and C5 ( left panel ), C3a and C5a ( right panel ) concentrations in cell lysates from steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. **** p < 0.0001. ( e ) Immunofluorescence analysis of C3aR and C5aR1 expression in unstimulated ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM ( n = 3). Data were analyzed using one-way ANOVA with Holm–Šidák’s multiple comparisons test. * p < 0.05; ** p < 0.01. The number of experiments shown in ( a , c – e ) refers to biological replicates derived from 6 independent preparations of trachea, each representing a separate cell isolation and culture.

Article Snippet: Briefly, we used neo-epitope-specific anti-hC3a mAb (Hycult Biotech #HM2074—Uden, The Netherlands) as capture and biotinylated anti-hC3/C3a mAb (Hycult Biotech #HM2073—Uden, The Netherlands) as detection antibody.

Techniques: Activation Assay, Expressing, Comparison, Immunofluorescence, Derivative Assay, Cell Isolation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: Proteomic analysis of HEV-1-infected cells. a , Confocal microscopy of mock- and HEV-1-infected HepG2/C3a-MAVS-KD cells stained for ORF2 protein (red) and nuclei (blue). Scale bar, 20 μm. Right: Quantification of infection rate. Each data point represents the percentage of ORF2-positive cells from 16 microscopic areas (~ 300 cells) across three independent experiments. Statistical significance was assessed using a nonparametric Student’s t-test, p < 0.0001 (****). b , Venn diagram of identified proteins (left) and volcano plot (right) showing differential protein abundance between HEV-1-infected and mock-infected cells (EdgeR). Dashed line indicates statistical significance threshold (− log₁₀ p-value); vertical lines denote significantly upregulated (magenta) and downregulated (blue) proteins. c , Heatmap with hierarchical clustering of significantly altered proteins. Data represent mean values from three independent experiments. Z-score–normalised expression is colour-coded (red, upregulation; blue, downregulation). d , Sankey plot of significantly enriched canonical pathways grouped by biological function (e.g., immune response, metabolism), as identified by Ingenuity Pathway Analysis (IPA). Each ribbon represents a pathway, colour-coded by functional category. Right, bubble plot showing pathway activation status: bubble colour indicates predicted z-score (red, activated; blue, inhibited; gray, no available pattern), with bubble size reflecting the number of overlapping proteins and x-axis showing − log₁₀ p -value. e , Chord diagram highlighting proteins contributing to two or more enriched pathways enriched pathways related to mitochondrial function, stress responses, and lipid metabolism, as well as autophagy and apoptosis. Connections indicate protein membership in the leading-edge subsets of each pathway (circlize R package). f , Network diagram showing functional interactions between enriched pathways based on co-enrichment and shared proteins. Nodes represent pathways, and edges indicate functional similarity or shared components, illustrating coordinated host cell reprogramming

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Confocal Microscopy, Staining, Quantitative Proteomics, Expressing, Functional Assay, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: HEV infection reprograms the neutral lipid metabolism. a , HepG2/C3a-MAVS-KD cells were infected with HEV-1 or HEV-3 genotypes for three weeks, to reach at least 50%. Large field micrographs showing nuclei (blue) and ORF2 staining in infected cells (green), scale bar 50 µM (left). Bar graph showing quantification of infection rate using large field microphaphs and (right). Data points represent percentage of ORF-2 positive cells from 25 microscopic areas from three independent experiment (10 4 cells) using StarDist tool. b , Neutral lipid (NL) were extracted from mock- (black), HEV-1- (red) and HEV-3-infected (blue) HepG2/C3a-MAVS-KD cells and quantified by comparing the peak area of individual species to class-specific internal standards (left panel). Left, total NL abundance (sum of CL, CE, and TG per mg protein). Right, The three main NL classes—cholesterol (CL), cholesteryl esters (CE), and triglycerides (TG) - relative proportions of CL, CE, and TG as percentages of total NL. c - d , Relative abundance (per mg protein) of the most significantly altered cholesterol (CL-C18, c ) and triglyceride subspecies ( d ). e , Heatmap showing the relative abundance profiles of individual NL subspecies. Each column represents a biological replicate; each row corresponds to a lipid species. f , Lipid droplet (LD) content visualised by Bodipy 493 − 503 staining using confocal microscopy. g , Representative FACS histograms gated on bodipy positive cells (left). Bar graph showing mean fluorescence intensity (MFI) from six independent experiments (right). Lipid amounts were normalised to protein level All the results are presented as mean values ± SEM from at least three independent experiments. Statistical significance was assessed using ANOVA for multiple comparisons. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Staining, Confocal Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: HEV infection impairs oxylipin metabolism. HepG2/C3a-MAVS-KD cells were infected with HEV-1 or HEV-3 for at least three weeks until infection rates exceeded 50%. The abundance of 20 major oxylipins in mock- (black), HEV-1- (red), and HEV-3-infected (blue) cells was quantified and normalised to protein amounts. a , Relative abundance of metabolites derived from omega-6 linoleic acid (LA) via the LOX pathways. b , Relative abundance of oxylipins derived from arachidonic acid. c , Relative abundance of bioactive metabolites derived from the omega-3 polyunsaturated eicosapentaenoic acid and docosahexaenoic acid Data represent mean values ± SEM of three independent experiments Statistical significance was assessed by repeated measures ANOVA with Tukey post hoc test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: HEV infection does not implicate glycolysis metabolic pathway. HepG2/C3a-MAVS-KD cells were uninfected (Mock, black) or infected with HEV-1 (red) or HEV-3 (blue) for at least three weeks until infection rates exceeded 50%. a , Extracellular acidification rate (ECAR) was measured using the XF Glycolysis Stress Test and the Seahorse XFp analyzer. Time points of glucose, Oligomycin (Oligo) and 2-Deoxy-D-Glucose (2-DG) are indicated by dotted arrow. Data are presented as mean values ± SEM from six independent experiments. b , Violin plots comparing glycolytic flux parameters: Glycolysis, Glycolytic Capacities, Glycolytic Reserve and non-glycolytic acidification rate. Data are presented as mean values ± SEM from repeated measures of six independent experiments. c , Lactate concentration quantified in culture supernatants. Data represent mean values ± SEM of at least six independent experiments Statistical significance was assessed by repeated measures ANOVA with Tukey post hoc test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Concentration Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: HEV infection increases mitochondrial function. HepG2/C3a-MAVS-KD cells were uninfected (Mock, black) or infected with HEV-1 (red) or HEV-3 (blue) for at least three weeks until infection rates exceeded 50%. a , Mitochondrial Oxygen consumption rate (OCR) measured in Mock- (black dots) and HEV-1-infected (red dots) cells. Time point of oligomycin (Oligo), FCCP and rotenone/antimycin A (Rot/Ant) are indicated by the dotted arrow. Data are presented as mean values ± SEM from six independent experiments. b , Violin plots comparing the parameters of mitochondrial function - basal respiration, ATP production, proton leak, maximal respiration, coupling efficiency, spare respiratory capacities, and non-mitochondrial respiration (non-mito O2 consumption). Data are presented as mean values ± SEM from repeated measures of six independent experiments. c , Representative confocal micrographs showing immunostaining of mitochondria (Tom22-Green), ORF2 (red) and nucleus (blue). Scale bar 20 µM. d , Mitochondria mass and polarization were analyzed using mitotracker green and TMRM, respectively. The ratio mitotracker/TMRM denoting mitochondria polarization/mitochondria mass is presented (right panel). e , Cellular ROS production measured by CellRox probe. Flow cytometry data are presented as geometric mean fluorescence intensity (MFI) Mock-infected cells (black dots), HEV-1 infected cells (red dots), HEV-3 infected cells (blue dots). d - e , Data are presented as mean values ± SEM from six independent experiments Statistical significance was assessed by repeated measures ANOVA with post hoc test p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Immunostaining, Flow Cytometry, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Mitochondrial and lipid metabolism rewiring during HEV infection

doi: 10.1007/s00018-025-05994-1

Figure Lengend Snippet: Fatty acid oxidation rather than glycolysis is required for HEV infection. HepG2/C3a-MAVS-KD cells were pre-treated for 1 h with the indicated inhibitor before being infected with HEV-1 or HEV-3 for 48 h in presence of the same inhibitor. a , Confocal micrographs of HEV replication in the presence of DMSO vehicle (CTRL) or different inhibitors of metabolic pathways: CD36 inhibitor (CD36-Inh), Etomoxir and Oligomycin (Oligo). ORF2 (green) and nuclei (dapi, blue). Scale bar 100 µM. b , Quantification of HEV-1(red) and HEV-3 (blue) replication under different culture conditions. Data points represented percentage of control and given as mean values ± SEM from six independent experiments. p values are computed using one-way ANOVA. p < 0.01 (**), p < 0.001 (***), p < 0.0001(****). c , Left: Confocal micrographs showing HEV-1 and HRV-3 replication in complete medium (CTRL), in the presence of 2DG inhibitor of glycolysis or under glucose restriction (ΔGlu). ORF2 staining (green) and DAPI-stained nuclei (blue). Scale Bar 100 µM Right: Quantification of HEV ORF2 positive cells. Data points represented percentage of control. Data are presented as mean values ± SEM from at least six independent experiments Statistical significance was assessed by repeated measures ANOVA with post hoc test p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Article Snippet: HepG2/C3a, a subclone of the human hepatocarcinoma cell line HepG2, was obtained from ATCC (CRL-3581) and used for viral production.

Techniques: Infection, Control, Staining

Journal: BMC Complementary Medicine and Therapies

Article Title: In vitro anti-diabetic activity and molecular docking studies of Amaranthus cruentus , Amaranthus hybridus and isolated compounds

doi: 10.1186/s12906-025-05169-2

Figure Lengend Snippet: Glucose utilisation (%) after 24 h of treatment in C3A hepatocytes. Results were normalised to cell viability as determined using the MTT assay. Error bars indicate the standard deviation of the mean of four replicates from a single experiment: αS- α -spinasterol, PA-palmitic acid, AH- A. hybridus and AC- A.s cruentus . The asterisk (*) is used to represent a statistically significant result. The values were considered significant at p < 0.05

Article Snippet: Human hepatoma-derived C3A hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

Techniques: MTT Assay, Standard Deviation

Journal: BMC Complementary Medicine and Therapies

Article Title: In vitro anti-diabetic activity and molecular docking studies of Amaranthus cruentus , Amaranthus hybridus and isolated compounds

doi: 10.1186/s12906-025-05169-2

Figure Lengend Snippet: Glucose uptake (%) after four hours in C3A hepatocytes following a 24-hour treatment period. A. hybridus showed significant glucose utilisation (20.86%) while palmitic acid increased glucose uptake by 24.52% and 25.13% at 7.8 and 31.3 µg/mL, respectively. Results were normalised to cell viability as determined using the MTT assay. Error bars indicate the standard deviation of the mean of four replicates from a single experiment: αS- α -spinosterol, PA-palmitic acid, AH- A. hybridus and AC- A. cruentus . The asterisk (*) is used to represent a statistically significant result. The values were considered significant at p < 0.05

Article Snippet: Human hepatoma-derived C3A hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

Techniques: MTT Assay, Standard Deviation

Journal: BMC Complementary Medicine and Therapies

Article Title: In vitro anti-diabetic activity and molecular docking studies of Amaranthus cruentus , Amaranthus hybridus and isolated compounds

doi: 10.1186/s12906-025-05169-2

Figure Lengend Snippet: Glucose utilisation (%) after 48 h of treatment in C3A hepatocytes. Results were normalised to cell viability as determined using the MTT assay. Error bars indicate the standard deviation of the mean of four replicates from a single experiment: αS- α -spinasterol, PA-palmitic acid, AH- A. hybridus and AC- A. cruentus . The asterisk (*) is used to represent a statistically significant result. The values were considered significant at p < 0.05

Article Snippet: Human hepatoma-derived C3A hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

Techniques: MTT Assay, Standard Deviation

Journal: BMC Complementary Medicine and Therapies

Article Title: In vitro anti-diabetic activity and molecular docking studies of Amaranthus cruentus , Amaranthus hybridus and isolated compounds

doi: 10.1186/s12906-025-05169-2

Figure Lengend Snippet: Glucose uptake (%) after four hours in C3A hepatocytes following a 48-hour treatment period. At concentrations of 15.6 and 31.3 µg/mL for α -spinasterol and 15.6 µg/mL for A. hybridus , glucose absorption was 53.68%, 31,25%, and 52.16%, respectively. At 10 µg/mL, insulin significantly increased glucose utilisation by 51.5%. Results were normalised to cell viability as determined using the MTT assay. Error bars indicate the standard deviation of the mean of four replicates from a single experiment: αS- α -spinasterol, PA-palmitic acid, AH- A. hybridus and AC- A. cruentus . The asterisk (*) is used to represent a statistically significant result. The values were considered significant at p < 0.05

Article Snippet: Human hepatoma-derived C3A hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

Techniques: MTT Assay, Standard Deviation