Journal: iScience

Article Title: A human-specific, concerted repression of microcephaly genes contributes to radiation-induced growth defects in cortical organoids

doi: 10.1016/j.isci.2025.111853

Figure Lengend Snippet:

Article Snippet: The STO MEF cells (ATCC, #CRL-1503, RRID:CVCL_3420) were cultured in DMEM (ATCC 30-2002) supplemented with 10% fetal bovine serum (Gibco, A5670701).

Techniques: Recombinant, Selection, Modification, Transfection, Knock-Out, Lysis, TUNEL Assay, In Situ, Reverse Transcription, Irradiation, Control, Software, Functional Assay, Gene Expression

Journal: Journal of visualized experiments : JoVE

Article Title: Monitoring cancer cell invasion and T-cell cytotoxicity in 3D culture

doi: 10.3791/61392

Figure Lengend Snippet: Concept and Approach described here:

Article Snippet: We adapted this 3D system for dynamic cell-based functional studies as well as endpoint molecular and biochemical assays that include Fluorescence Activated Cell Sorting (FACS), immunofluorescence (IF) or immunohistochemistry (IHC) staining as well as gene expression analysis of the intact 3D co-culture: table ft1 table-wrap mode="anchored" t5 Name of Material/Equipment Company Catalog Number Comments/Description 3D Petri Dishes Microtissues Inc Z764019 & Z764051 referred to as "rubber molds" in the protocols; 81-microwell & 35-microwell molds 8-well Chamber Slides Lab-Tek 154534 Agarose Type I, low EEO Sigma-Aldrich A6013 anti-rabbit-HRP conjugated secondary antibody Agilent K4003 ready to use Collagen Type I, Rat Tail, 100 mg Millipore 08–115 Collagenase Type 4, 1 g Worthington LS004188 DMEM, fetal bovine serum ThermoFisher 11965092, 16000044 referred to as "cell culture medium" in the protocols Harris hematoxylin ThermoFisher SH30–500D HistoGel ThermoFisher HG-4000–012 referred to as "Hydroxyethyl agarose processing gel" in the protocols Hoechst Life Technologies H1399 1/1000 dilution Phalloidin 546 Invitrogen 486624 1/200 dilution rabbit anti-CD8 antibody Cell Signaling 98941 1/25 dilution rat anti-keratin 8 DSHB TROMA-I AB_531826 1/500 dilution RNeasy Mini Kit Qiagen 74104 referred to as "RNA extraction kit" in the protocols RPMI ThermoFisher 11875093 for T-cell culture medium Triton X-100 BioRad 1610407 referred to as "Octoxynol" in the protocols Trizol ThermoFisher 15596026 referred to as "guanidinium thiocyanate with phenol" in the protocols Tween 20 Sigma-Aldrich P1379 referred to as "polysorbate 20" in the protocols TypLE ThermoFisher 12604013 referred to as "cell dissociation enzymes solution" in the protocols Open in a separate window Concept and Approach described here: For functional studies: Embedding spheroids in type I collagen within the agarose casts results in invasion of cancer cells from equidistant spheroids and permits monitoring essential cell line-specific features, such as single cell vs. collective cell migration 18 , 19 .

Techniques: Cell Culture, RNA Extraction

Journal: Frontiers in Ecology and Evolution

Article Title: Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

doi: 10.3389/fevo.2021.795994

Figure Lengend Snippet: FIGURE 6 | Screening of candidate ligands on OR1. Multiple traces of fluorescence variation associated with Ca++i increase in HEK293A cells transfected with CpomOrco + PmachOR1 (left) and CpomOrco + PhospOR1 (right). (A) Application of 500 µM doses of VUAA1, L-nicotine, caffeine, D-(-)-salicin, quercetin, and AITC. (B) Two consecutive applications of AITC (500 µM) to non-transfected HEK293A. A reversible effect on non-transfected cells was observed, excluding its relations with the human TRPA1 (Cattaneo et al., 2017b), which is in accordance with the documented absence of TRPA1-transcripts in HEK cells (BioGPS Cell Line Gene Expression Profiles—HEK293). N = 67 for all experiments; black bar: stimulus (10 s for VUAA1, 20 s for ligands).

Article Snippet: Human Embryonic Kidney (HEK293A) cells were grown to semi-confluence in 35-mm Petri dishes containing HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, United States), 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Invitrogen)] at 37◦C and 5% CO2.

Techniques: Transfection, Gene Expression

Journal: Frontiers in Ecology and Evolution

Article Title: Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

doi: 10.3389/fevo.2021.795994

Figure Lengend Snippet: FIGURE 7 | Functional studies on TRP Painless. (A) Mean ± SE of fluorescence variation associated with PhospPain (N = 33) and PmachPain (N = 28) upon stimulation with 500 µM allyl-isothiocyanate (AITC, two consecutive applications). (B) Application of 500 µM doses of L-nicotine, caffeine, D-(-)-salicin, quercetin on HEK-cells transfected with PhospPain that were found responsive to AITC. Black bars: stimulus (20 s). (C) Mean ± SE of fluorescence variation associated with PhospPain (N = 94) and PmachPain (N = 107) upon stimulation with heat (T ∼42–44◦C). Right: positive control experiment stimulating HEK293A cells transfected with dTRPA1(B) (N = 101). Red line: time point for thermal experiments preceding an endogenous-effect observed in the phase of testing HEK-cells transfected for Papilio Painless. The demonstrated Ca++-dependency of this effect on HEK cells (Ong et al., 2015; Shalygin et al., 2015) may associate the activation of cation channels most probably belonging to the asset of thermal-gated channels selective for Calcium (Xiao et al., 2011).

Article Snippet: Human Embryonic Kidney (HEK293A) cells were grown to semi-confluence in 35-mm Petri dishes containing HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, United States), 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Invitrogen)] at 37◦C and 5% CO2.

Techniques: Functional Assay, Transfection, Positive Control, Activation Assay

Journal: The Journal of Experimental Biology

Article Title: Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti

doi: 10.1242/jeb.250150

Figure Lengend Snippet: Functional heterologous receptor assay involving CHO-K1 cells expressing the A. aegypti CAPA receptor to determine CAPA titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13– 10 −5 mol l −1 doses of Aedes CAPA-1 peptide (EC 50 =1.83 nmol l −1 ). Luminescence was normalized to BSA medium and plotted relative to the maximal response (10 −5 mol l −1 ); data represent the means±s.e.m. ( n =4). (B) Measured concentrations of CAPA peptide levels in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed). Significantly different CAPA peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post-hoc test (* P <0.05); data represent means±s.e.m. ( n =8–11).

Article Snippet: Approximately 48 h post-transfection, cells were dislodged from the culture flasks using 5 mmol l −1 ethylenediaminetetraacetic acid (EDTA; Life Technologies, Burlington, ON, Canada) in DPBS and cells were resuspended in BSA medium (DMEM-F12 medium containing 0.1% bovine serum albumin, 1X antimycotic-antibiotic) at a concentration of 10 6 –10 7 cells ml −1 quantified using a Countess II FL cell counter (Thermo Fisher Scientific, Burlington, ON, Canada).

Techniques: Functional Assay, Expressing, Comparison

Journal: The Journal of Experimental Biology

Article Title: Dynamics of release and activity of select neuropeptides post-bloodmeal in the female mosquito, Aedes aegypti

doi: 10.1242/jeb.250150

Figure Lengend Snippet: Functional heterologous receptor assay of CHO-K1 cells expressing the A. aegypti kinin receptor to determine Aedae kinin titres in the haemolymph of blood-fed female A. aegypti . (A) Normalized dose-response curve after the addition of 10 −13 to 10 −5 mol l −1 doses of CDP (EC 50 =11.47 nmol l −1 ) and leucokinin (LK) (EC 50 =4.34 nmol l −1 ). (B) Raw luminescent response following addition of 10 −6 mol l −1 dose of representative neuropeptides belonging to several insect peptide families; different letters denote bars that are significantly different from control (BSA assay medium) as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test ( P <0.0001). For peptide sequence information, see Table 2 . Individual data points ( n =3) from each experimental replicate are shown. (C) Concentration of kinin peptides in female A. aegypti haemolymph following a bloodmeal (nBF=non-blood-fed; relative to LK standard curve). Significantly different kinin peptide levels in the haemolymph compared with nBF are denoted by asterisks as determined by a one-way ANOVA and Dunnett's multiple comparison post hoc test (** P <0.001); data represent means±s.e.m. ( n =10–15).

Article Snippet: Approximately 48 h post-transfection, cells were dislodged from the culture flasks using 5 mmol l −1 ethylenediaminetetraacetic acid (EDTA; Life Technologies, Burlington, ON, Canada) in DPBS and cells were resuspended in BSA medium (DMEM-F12 medium containing 0.1% bovine serum albumin, 1X antimycotic-antibiotic) at a concentration of 10 6 –10 7 cells ml −1 quantified using a Countess II FL cell counter (Thermo Fisher Scientific, Burlington, ON, Canada).

Techniques: Functional Assay, Expressing, Control, Comparison, Sequencing, Concentration Assay

Journal: Materials

Article Title: Evaluation of Functionalized Porous Titanium Implants for Enhancing Angiogenesis in Vitro

doi: 10.3390/ma9040304

Figure Lengend Snippet: Migration Assay with GM7373 and supernatants from functionalized implants. Comparison of chemotactic behavior of the endothelial cell line (GM7373) using supernatants from implants functionalized with VEGF (vascular endothelial growth factor), HMGB1 (high mobility group box 1) and a combination of HMGB1/VEGF. All of the functionalized implants showed significantly higher chemotaxis than DMEM with 20% FCS or 0.1% FCS. VEGF was significantly more chemotactic than the combination of VEGF + HMGB1. F -test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used ( p ≤ 0.05).

Article Snippet: GM7373 cells showed significantly higher chemotaxis using supernatants from functionalized implants compared to the control DMEM (Dulbecco’s Modified Eagle Medium) (Biochrom AG, Berlin, Germany) with 20% FCS (fetal calf serum) (PAA, Coelbe, Germany) or 0.1% FCS ( ).

Techniques: Migration, Comparison, Chemotaxis Assay

Journal: Cell Reports Medicine

Article Title: Optimized arenaviruses with tumor-tropic mutations promote safe anti-tumor efficacy via sustainable immune modulatory properties

doi: 10.1016/j.xcrm.2025.102411

Figure Lengend Snippet: Fast-evolution LCMV mutations (A) Total numbers of AA positions and the numbers of AA positions that were identified as tumor-tropic in the fast evolution platform, sorted by structural regions (upper graph) or functional regions (lower graph). ∗ shows a significant difference in the mutation frequency measured in the chi-square test. (B) Infection assays (MOI = 0.1, 16 h) of human cancer cells (lung adenocarcinoma: H1975 and A549; primary sarcoma: Gist-T1; melanoma metastasis: MaMel86a and MaMel51) ( n = 4–6; duplicates in 2–3 independent experiments), primary neurons ( n = 4; duplicates in 2 experimental set ups), myotubes and human skeletal muscle myoblasts (HSMM) ( n = 6; duplicates in 3 experimental replicates) with recombinant viruses carrying point mutations at respective positions. (C) Entry assays on lung adenocarcinoma (A549) cells of recombinant viruses that carry the mentioned mutations from the fast evolution platform ( n = 6/mutation; 3 independent experimental replicates). The structural region where the mutation is located is given. Mutated viruses (colored line) compared to the recombinant WE-CL13 un-mutated control. (D) Infection assays (MOI = 0.1, 16 h) of different human cancer cells (lung adenocarcinoma: H1975 and A549; primary sarcoma: Gist-T1; melanoma metastasis MaMel86a and MaMel51) ( n = 4–6; duplicates in 2–3 independent experiments), primary neurons ( n = 4; duplicates in 2 experimental set ups), myotubes and HSMM ( n = 6; duplicates in 3 experimental replicates) with different recombinant viruses carrying a point mutation in the respective position. Data are presented as the mean ± SEM; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by t test (C).

Article Snippet: A549 (ATCC: CCL-185; DMEM with 10% FBS) is a human lung adenocarcinoma cell line.

Techniques: Functional Assay, Mutagenesis, Infection, Recombinant, Control

Journal: Cell Reports Medicine

Article Title: Optimized arenaviruses with tumor-tropic mutations promote safe anti-tumor efficacy via sustainable immune modulatory properties

doi: 10.1016/j.xcrm.2025.102411

Figure Lengend Snippet: Analysis of different combinations of fast evolution mutations (A) Infection assays (MOI = 0.1, 16 h) of different human cancer cells (lung adenocarcinoma: H1975 and A549; primary sarcoma: Gist-T1; melanoma metastasis: MaMel86a and MaMel51), myotubes and human skeletal muscle myoblasts (HSMM) ( n = 4–6; duplicates in 2–3 experimental replicates) with different recombinant viruses carrying a point mutation in the respective position. Notably, the GP mutation 492I always occurred alongside one single NP mutation. Hence, in all further analyses, the mention of mutation 492I mutation inherently includes the NP mutation, even if not explicitly stated. (B) Infection assays (MOI = 0.1, 16 h) of different murine cancer cells (oropharyngeal carcinoma: MOPC; Lewis lung carcinoma: LLC; prostate adenocarcinoma: TrampC2; colon adenocarcinoma: MC38) and human cancer cells (lung adenocarcinoma: H1975 and A549; melanoma metastasis: MaMel86a and MaMel51) ( n = 6; duplicates in 3 experimental replicates) using different recombinant viruses carrying specific point mutations. (C) Spider plots showing the factor of acceleration in propagation of the mutations tested in various human and murine tumor cells. The mean ratio for each mutated virus is given ( n = 4–6; duplicates in 2–3 experimental set ups). (D) Entry assay on human lung adenocarcinoma (A549) cells and A549-αDG knockout cells of recombination virus GP181M-185W-492I and control virus ( n = 6; duplicates in 3 independent experiments). (E) A549 lung adenocarcinoma cells were treated with CD164 blocking antibody or isotype control for 1 h and subsequently infected with the recombination virus GP181M-185W-492I (MOI 10) for 1, 5, and 15 min. The number of viral particles outside the cells per one cell is shown ( n = 6 cells analyzed per sample, ∗∗ p < 0.01). (F and G) Representative pictures for cells treated with isotype control (scale bars: left = 2 μm, right = 200 nm) (F) and CD164 blocking antibody (scale bars: left = 2 μm, right = 200 nm) (G) for 1 min of infection are shown. (H) Infection assays (MOI = 0.1, 16 h) of different human cancer cells (thyroid anaplastic carcinoma: Cal62, C643, 8305C, and 8505C; epidermoid carcinoma: A431; lung adenocarcinoma: KRAS-mutated: A549 and H23; EGFR-mutated: H1975, Alk-rearranged: H2228, WT/other: H1299, H1355, H1792, and H1373; small cell lung cancer: HCC-44; endocervical adenocarcinoma: HeLa; fibroblast liposarcoma: SW-872; colon adenocarcinoma: SW-480; bronchiole lung carcinoma: H358; hepatocellular carcinoma: HepG2) ( n = 6–8; duplicates in 3–4 experimental set ups) comparing WE, recombinant WE-CL13 and a recombinant virus carrying three point mutations as shown. For statistical analysis, WE-CL13-GP181M-185W-492I was compared to both WE and WE-CL13. (I) Hepatocytes ( n = 3; biological replicates; separate flasks), melanocytes ( n = 3; biological replicates; separate flasks), epithelial cells (InEpc, n = 3; biological replicates; separate flasks) and alveolar cells (ALI-cultures, n = 6; duplicates of 3 different patients) were infected with WE or WE-CL13-GP181M-185W-492I. Number of infected cells was determined 24 h with flow cytometry. For statistical analysis, the WE-CL13-GP181M-185W-492I virus was compared to the mock-infected control, but no significant difference was observed. However, a statistical difference between WE and WE-CL13-GP181M-185W-492I was detected. Data are presented as the mean ± SEM; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by t test (D, E, H, and I).

Article Snippet: A549 (ATCC: CCL-185; DMEM with 10% FBS) is a human lung adenocarcinoma cell line.

Techniques: Infection, Recombinant, Mutagenesis, Virus, Knock-Out, Control, Blocking Assay, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Optimized arenaviruses with tumor-tropic mutations promote safe anti-tumor efficacy via sustainable immune modulatory properties

doi: 10.1016/j.xcrm.2025.102411

Figure Lengend Snippet: LCMV-GP mutations affect the entry mechanism of the virus and increase virus replication in tumor cells (A) Lung adenocarcinoma (A549) spheroids were infected with LCMV-WE or WE-CL13-GP181M-185W-492I (scale bars: 100 μm, n = 2, blue: DAPI, red: LCMV NP). (B) Virus titers in mice carrying a melanoma (B16F10-OVA) that were treated intravenously on day 0 with WT LCMV-WE or a recombinant virus (WE-CL13), which carries the indicated combined mutations is shown ( n = 3 mice/group). (C) RT-PCR for LCMV in different organs seven days after intravenous infection with a virus harboring the combined mutations in lung cancer-bearing mice (TC-1) (left: 2 × 10 5 FFU infection dose, n = 5–10; right panel: 2 × 10 7 FFU infection dose, n = 5–6 mice/group). Significance levels were measured comparing the virus titer in tumor to the other organs. Data are presented as the mean ± SEM; ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by two-way ANOVA (B) or t test (C).

Article Snippet: A549 (ATCC: CCL-185; DMEM with 10% FBS) is a human lung adenocarcinoma cell line.

Techniques: Virus, Infection, Recombinant, Reverse Transcription Polymerase Chain Reaction