Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: Representative HiBiT blots were obtained from three experimental conditions: (1) SPI-1–permissive LB conditions, including total lysates (TL) from early stationary phase cultures (OD 600 = 2.0) or overnight cultures, along with the corresponding secreted fractions (secretomes); (2) SPI-2–inducing conditions using mildly acidic LPM medium (pH 5.8); and (3) infection-derived fractions from HeLa cells infected at a multiplicity of infection (MOI) of 50, separated into TX-100–insoluble pellets (representing bacteria-associated effectors) and TX-100– soluble supernatants (representing translocated effectors). For each effector, a representative blot from a condition in which a clear HiBiT signal was obtained is shown. The accompanying six-square panel indicates detection (green), detection at higher protein load (orange), no detection (red), or not determined (black) across: (A) LB TL (OD 600 = 2.0 and/or LB O/N), (B) LB secretomes (O/N), (C) SPI-2–inducing conditions TL, (D) TX-100-insoluble pellet fraction (infected HeLa cells) TL, (E) TX-100-soluble supernatant (translocated effectors), and (F) real-time HiBiT translocation readout (infected HeLa cells). Representative blots obtained from LB OD 600 = 2.0 TL: AvrA (PVDL0187), GtgA (PVDL0313), GtgE (PVDL0246), SipB (PVDL0183), SipC (PVDL0028), SlrP (PVDL0184), SopB (PVDL0024), SopD (PVDL0185), SptP (PVDL0034), and SteC (PVDL0263). Representative blots obtained from LB O/N TL: CigR (PVDL0045), SifB (PVDL0041), SipA (PVDL0328), SopD2 (PVDL0299), SopE (PVDL0267), SopE2 (PVDL0033), SopF (PVDL0278), SpvB (PVDL0425), SseF (PVDL0257), SseK1 (PVDL0248), SseL (PVDL0249), SseM (PVDL0046), and SteB (PVDL0311). Representative blots obtained from SPI-2–inducing conditions: GogB (PVDL0298), PipB (PVDL0251), PipB2 (PVDL0040), SifA (PVDL0312), SpvC (PVDL0426), SpvD (PVDL0427), SrfJ (PVDL0280), SseD (PVDL0270), SseG (PVDL0258), SseI (PVDL0259), SseJ (PVDL0275), SseK2 (PVDL0260), SseK3 (PVDL0289), SspH2 (PVDL0261), and SteD (PVDL0271). Representative blots obtained from additional conditions: SopA from infection-derived Triton X-100–insoluble pellet fractions from infected HeLa cells (PVDL0032; 8 h post-infection (hpi)) and SteA (PVDL0029; LB O/N secretome).

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Infection, Derivative Assay, Bacteria, Translocation Assay

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: Representative HiBiT blots illustrating the secretion potential of all 39 Salmonella typedetected green fluorescent objects III effectors in overnight (O/N) LB cultures, which mainly supports SPI-1–dependent secretion. For each effector, total lysates (TL) are shown alongside their corresponding secretome (S) fractions, providing a comparative map of basal, SPI-1–mediated secretion capacity. TL samples are included to demonstrate expression relative to secretion under these conditions. As in , the accompanying six-square panel summarizes detection outcomes across the major experimental categories, with green indicating detection, orange detection at higher protein load, red no detection, and black not determined, across: (A) LB TL (OD 600 = 2.0 and/or LB O/N), (B) LB secretomes (O/N), (C) SPI-2–inducing conditions TL, (D) TX-100-insoluble pellet fraction (infected HeLa cells) TLs, (E) TX-100-soluble supernatants (translocated effectors), and (F) real-time HiBiT translocation readout (infected HeLa cells). All HiBiT-tagged effectors were expressed from endogenously modified strains in the GFP-expressing SL1344 background ( hisG 46 P tet :: gfp ; PVD0002), except for SipA, SpvB, SpvC, and SpvD, which were expressed in the wild-type SL1344 background (PVD0001). Individual strain identifiers are listed in Supplementary Table S2 .

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Expressing, Infection, Translocation Assay, Modification

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HiBiT blotting analysis of 10 selected Salmonella type III effectors—SopA, SipC, SopE2, SteA, SopB, SptP, SseM, PipB2, SifB, and CigR—during infection of HeLa cells. CigR data are not shown due to the absence of a detectable HiBiT signal. (A) Representative HiBiT blots of Triton X-100–soluble (host cell cytosolic; translocated effectors) and Triton X-100–insoluble (bacteria-associated; expressed effectors) fractions collected at the indicated hours post-infection (hpi). GAPDH and GFP immunoblots are shown as representative and reproducible controls from the SopB experiment and were used as markers of the TX-100–soluble host cell fraction and as a normalization control for bacterial load, respectively. The black arrowhead denotes the mono-ubiquitinated form of the effector SopB, indicative of host-cell translocation. SopA, SipC, and SopE2 were detectable exclusively in the TX-100–insoluble fraction under the infection conditions tested (8–16 hpi), indicating bacterial expression but no detectable host cell translocation. (B) Densitometric quantification of HiBiT signals over time (background-corrected signal intensity; a.u.: arbitrary units). Upper panels depict translocated effector levels (tr.), quantified from the TX-100–soluble fraction. Middle panels show bacteria-associated effector levels (expr.), quantified from the TX-100–insoluble fraction. Lower graphs represent the relative translocation efficiency (tr./expr.), calculated as the ratio of translocated (TX-100–soluble) to bacteria-associated (TX-100–insoluble) HiBiT signal. Quantification is shown for those effectors displaying detectable host-cell translocation under the infection conditions tested (8–16 hpi) (SopB, SptP, SifB, PipB2, and SseM). HeLa cells were infected at an MOI of 50, and samples were processed as described in Methods. Abbreviations: tr., translocated effector (TX-100–soluble fraction); expr., bacteria-associated (expressed) effector (TX-100–insoluble fraction).

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Infection, Bacteria, Western Blot, Control, Translocation Assay, Expressing

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells stably expressing LgBiT were infected with a constitutively eGFP-expressing Salmonella enterica serovar Typhimurium SL1344 strain, either wild-type (WT eGFP+, left panels) or expressing chromosomally integrated sopB-HiBiT (SopB-HiBiT eGFP+, right panels), at multiplicities of infection (MOIs) of 10 (green), 50 (red), or 100 (blue). Infections were performed in quadruplicate, and real-time acquisition was conducted for 24 h using a multimode plate reader under environmental control. Data acquisition started 2 h post-infection (hpi). Top-to-bottom panels show: (i) fold change in HeLa cell count based on label-free automated brightfield imaging (10x objective); (ii) green fluorescence (RFU) as a proxy of bacterial load; (iii) average size (µm 2 ) of detected green fluorescent objects; and (iv) luminescence signal (RLU) either background levels or SopB-HiBiT translocation into LgBiT-expressing HeLa cells via NanoLuc complementation. Vivazine Live Cell Substrate was added following the gentamicin protection step and prior to acquisition to enable continuous luminescence detection throughout the infection course, starting at 2 hpi. Data points (every 30’) represent the mean ± SD of quadruplicates. WT-infected controls (left) confirm minimal background luminescence in the absence of HiBiT-tagged effectors.

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Stable Transfection, Expressing, Infection, Control, Cell Characterization, Imaging, Fluorescence, Translocation Assay

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells were infected with constitutively eGFP-expressing S. Typhimurium SL1344 wild-type (WT) or mutant strains deficient in key virulence factors: ΔinvA (T3SS-1-deficient), ΔssaV (T3SS-2-deficient), or a translational knockout of the SPI-2 effector SifA (ΔsifA, tko). Infections were performed at a multiplicity of infection (MOI) of 50, and real-time monitoring was conducted over 24 hours using automated label-free imaging in a multimodal plate reader under environmental control. Fluorescence parameters were normalized to their respective maxima (% max). Total green fluorescence (RFU, green) and average green object size (µm 2 , red) serve as proxies for intracellular bacterial load and bacterial distribution within infected cells, respectively. Data points represent mean ± SD of technical quadruplicates.

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Infection, Expressing, Mutagenesis, Knock-Out, Imaging, Control, Fluorescence

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells stably expressing LgBiT were infected with a panel of 39 endogenously HiBiT-tagged Salmonella enterica serovar Typhimurium SL1344 strains, all generated in a constitutively eGFP-expressing background ( hisG 46 P tet :: gfp ; PVD0002). Infections were performed at a multiplicity of infection (MOI) of 50, and real-time monitoring was conducted for 24 hours (from 2 to 26 hpi) using automated multimodal imaging in a plate reader under environmental control. Total green fluorescence (RFU, green) and average green object size (µm , red) serve as proxies for intracellular bacterial load and bacterial distribution within infected cells, respectively. Luminescence signal (RLU, blue) reflects NanoLuc complementation following translocation of HiBiT-tagged effectors into the host cell cytosol. Fluorescence and luminescence parameters were normalized to their respective maxima (% max) to facilitate comparison across strains. Data points represent mean ± SD of technical quadruplicates. Individual strain identifiers and effector annotations are listed in Supplementary Table S2 .

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Stable Transfection, Expressing, Infection, Generated, Imaging, Control, Fluorescence, Translocation Assay, Comparison

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells stably expressing LgBiT were infected with endogenously HiBiT-tagged Salmonella enterica serovar Typhimurium SL1344 effector strains in three genetic backgrounds: wild-type (WT) SL1344 carrying constitutive eGFP + ( hisG 46 P tet ::gfp; PVD0002; green), the T3SS-1–deficient ΔinvA::cat mutant (PVD0316; blue), and the T3SS-2–deficient ΔssaV::cat mutant (PVD0317; red). Luminescence profiles (RLU), reflecting NanoLuc complementation following effector translocation into the host cytosol, were recorded from 2 to 26 hours post-infection (hpi). Panels show a representative subset of effectors grouped by secretion system dependence—SPI-1 (SopB, SptP, SopD; left), SPI-1/2 (GogB [25% SPI-2 contribution], SseM [56%], SseJ [95%]; middle), and SPI-2 (SteD, SseF, SspH2; right)—as determined by phase-resolved AUC analysis (see Methods and Supplementary Table S5 ). The complete set of translocation profiles for all 39 effectors is shown in . Infections were performed at a multiplicity of infection (MOI) of 50, and real-time luminescence monitoring was conducted using a Spark Cyto 400 multimodal plate reader under environmental control in the presence of Nano-Glo Vivazine. Luminescence values were background-subtracted (non-infected HeLa). Data points represent mean ± SD of technical quadruplicates. Individual strain identifiers and effector annotations are provided in Supplementary Table S2.

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Stable Transfection, Expressing, Infection, Mutagenesis, Translocation Assay, Control

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells stably expressing LgBiT were infected with endogenously HiBiT-tagged Salmonella enterica serovar Typhimurium SL1344 effector strains in three genetic backgrounds: wild-type SL1344 carrying constitutive eGFP + ( hisG 46 P tet :: gfp ; PVD0002; green), the T3SS-1–deficient ΔinvA::cat mutant (PVD0316; blue), and the T3SS-2–deficient ΔssaV::cat mutant (PVD0317; red). Luminescence profiles (RLU), reflecting NanoLuc complementation following effector translocation into the host cytosol, were recorded from 2 to 26 hours post-infection (hpi). Infections were performed at a multiplicity of infection (MOI) of 50, and real-time luminescence monitoring was conducted using a Spark Cyto 400 multimodal plate reader under environmental control in the presence of Nano-Glo Vivazine. Luminescence values were background-subtracted (non-infected HeLa). Effectors are displayed alphabetically and distributed across panels (A–C). Data points represent mean ± SD of technical quadruplicates. Individual strain identifiers and effector annotations are provided in Supplementary Table S2 .

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Stable Transfection, Expressing, Infection, Mutagenesis, Translocation Assay, Control

Journal: bioRxiv

Article Title: Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

doi: 10.64898/2026.03.06.710167

Figure Lengend Snippet: HeLa cells stably expressing cytosolic LgBiT were infected with selected HiBiT-tagged S. Typhimurium SL1344 strains in a constitutively eGFP-expressing background ( his G46 P tet ::gfp; PVD0002), including T3SS-1–deficient ( ΔinvA; blue) and T3SS-2–deficient ( ΔssaV; red) derivatives. Luminescence was measured at 24 hours post-infection (hpi), corresponding to the indicated timepoint from continuous real-time acquisition using (A) furimazine, added shortly before readout, or (B) vivazine, present throughout infection. Bars represent raw luminescence values (RLU; mean ± SD of technical replicates). Strain identifiers and effector annotations are listed in Supplementary Table S2 .

Article Snippet: HeLa cells (human cervical adenocarcinoma, American Type Culture Collection, Manassas, VA, USA; ATCC ® CCL-2TM) and a stable HeLa cell line expressing LgBiT (HeLa-LgBiT) , kindly provided by Prof. Samuel Wagner, were maintained in GlutaMAX TM -supplemented Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 31966047) containing 10% foetal bovine serum (FBS; Invitrogen, cat. no. 10270106) and antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin; Gibco, cat. no. 15070063).

Techniques: Stable Transfection, Expressing, Infection

Journal: International Journal of Molecular Sciences

Article Title: A Simple Ionic-Gelation Method for Chitosan Nanoparticle Synthesis and Standardized Protocols for Biological Safety Assessment: Antibacterial Activity, Phytotoxicity, and Biocompatibility

doi: 10.3390/ijms27083673

Figure Lengend Snippet: Cell viability of HDF cells treated with Ch NPs at concentrations of 2.5–160 µg/mL for 24 h. Viability was determined using the MTT assay and expressed as mean ± SE (n = 3). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: The biocompatibility of Ch NPs was evaluated using primary HDF (ATCC ® PCS-201-012TM [ ], American Type Culture Collection, Manassas, VA, USA) and HaCaT cells (Cat. No. T0020001, AddexBio, San Diego, CA, USA).

Techniques: MTT Assay

Journal: Cells

Article Title: Fixed-Bed Bioreactor Culture Enhances Yield and Reparative Properties of hTERT Mesenchymal Stem Cell Extracellular Vesicles

doi: 10.3390/cells15070654

Figure Lengend Snippet: EV function and signaling activity. ( a ) Primary keratinocytes were exposed to IR and treated with EVs from either flask or bioreactor cultures. Cell viability was assessed at 24, 48, and 72 h using the CellTiter-Glo assay. ( b ) Non-irradiated keratinocytes were treated with EVs from flask or bioreactor cultures, and viability was assessed after 72 h using the same assay. * = p < 0.05; *** = p < 0.001; **** = p < 0.0001. ( c ) Western blot of cell cycle proteins associated with hTERT MSC EVs produced from both the bioreactor and flask-based cultures. ( d ) In vitro kinase assay of EV-associated CDK2. EVs were immunoprecipitated with CDK2 antibodies or IgG control and kinase activity was assessed using Histone H1 as substrate.

Article Snippet: Normal primary epidermal keratinocytes (ATCC ® PCS-200-011TM) were maintained in Dermal Cell Basal Medium (ATCC ® PCS-200-030TM) supplemented with Keratinocyte Growth Kit (ATCC ® PCS-200-040TM).

Techniques: Activity Assay, Glo Assay, Irradiation, Western Blot, Produced, In Vitro, Kinase Assay, Immunoprecipitation, Control

Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: B2AR V34A 1.33 and S41A 1.40 mutations alter the organization of receptor homomers (A) Schematic showing the experimental design of BRET for receptor-receptor associations. HEK 293 cells were transiently transfected with equal amounts of Rluc8-tagged receptor, either wild-type or with mutations, and increasing amounts of Venus-tagged receptor, either wild-type or with mutations. BRET saturation curves were used to determine the (B) BRET max and (C) the BRET 50. Data are the mean ± SEM; one-sample t test: ∗ p < 0.05. (D) Curve plotted using one-site specific binding saturation equation, representative curves shown, n = 6–8.

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Transfection, Binding Assay

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: The effect of B2AR mutations on basal and ligand-induced cAMP (A) cAMP accumulation in HEK 293 cells transiently transfected with HA-B2AR WT and mutants with isoproterenol treatment (1 pM-10 μM, 5-min). (B) cAMP production at the maximum isoproterenol dose (10 μM) and (C) basal cAMP production. Data are the mean ± SEM from n = 5–6 independent experiments. One-sample t test: ∗∗∗ p < 0.001. (D) EC 50 values for curves in A.

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Transfection

Journal: iScience

Article Title: Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

doi: 10.1016/j.isci.2026.115033

Figure Lengend Snippet: Double mutation decreases basal activity by increasing constitutive internalization (A and B) Cell surface expression of HEK 293 cells transiently transfected with HA-B2AR WT or mutant receptors. (A) Percent cells expressing receptor shown as fold change to the WT and (B) the amount of receptor at the plasma membrane shown as fold change to the WT. n = 5, one sample t test: ∗ p < 0.05, ∗∗ p < 0.01. (C) Whole-cell expression of B2AR-Rluc8 mutant receptors shown as fold change to the WT receptor, n = 6–10. One-sample t test: ∗∗ p < 0.01. (D and E) cAMP production in (D) basal and (E) isoproterenol (1 pM-10 μM, 5-min) treated HEK 293 cells transiently transfected with WT and V34A-S41A B2AR and matched for receptor expression at the plasma membrane . (F) Schematic demonstrating antibody conditions for labeling endocytic/constitutively internalized receptor pools and the total pool of receptor (endocytic + biosynthetic). (G) Confocal microscopy images of HEK 293 cells transiently transfected with HA-B2AR WT and mutant receptors, either fed live with anti-HA antibody (endocytic imaging) or incubated with anti-HA antibody post-fixation and permeabilization (endocytic + biosynthetic imaging). Representative images shown, n = 10 cells. Scale bars, 10 μm; inset = 3 μm. (H) Constitutive internalization measured in HEK 293 cells transiently transfected with HA-B2AR, HA-V34A-S41A B2AR, or HA-B1AR live labeled with anti-HA antibody at 4°C with/without 1-h incubation at 37°C. Cell surface expression measured via flow cytometry. Data shown as percentage change from surface expression in cells kept at 4°C, mean ± SEM. n = 4. One-way ANOVA with Šídák’s multiple comparisons test: ∗ p < 0.05; ∗∗ p < 0.01. See also .

Article Snippet: HEK 293 , ATCC , CRL-1573; RRID:CVCL_0045.

Techniques: Mutagenesis, Activity Assay, Expressing, Transfection, Clinical Proteomics, Membrane, Labeling, Confocal Microscopy, Imaging, Incubation, Flow Cytometry

Journal: STAR Protocols

Article Title: Protocol for validating computationally predicted splice-altering variants using full-length gene reporter assays

doi: 10.1016/j.xpro.2026.104433

Figure Lengend Snippet: Transient splicing reporter assay using the genomic locus sequence reproduces the endogenous TREM2 and APOE isoform patterns (A) Schematic representation of the genomic TREM2 and APOE sequences cloned into the pcDNA3.1(+) vector. (B) Human cell lines expressing endogenous TREM2 (THP-1, HMC3), non-expressing HEK293T cells, and mouse microglial BV2 cells were transfected with the full-length (FL) TREM2 reporter. (C) HMC3 and HEK293T cells expressing endogenous APOE were transfected with the FL APOE reporter. HB, endogenous TREM2 (B) and APOE (C) transcripts from human brain (frontal cortex). cDNA synthesis was primed with either oligo(dT) or a plasmid-specific RT primer (P) positioned downstream of the gene insert. Arrows mark RT-PCR primer positions. Amplified products were resolved by agarose gel electrophoresis.

Article Snippet: HEK293T , ATCC , CRL-3216.

Techniques: Reporter Assay, Sequencing, Clone Assay, Plasmid Preparation, Expressing, Transfection, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis