Journal: Aging Cell

Article Title: Decreased PTGES2 Farnesylation in Granulosa Cells Compromises PGE2 ‐Dependent Cumulus Expansion and Oocyte Maturation During Ovarian Aging

doi: 10.1111/acel.70374

Figure Lengend Snippet: Inhibition of farnesylation leads to cumulus expansion and oocyte maturation impairment in young COCs in vitro. (A) A schematic diagram showing young COCs collection and FTI‐277 treatment for analyzing cumulus expansion and oocyte maturation. (B) Representative COC images before and after cumulus expansion in the CTL and FTI‐277 groups. Scale bars, 100 μm. (C) COC diameter analysis before cumulus expansion in the CTL ( n = 19) and FTI‐277 ( n = 25) groups. (D) Fold change of COC diameter before and after cumulus expansion in the CTL ( n = 19) and FTI‐277 ( n = 25) groups. (E) Representative oocyte images in the CTL and FTI‐277 groups. Scale bars, 100 μm. (F) PBE rates of oocytes in the CTL ( n = 36) and FTI‐277 ( n = 39) groups. (G) Representative images of spindle morphologies and chromosome alignment of oocytes in the CTL and FTI‐277 groups. Scale bars, 25 μm. (H) Meiotic defect rates of oocytes in the CTL ( n = 33) and FTI‐277 ( n = 34) groups. COCs, cumulus‐oocyte complexes. IVM, in vitro maturation. PBE, polar body extrusion. CTL (control) group: Young COCs cultured in MEMα. FTI‐277 group: Young COCs cultured in MEM supplemented with 50 μM FTI‐277. Data are shown as means ± SD from at least three independent repeats. Statistical analysis was performed via an unpaired Student's t‐test. ** p < 0.01, **** p < 0.0001, ns, not significant.

Article Snippet: The concentration of FOH, FTI‐277 (Aladdin, F331609), and PGE2 (MCE, HY‐101952) used in this study was 50, 50, and 1 μM, respectively.

Techniques: Inhibition, In Vitro, Control, Cell Culture

Journal: Aging Cell

Article Title: Decreased PTGES2 Farnesylation in Granulosa Cells Compromises PGE2 ‐Dependent Cumulus Expansion and Oocyte Maturation During Ovarian Aging

doi: 10.1111/acel.70374

Figure Lengend Snippet: FOH supplementation improves cumulus expansion and oocyte maturation in aged COCs via farnesylation in vitro. (A) A schematic diagram showing old COCs collection and treatment with FOH or FOH + FTI‐277 for analyzing cumulus expansion and oocyte maturation. (B) Representative COC images before and after cumulus expansion in the CTL, FOH, and FOH + FTI‐277 groups. Scale bars, 100 μm. (C) COC diameter analysis before cumulus expansion in the CTL ( n = 14), FOH ( n = 14), and FOH + FTI‐277 ( n = 12) groups. (D) Fold change of COC diameter before and after cumulus expansion in the CTL ( n = 14), FOH ( n = 14), and FOH + FTI‐277 ( n = 12) groups. (E) Representative oocyte images in the CTL, FOH, and FOH + FTI‐277 groups. Scale bars, 100 μm. (F) PBE rates of oocytes in the CTL ( n = 36), FOH ( n = 42), and FOH + FTI‐277 ( n = 40) groups. (G) Representative images of spindle morphologies and chromosome alignment of oocytes in the CTL, FOH, and FOH + FTI‐277 groups. Scale bars, 25 μm. (H) Meiotic defect rates of oocytes in the CTL ( n = 30), FOH ( n = 37), and FOH + FTI‐277 ( n = 44) groups. COCs, cumulus‐oocyte complexes. FOH, farnesol. IVM, in vitro maturation. PBE, polar body extrusion. CTL (control) group: Old COCs cultured in MEMα; FOH group: Old COCs cultured in MEM supplemented with 50 μM FOH; FOH + FTI‐277 group: Old COCs cultured in MEMα supplemented with 50 μM FOH + 25 μM FTI‐277. Data are shown as means ± SD from at least three independent repeats. Statistical analysis was performed via one‐way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.

Article Snippet: The concentration of FOH, FTI‐277 (Aladdin, F331609), and PGE2 (MCE, HY‐101952) used in this study was 50, 50, and 1 μM, respectively.

Techniques: In Vitro, Control, Cell Culture

Journal: Aging Cell

Article Title: Decreased PTGES2 Farnesylation in Granulosa Cells Compromises PGE2 ‐Dependent Cumulus Expansion and Oocyte Maturation During Ovarian Aging

doi: 10.1111/acel.70374

Figure Lengend Snippet: PTGES2 farnesylation facilitates localization to the endoplasmic reticulum and PGE2 production. (A) Western blotting showing PTGES2 expression in the membrane fractions in the CTL, FOH, and FOH + FTI‐277 groups. (B) Mean gray value of PTGES2 in the CTL, FOH, and FOH + FTI‐277 groups. (C) Immunofluorescence co‐staining of PTGES2 and calnexin in the CTL, FOH, and FOH + FTI‐277 groups. Scale bars, 10 μm. (D) Relative fluorescent intensity of PTGES2 and calnexin co‐staining in the CTL, FOH, and FOH + FTI‐277 groups. CTL (control) group: KGN cells without treatment; alk‐FOH group: KGN cells treated with 50 μM alk‐FOH; alk‐FOH + FOH group: KGN cells treated with 50 μM alk‐FOH + 50 μM FOH. (E) Western blotting showing PTGES2 expression in the membrane fractions in the CTL, OE, and C16S groups. (F) Mean gray value of PTGES2 in the CTL, OE, and C16S groups. CTL (control) group: 293 T cells transfected with vehicle plasmid; OE group: 293 T cells transfected with the PTGES2 overexpressing plasmid and treated with 50 μM alk‐FOH; C16S group: 293 T cells transfected with the PTGES2 C16S mutant plasmid and treated with 50 μM alk‐FOH. (G) ELISA showing PGE2 levels in the conditional culture media of KGN cells in the CTL, FOH, and FOH + FTI‐277 groups. FOH, farnesol. PTGES2, prostaglandin E2 synthase 2. OE, PTGES2‐overexpression plasmid. C16S, single‐point mutation plasmid of the CaaX motif in PTGES2. Data are shown as means ± SD from at least three independent repeats. Statistical analysis was performed via one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The concentration of FOH, FTI‐277 (Aladdin, F331609), and PGE2 (MCE, HY‐101952) used in this study was 50, 50, and 1 μM, respectively.

Techniques: Western Blot, Expressing, Membrane, Immunofluorescence, Staining, Control, Transfection, Plasmid Preparation, Mutagenesis, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: Lab on a chip

Article Title: Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

doi: 10.1039/d4lc00331d

Figure Lengend Snippet: Fig. 1 Overview of the high-throughput digital assay for quantifying PD-L1+/CD81+ EVs. (a) Diagram illustrating the process of PD-L1+/CD81+ EV quantification using the digital assay. Workflow depicting the generation of a digital signal from a small volume of blood (less than 10 μl of plasma). The workflow consists of (i) acquisition of plasma, (ii) capture of target EVs onto antibody functionalized microbeads, (iii) labeling of captured EVs with labeling antibody, (iv) tagging the labeling antibody with HRP enzyme, and finally (vi) digital encapsulation of beads into substrate filled droplets for fluorescence read-out. (b) Scanning Electron Microscopy (SEM) image demonstrating that typically no more than one EV is bound to each microbead. (c) Fluorescence microscopy image illustrating the digital signal obtained at various mel-B7H1 EV concentrations. (d) Image of the microfluidic chip designed for droplet generation, incubation, and video recording. (e) Illustration of a parallel droplet-generator system, which produces droplets averaging 20.4 μm in diameter with a coefficient of variation (CV) of 9.8%. (f) Conceptual diagram of signal generation in DEVA, highlighting the use of laser diodes to excite microbead and ELISA signals which are modified with maximum length sequences (MLS) to create patterned streaks as droplets with beads and/or positive ELISA signal move across the imaging area. (g) Bead and ELISA signals can be distinguished through correlation-based analysis, with coinciding bead and ELISA signals indicating dual PD-L1+/CD81+ EV. (h) Representative image showcasing the imaging area of DEVA.

Article Snippet: The CD81 antibody underwent biotinylation via the One-Step Antibody Biotinylation Kit (Miltenyi), enabling the attachment of HRPstreptavidin.

Techniques: High Throughput Screening Assay, Clinical Proteomics, Labeling, Encapsulation, Fluorescence, Electron Microscopy, Microscopy, Incubation, Enzyme-linked Immunosorbent Assay, Modification, Imaging

Journal: Lab on a chip

Article Title: Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

doi: 10.1039/d4lc00331d

Figure Lengend Snippet: Fig. 2 Comparative analysis of PD-L1+ EV quantification using DEVA versus conventional ELISA in in vitro samples: (a) methodology for establishing titration curves for both conventional sandwich ELISA and DEVA, utilizing cell culture media from the mel-B7H1 cell line. (b) Analysis of surface tetraspanin markers on mel-B7H1 EVs via ExoView, highlighting the absence of CD9 expression and identifying CD81 as having the superior signal-to-background ratio (n = 3 replicates). Light gray bars indicate background EVs detected by ExoView's interferometric reflectance imaging sensor but not labeled with any fluorescent detection antibody. (c) Titration curves for DEVA and conventional ELISA, demonstrating DEVA's significantly enhanced LOD and LOQ (n = 3 replicates for DEVA's blank sample, n = 2 otherwise). (d) Sample control using mel-624 EVs and isotype controls with non-specific capture antibodies at high EV input concentrations on DEVA maintained low background levels below the LOD (n = 2 replicates). All error bars represent the standard error of the mean.

Article Snippet: The CD81 antibody underwent biotinylation via the One-Step Antibody Biotinylation Kit (Miltenyi), enabling the attachment of HRPstreptavidin.

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Titration, Sandwich ELISA, Cell Culture, Expressing, Imaging, Labeling, Control

Journal: Pigment Cell & Melanoma Research

Article Title: PLX4032, a selective BRAF V600E kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAF WT melanoma cells

doi: 10.1111/j.1755-148X.2010.00685.x

Figure Lengend Snippet: Inactivation/activation of downstream ERK targets in response to PLX4032. (A, B) Western blots showing changes in p90 RSK and CREB activation states, respectively, employing phospho-specific antibodies. (C) Changes in c-FOS and JUNB gene transcripts in response to PLX4032 as evaluated by Real Time RT-PCR. Data are averages of three replicates ± STDV. (D) Heatmap showing upregulation of gene expression by at least 3-fold and above in YUDOSO-BRAF WT melanoma cells in response to treatment with PLX4032 for 8 and 24 h employing the NimbleGen whole genome expression arrays. Marked in red circles are IL8 and LIF. (E) ELISA assay confirming an increase in secreted IL8 levels after 24 h incubation with PLX4032 in BRAF WT (YUDOSO, YUFIC and YUKIM), but not in mutant melanoma cells (YULAC). Error bars represent STDV of six wells.

Article Snippet: The antibodies used were phospho-Mek1/2 pSer217/221, MEK1/2, phospho-Erk2 pThr202/Tyr204 (mAb), ERK1/2 (ERK 1/2, 137F5), phospho-p90RSK Ser380 (9D9 Rabbit mAb), phospho-p90RSK Thr359/Ser363, phospho-p90RSK Thr573, RSK1/RSK2/RSK3 (32D7), FAK (all from Cell Signaling Technology, Beverly, MA, USA), anti-actin (Sigma; mouse mAb), pFAK (S910; BioSourceTM, Invitrogen Corporation, Carlsbad, CA, USA, Cat # 44-596G), BRAF (goat AF3424; R&D Systems, Minneapolis, MN, USA), CREB-1 (24H4B; Santa Cruz Biotechnologies, Inc, Santa Cruz, CA, USA) and phospho-Ser133 CREB-1 rabbit polyclonal antibodies (20), and others described in .

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Mutagenesis

Journal: Nature Cancer

Article Title: CAR-adapted PIK3CD base editing enhances T cell anti-tumor potency

doi: 10.1038/s43018-025-01099-7

Figure Lengend Snippet: a . Tumor growth in the Nalm6 experiment described in Fig. was monitored by bioluminescence imaging (E81K 19BBz: n = 12, control 19BBz: n = 13 mice per group, UT: n = 3 mice per group; T cells from n = 3 biologically independent healthy donors). b . Relative cell growth of E81K-modified and mock 19BBz CAR T cells without (left panel) and with (right panel) IL-7/IL-15 support (technical triplicates; mean ± SD). c . Body weight of NSG mice that were i.v. injected with Nalm6 cells followed by treatment with 3e6 19BBz CAR T cells (TRBC KO) with and without E81K editing during an observation period of 150 days. Data represent n = 19 mice per group treated with CAR T cells from n = 4 biologically independent healthy donors. d . Representative H&E stainings from livers, lungs, kidneys, brains, and bone marrows 66 days after i.v. infusion of E81K-modified or unmodified 19BBz CAR T cells (TRBC KO) in Nalm6-bearing NSG mice (n = 4 mice per group; scale bar size as indicated). e . Flow cytometric analysis of absolute CAR T cell numbers in brain, lung, liver, kidney, spleen and bone marrow 66 days after administration of E81K-modified or unmodified 19BBz CAR T cells (TRBC KO) in Nalm6-bearing mice (n = 4 mice per group; mean + SD; two-sided unpaired Student’s t -test). f ., g . Serum levels of AST ( f .) and ALT ( g .) 66 days after infusion of 19BBz CAR T cells with and without E81K modification in Nalm6-bearing mice (n = 4 mice; mean ± SD; two-sided unpaired Student’s t -test). h . Cytokine serum levels 16 days after CAR T cell infusion in Nalm6-bearing mice as measured by multiplexed ELISA (n = 5 mice, mean ± SD; two-sided unpaired Student’s t -test).

Article Snippet: T cells were activated at a cell-to-bead-ratio of 1:1 with human T activator CD3/CD28 Dynabeads (Thermo Fisher, 11161D) in the presence of 5 ng ml −1 IL-7 and IL-15 (Miltenyi Biotec, 130-095-363 and 130-095-765).

Techniques: Imaging, Control, Modification, Injection, Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Detection of mpox and other orthopoxviruses using a lateral flow device as a point-of-care diagnostic

doi: 10.1128/spectrum.02456-24

Figure Lengend Snippet: Characterization of A27 monoclonal antibodies. ( A ) Titration of two mAbs; Rub.2A12.B7.C6 (filled symbols) and Rub.2.B2.E6 (open symbols) against vaccinia virus (circles) and mpox virus (squares). ELISA plates were coated with cell culture supernatant containing either titrated VACV, MPXV, or control supernatant. ( B ) Capture ELISA showing mAb binding to orthopoxviruses (CMLV, CPXV, MPXV, and VACV). ( C ) Capture ELISA showing mAb binding to other poxviruses (SFV, FWPV, Milkers nodule virus, MYXV, SWPV, TANV, and VACV).

Article Snippet: VACV strain IHD-J (VR-156), rabbit (Shope) fibroma virus (RFV) strain OA (VR112), fowlpox virus (FWPV) strain FH (VR-229), pseudocowpox virus (PCPV) strain TJS (VR-634), myxoma virus (MYXV) strain Lausanne (VR-115), swinepox virus (SWPV) strain Kasza (VR-363), and Tanapox virus (TANV) strain Davis (VR-937) were purchased from the American Type Culture Collection.

Techniques: Bioprocessing, Titration, Virus, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Binding Assay