Journal: Chemmedchem

Article Title: Evaluation of a Radiolabeled Macrocyclic Peptide as Potential PET Imaging Probe for PD−L1

doi: 10.1002/cmdc.202200091

Figure Lengend Snippet: Membrane bound and cell internalized fractions in CHO−K1 and CHO−K1 hPD−L1 cell line for [68Ga]Ga‐WL12 and [68Ga]Ga‐NJMP1 after 90 min. Data are presented as mean values±SD (n=3 in triplicate).

Article Snippet: Recombinant CHO−K1 cells stably expressing human PD−L1 (hPD−L1), henceforth referred to as CHO−K1 hPD−L1, were purchased from BPS Bioscience (San Diego, California) and were maintained in F‐12 K medium with 10 % FBS, 1 % P/S, and 2 mg/mL G418.

Techniques: Negative Control

Journal: The Journal of Infectious Diseases

Article Title: Evaluation of a Single-Dose Nucleoside-Modified Messenger RNA Vaccine Encoding Hendra Virus-Soluble Glycoprotein Against Lethal Nipah virus Challenge in Syrian Hamsters

doi: 10.1093/infdis/jiz553

Figure Lengend Snippet: Survivors among recipients of soluble Hendra virus glycoprotein mRNA LNP vaccine (10 µg [VAX LO] or 30 µg [VAX HI]) in NiV challenge show robust anti–NiV-specific antibody responses. (A), Adjusted absorbance readings from 4-fold dilutions of hamster plasma in a NiV-specific immunoglobulin G (enzyme-linked immunosorbent assay. Horizontal bars represent means for survivor groups and non-survivors (from all groups); vertical lines, standard deviations; dotted line, cutoff value for diagnostic determination of a positive sample. *P < .01; †P < .001 (2-way analysis of variance [ANOVA] with Tukey multiple comparisons test). (B), NiV neutralization by plasma samples from individual hamsters. Reciprocal plasma dilutions in which hamster plasma completely neutralized 200 times the median tissue culture infective dose (TCID50) of NiV genotype M (NiV M) across 4 replicates were plotted according to group-specific indications. Horizontal bars represent geometric means for survivor groups and non-survivors (from all groups); vertical lines, standard deviation; dotted line at 10, limit of detection for virus neutralization assay. Plasma samples with undetectable neutralizing activity were plotted as having a titer of 5. Owing to insufficient samples, only 3 plasma samples were tested from recipients of phosphate-buffered saline vehicle control. *P < .01; †P < .001 (1-way ANOVA with multiple comparisons Kruskal-Wallis test). (C), Immunofluorescence assay depicted with representative micrographs from a VAX HI survivor. Plasma samples collected from representative NiV challenge survivors, diluted 1:40 in phosphate-buffered saline with 5% skim milk, were incubated with either NiV nucleoprotein (NiV N), NiV attachment glycoprotein (NiV G), or mock-transfected CHO-K1 cells. Goat anti-hamster Dylight 488–conjugated antibodies were used to detect primary hamster antibodies, and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bar represent 100 µm.

Article Snippet: Finally, we used representative post-vaccination, pre-challenge, and post-challenge plasma samples from each group to perform immunofluorescence assay against Chinese hamster ovary cells (CHO-K1; CCL-61 from ATCC) that were either mock-transfected or transfected with a plasmid expressing the NiV nucleoprotein (NiV N) or the attachment glycoprotein (NiV G).

Techniques: Virus, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Neutralization, Standard Deviation, Activity Assay, Saline, Control, Immunofluorescence, Incubation, Transfection, Staining

Journal: Developmental cell

Article Title: The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation.

doi: 10.1016/j.devcel.2023.07.009

Figure Lengend Snippet: Figure 1. MBs and MBRs are sites of RNA storage MKLP1 and ARC are necessary for mRNA localization and maintenance in the dark zone. (A–C) (A) RNA-seq analysis of the MB transcriptome. mRNA was sequenced from three stages of the cell cycle: interphase, metaphase, and late cytokinesis (or ‘‘MB stage’’). Tubulin structures were purified, and associated RNAs were isolated and analyzed by RNA-seq. Of 21,607 distinct CHO transcripts identified, 20,821 could be annotated by gene ontology. Of those, 7,986 had R100 reads in all cell cycle stages and were further analyzed as plotted in (B). Raw data can be found in Tables S1, S2, S3, and S4. (B) Transcripts with R100 reads in all three populations were compared and plotted based on their log2 enrichment scores (RPKM/RPKM). Dotted lines at x = 1 and y = 1 indicate minimum values for 2-fold enrichment. The 22 transcripts enriched in the MB relative to both interphase and metaphase are highlighted in red. (C) Enrichment score (relative diameter) and gene ontology groups of the 22 MB-enriched transcripts; colors correspond to gene ontology biological process terms (Figure 1; see also Tables S1, S2, S3, and S4). (D) Single-molecule RNAscope (RNA in situ) hybridization revealed mRNA enrichment in the MB and released MBRs. Poly(A)-positive mRNAs (red) localized to mitotic MBs and post-mitotic MBRs in both CHO and HeLa Kyoto cells, in contrast with the bacterial DapB negative control (E). (E) The bacterial DapE was not found at the MB in HeLa Kyoto or CHO cells MBs or MBRs. (F and G) Two mRNAs. EPEMP1 and CNCL5, identified from interphase enriched RNA-seq data (see Table S4) were not enriched in the MB. Significance was determined and denoted by *. n.s., not significant. (H) Localization of poly(A), MKLP1, KLF4, JUN, and ANXA11 to the MB in HeLa Kyoto cells. Here we observed an enrichment of poly(A) and MKLP1 RNAs but less so the other transcription factors, KLF4 and JUN, and ANXA11 (plot). *Significance was determined by comparing data to DapB (Figure 1E). n.s. denotes not significant. See Figure S2C for CHO cell RNAscope quantification for similar probes.

Article Snippet: Chinese Hamster Ovary (CHO) cells (ATCC CCl-61 ) were maintained at 37 C and 5% CO2 in DMEM/F-12 (Thermo Fisher, Cat# 11330057) with 10% FBS (Thermo Fisher, Cat# 26140079) and 1% Penicillin-Streptomycin (Thermo Fisher, Cat# 15140-122).

Techniques: RNA Sequencing, Isolation, RNAscope, RNA In Situ Hybridization, Negative Control

Journal: Developmental cell

Article Title: The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation.

doi: 10.1016/j.devcel.2023.07.009

Figure Lengend Snippet: Figure 6. The MB is a site of spatiotemporally regulated translation, which also occurs in different cell types (A) Translational onset (a-Puro; arrowheads at MB) occurred precisely as cells formally exited mitosis at the G1 transition, coincident with the mature reformation of the nuclear envelope (detected by lamin A/C) and the decondensation of chromatin (DNA detected by DAPI staining). DAPI, 40,6-diamidino-2-phenylindole. Quantification is noted at each stage in figure; 83% ET (n = 10/12), 100% LT (n = 3/3), and 100% MBR (n = 3/3). (B) Quantification of the number of distinct puromycin rings observed at different points during the late stages of mitosis, namely ET, LT, and MBR. The a-Puro label was primarily found in LT/G1 and continued in the MBR stage after MBR release. Quantification is noted next to each stage. Line scans denoted by the dotted line in each schematic were quantified for datasets and plotted (n = 10). Here, the puromycin ring is seen prominently during LT and MBR stages. Asterisk denotes significance. (C) Retinal pigmented epithelial (RPE) cells (n = 5), HeLa CCL2 cells (n = 5), CHO cells (n = 4), and neural stem/progenitor cells (NSPCs) (n = 4) all had puromycin rings labeled with MKLP1 within the bridge (tubulin). DAPI, 40,6-diamidino-2-phenylindole. Quantification is noted next to each cell type, which is 100% for each cell type. Scale bars are 1 mm unless noted.

Article Snippet: Chinese Hamster Ovary (CHO) cells (ATCC CCl-61 ) were maintained at 37 C and 5% CO2 in DMEM/F-12 (Thermo Fisher, Cat# 11330057) with 10% FBS (Thermo Fisher, Cat# 26140079) and 1% Penicillin-Streptomycin (Thermo Fisher, Cat# 15140-122).

Techniques: Staining, Labeling

Journal: Reproduction (Cambridge, England)

Article Title: EP3 Receptor Isoforms are Differentially Expressed in Subpopulations of Primate Granulosa Cells and Couple to Unique G-Proteins

doi: 10.1530/REP-13-0274

Figure Lengend Snippet: Subcellular localization of each EP3 receptor isoforms. pcDNA3.1 (upper panels) and pSI (bottom panels) were used to determine the location of each monkey EP3 receptor isoform within CHO cells. Transient transfection with empty vector or vector containing a single EP3 receptor isoform with HA tag was performed. Cells were then fixed and stained with anti-HA tag antibody (green). Nuclei were stained with propidium iodide (red). No staining was observed when the primary antibody was omitted (insets). The results shown are representative of two independent experiments. All images are at the same magnification, with scale bar in upper left panel and inset (Scale bars = 5 μm).

Article Snippet: Cell line culture and Transfection Chinese Hamster Ovary (CHO) cells (ATCC, Manassas, VA) were cultured in F-12K (Kaighn's Modification of Ham's F-12) medium (ATCC) supplemented with 10% fetal bovine serum (Bench Mark, Millwood, WV) and 1% antibiotic-antimycotic solution (Gibco, Langley, OK) containing 10,000 μg/ml streptomycin, 10,000 units/ml penicillin and 25 μg/ml amphotericin B.

Techniques: Transfection, Plasmid Preparation, Staining

Journal: Reproduction (Cambridge, England)

Article Title: EP3 Receptor Isoforms are Differentially Expressed in Subpopulations of Primate Granulosa Cells and Couple to Unique G-Proteins

doi: 10.1530/REP-13-0274

Figure Lengend Snippet: EP3 receptor isoforms alter cAMP. CHO cells transiently transfected with EP3-5 (A), EP3-9 (B), or EP3-14 (C) were treated with 0.01% DMSO (control) or sulprostone (Sulp) for 4 h. Cultured media were assessed for cAMP by EIA. Some cultures were pretreated with PTX or GDP-β-S (GDP) before addition of sulprostone. cAMP levels are presented relative to control, which is set equal to 1.0 for each independent experiment. Data are expressed as mean + SEM; n = 4-10 independent experiments. Within each panel, data were assessed by ANOVA with 1 repeated measure, followed by Duncan's post-hoc test; groups with no common superscripts are different, p < 0.05.

Article Snippet: Cell line culture and Transfection Chinese Hamster Ovary (CHO) cells (ATCC, Manassas, VA) were cultured in F-12K (Kaighn's Modification of Ham's F-12) medium (ATCC) supplemented with 10% fetal bovine serum (Bench Mark, Millwood, WV) and 1% antibiotic-antimycotic solution (Gibco, Langley, OK) containing 10,000 μg/ml streptomycin, 10,000 units/ml penicillin and 25 μg/ml amphotericin B.

Techniques: Transfection, Control, Cell Culture

Journal: Reproduction (Cambridge, England)

Article Title: EP3 Receptor Isoforms are Differentially Expressed in Subpopulations of Primate Granulosa Cells and Couple to Unique G-Proteins

doi: 10.1530/REP-13-0274

Figure Lengend Snippet: EP3-9 modulates intracellular calcium. CHO cells transiently transfected with the EP3-9 isoform were pretreated with no inhibitor (solid square), PTX (open square), or GDP-β-S (open circle); all cells were then incubated with the fluorescent calcium dye Fluo-4AM. Basal fluorescent emission was determined (0 second). Sulprostone was added, and fluorescent emission of the same cells was measured every 20 seconds. Finally, cells were exposed to the calcium ionophore A23187 (grey bar) as a positive control. Fluorescence is expressed relative to basal levels, which is set equal to 1.0 for each independent experiment. The mean of 7 independent experiments is shown. Statistics are shown on Table 3.

Article Snippet: Cell line culture and Transfection Chinese Hamster Ovary (CHO) cells (ATCC, Manassas, VA) were cultured in F-12K (Kaighn's Modification of Ham's F-12) medium (ATCC) supplemented with 10% fetal bovine serum (Bench Mark, Millwood, WV) and 1% antibiotic-antimycotic solution (Gibco, Langley, OK) containing 10,000 μg/ml streptomycin, 10,000 units/ml penicillin and 25 μg/ml amphotericin B.

Techniques: Transfection, Incubation, Positive Control, Fluorescence

Journal:

Article Title: Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal Targeting Sequence 1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

doi: 10.1016/j.jmb.2008.04.064

Figure Lengend Snippet: Peroxisomal import of hsEH requires PTS1. (a–f) GFP tagged WT and modified hsEH were expressed in CHO-K1 cells. All images were acquired 72 hour post-transfection. RFP-SKL was co-expressed in all cells as a peroxisomal marker. GFP (a), the GFP-hsEH N-terminal construct (d) and GFP-WT hsEH(SKV) (e) were localized exclusively in the cytosol. GFP-WT hsEH (b), the GFP-hsEH C-terminal construct (c) and GFP-PHYH (f) were co-localized with the peroxisomal marker RFP-SKL. (g) Lysates of CHO-K1 cells expressing GFP tagged WT and modified hsEH were analyzed by SDS-PAGE followed by immunoblotting with anti-GFP and anti-hsEH. 15μg total protein was loaded for each sample. Bar=10 μm

Article Snippet: Cell culture, transient transfection and stable cell line construction Chinese hamster ovary (CHO) K1 cells (ATCC) were maintained in F12K medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.5% antibiotics at 37°C in a humidified incubator under 95% air and 5% CO 2 .

Techniques: Modification, Transfection, Marker, Construct, Expressing, SDS Page, Western Blot

Journal:

Article Title: Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal Targeting Sequence 1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

doi: 10.1016/j.jmb.2008.04.064

Figure Lengend Snippet: Cellular fluorescence of GFP-WT hsEH was similar to that of GFP-R287Q hsEH. GFP-WT hsEH and GFP-R287Q hsEH were expressed in CHO-K1 cells. Fluorescence of 10,000 cells was then measured using a flow cytometer every 24 hours. The geometric mean of each sample was calculated using CellQuest. Fluorescence of GFP-WT hsEH at 24 hour post-transfection was set as 100%. Error bars indicate mean ± s.e.

Article Snippet: Cell culture, transient transfection and stable cell line construction Chinese hamster ovary (CHO) K1 cells (ATCC) were maintained in F12K medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.5% antibiotics at 37°C in a humidified incubator under 95% air and 5% CO 2 .

Techniques: Fluorescence, Flow Cytometry, Transfection

Journal:

Article Title: Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal Targeting Sequence 1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

doi: 10.1016/j.jmb.2008.04.064

Figure Lengend Snippet: Peroxisomal import of GFP-WT hsEH is modulated by protein expression level. (a) Immunoblot analysis of GFP-WT hsEH expression in CHO-K1 cells using anti-GFP antibody. β actin levels were probed as a loading control. (b) Confocal microscopy of GFP-WT hsEH expressed in CHO-K1 cells after transient transfection (left), stable transfection (middle) and retransfection in a stable cell line (right). (c) Confocal microscopy of GFP-R287Q hsEH expression in stably-transfected CHO-K1 cells. (d) and (e), CHO-K1 cells transiently-transfected with GFP-WT hsEH (d) and GFP-R287Q hsEH (e) were sorted into low (M1) and high (M2)-fluorescence pools by fluorescence-activated cell sorting, plated, and analyzed by confocal microscopy. Error bars indicate mean ± s.e. Bar=10 μm

Article Snippet: Cell culture, transient transfection and stable cell line construction Chinese hamster ovary (CHO) K1 cells (ATCC) were maintained in F12K medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.5% antibiotics at 37°C in a humidified incubator under 95% air and 5% CO 2 .

Techniques: Expressing, Western Blot, Control, Confocal Microscopy, Transfection, Stable Transfection, Fluorescence, FACS

Journal:

Article Title: Retroviral Vectors Produced in the Cytoplasmic Vaccinia Virus System Transduce Intron-Containing Genes

doi: 10.1128/JVI.76.3.1236-1243.2002

Figure Lengend Snippet: CFAs for different cell lines

Article Snippet: The African green monkey kidney cell line CV-1 (ATCC CCL-70), the NIH 3T3 cell line (ATCC CRL-1658), the Chinese hamster ovary cell line CHO (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Virus

Journal:

Article Title: Retroviral Vectors Produced in the Cytoplasmic Vaccinia Virus System Transduce Intron-Containing Genes

doi: 10.1128/JVI.76.3.1236-1243.2002

Figure Lengend Snippet: FIX expression (ELISA data) of transduced NIH 3T3 and CHO cell clones

Article Snippet: The African green monkey kidney cell line CV-1 (ATCC CCL-70), the NIH 3T3 cell line (ATCC CRL-1658), the Chinese hamster ovary cell line CHO (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Retroviral Vectors Produced in the Cytoplasmic Vaccinia Virus System Transduce Intron-Containing Genes

doi: 10.1128/JVI.76.3.1236-1243.2002

Figure Lengend Snippet: Western blot analysis of the FIX expression in the transduced 3T3 cell clones (A) and in the transduced CHO cell clones (B). (A and B) Lane 1, marker proteins; lane 2, FIX standard purified from plasma; lane 3, cell culture supernatants from nontransduced parental cell lines; lanes 4 to 6, cell culture supernatants from transduced cell clones. The arrowheads on the right point to the FIX band.

Article Snippet: The African green monkey kidney cell line CV-1 (ATCC CCL-70), the NIH 3T3 cell line (ATCC CRL-1658), the Chinese hamster ovary cell line CHO (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Clone Assay, Marker, Purification, Clinical Proteomics, Cell Culture

Journal:

Article Title: Retroviral Vectors Produced in the Cytoplasmic Vaccinia Virus System Transduce Intron-Containing Genes

doi: 10.1128/JVI.76.3.1236-1243.2002

Figure Lengend Snippet: Northern blot analysis of the transcripts derived from the transduced cell clones and the controls. (A) A schematic representation of the binding regions of probes 1 and 2. Probe 1 is complementary to the region upstream of the AATAAA signal (underlined) of the FIX transcripts. Probe 2 is complementary to the region downstream of the processing signal. (B and C) The Northern blots obtained with probe 1 (B) and probe 2 (C). The transcripts (t1-t2, left side) and their respective sizes in kilobases (right side) are shown. Lanes 1 to 3, analysis of RNAs from transduced cell clones 3T3FIX#1.1, 3T3FIX#2.4, and 3T3FIX#2.5; lane 4, negative control RNA from NIH 3T3 cells; lane 5, positive control RNA from a classically transfected and amplified CHO cell clone expressing human FIX; lane 6, control RNA from CV-1 cells infected with the vaccinia virus vR-Xi9pASN. (D) A control blot obtained with a GAPDH probe confirming equal RNA loading of the gel.

Article Snippet: The African green monkey kidney cell line CV-1 (ATCC CCL-70), the NIH 3T3 cell line (ATCC CRL-1658), the Chinese hamster ovary cell line CHO (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Northern Blot, Derivative Assay, Clone Assay, Binding Assay, Negative Control, Positive Control, Transfection, Amplification, Expressing, Control, Infection, Virus

Journal: Journal of medicinal food

Article Title: Spergularia marina induces glucagon-like peptide-1 secretion in NCI-H716 cells through bile acid receptor activation.

doi: 10.1089/jmf.2013.3091

Figure Lengend Snippet: FIG. 3. Inhibition of Takeda G- protein-coupled receptor 5 (TGR5) mRNA expression by small interfer- ence RNA (siRNA), and effect on AEE-SM-induced GLP-1 release and intracellular Ca2 + and cAMP in- creases from NCI-H716 cells. Trans- fections using nontargeting siRNA were used as controls. (A) TGR5 mRNA assessed by real-time quanti- tative PCR 48 h posttransfection. (B) Effect of TGR5 knockdown on AEE- SM-induced GLP-1 release. (C) Effect of TGR5 knockdown on AEE- SM-induced increases in intracellular Ca2 + levels. (D) Effect of TGR5 knockdown on AEE-SM-induced in- creases in intracellular cAMP levels. The data are mean – SEM (n = 12); values not sharing a common letter are significantly different (P < .05 by ANOVA and Tukey’s test). *P < .05, **P < .01, ***P < .001 versus control siRNA.

Article Snippet: TGR5 transfection in CHO-K1 cells Human TGR5 cDNA cloned to the pCMV6-XL5 expression vector was purchased from Origene Technologies, Inc. (Rockville, MD, USA) and amplified in chemically component cells by the heat shock method.

Techniques: Inhibition, Expressing, Knockdown, Control

Journal: Journal of medicinal food

Article Title: Spergularia marina induces glucagon-like peptide-1 secretion in NCI-H716 cells through bile acid receptor activation.

doi: 10.1089/jmf.2013.3091

Figure Lengend Snippet: FIG. 4. Effect of AEE-SM on intracellular cAMP levels in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5 expression plasmid or mock (without TGR5 plasmid). Data are mean – SEM (n = 12). Values not sharing a common letter are sig- nificantly different (P < .05 by ANOVA and Tukey’s test).

Article Snippet: TGR5 transfection in CHO-K1 cells Human TGR5 cDNA cloned to the pCMV6-XL5 expression vector was purchased from Origene Technologies, Inc. (Rockville, MD, USA) and amplified in chemically component cells by the heat shock method.

Techniques: Transfection, Expressing, Plasmid Preparation