Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 inhibition suppresses nerve growth factor signaling and nociception in pain models

doi: 10.1172/JCI183873

Figure Lengend Snippet: ( A ) Immunofluorescence detection of TrkA and NRP1 in mouse DRG. TrkA was largely intracellular (arrows), whereas NRP1 was localized to the plasma membrane (arrowheads). Scale bar: 50 μm. ( B ) RNAScope detection of Ntrk1 (TrkA) and Nrp1 (NRP1) mRNA in mouse DRG neurons identified by NeuN immunofluorescence. Arrowheads indicate neurons coexpressing Ntrk1 and Nrp1 . Scale bar: 50 μm. ( C ) Immunofluorescence detection of CGRP and RNAScope detection of Nrp1 mRNA in mouse DRG. Arrows indicate neurons coexpressing CGRP and Nrp1 . Scale bar: 20 μm. ( D ) Immunofluorescence detection of NRP1 and GS in mouse DRG. Arrows indicate satellite glial cells expressing NRP1. Scale bar: 50 μm. ( E ) RNAScope detection of NTRK1 and NRP1 mRNA in human DRG. Arrowheads indicate neurons coexpressing NTRK1 and NRP1 . Scale bar: 500 μm. ( F ) Immunofluorescence of P2X3 and CGRP and RNAScope detection of NRP1 mRNA in human DRG. Arrowheads indicate neurons coexpressing CGRP and NRP1 . Arrows indicate neurons expressing P2X3 but not NRP1 . Scale bar: 50 μm. *Denotes fluorescence in human neurons due to lipofuscin. Nuclei shown in blue. ( G ) Percentage of mouse DRG neurons expressing Ntrk1 or CGRP that coexpress Nrp1 . ( H ) Percentage of human DRG neurons expressing NTRK1 , CGRP, or P2X3 that coexpress NRP1 . A – F show representative images from n = 4–5 mice and n = 3 humans. G and H show hybridized positive neurons (%) from n = 3–4 mice and n = 3 humans.

Article Snippet: Human DRG suspension cells were from AnaBios Corp. and were studied within 96 hours.

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, RNAscope, Expressing, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 inhibition suppresses nerve growth factor signaling and nociception in pain models

doi: 10.1172/JCI183873

Figure Lengend Snippet: ( A – C ) RNAScope localization of Gipc1 mRNA in mouse DRG ( A ) and of NTRK1 and GIPC1 mRNA in human DRG ( B ). Arrows indicate mRNA expression within the same cell. Representative images, n = 5 mice and n = 3 humans. Scale bars: 500 μm. ( C ) Percentage of human DRG neurons expressing NTRK1 or GIPC1 that coexpress GIPC1 or NRP1 . Hybridized positive neurons (%) from n = 3 humans. ( D ) Effect of GIPC1 siRNA on BRET measurements of TrkA levels at the plasma membrane of HEK293T cells under basal conditions and after coexpression with NRP1. ( E and F ) Effect of 30 minutes preincubation of GIPC1 antagonist (300 μM CR1023 or inactive control, Ctrl) or myosin VI inhibitor (50 μM 2,4,6-triiodophenol, TIP) on NGF-induced TrkA-Rluc8 trafficking from a marker of the plasma membrane (RGFP-CAAX) in CAD cells. ( G ) Effect of GIPC1 siRNA on NGF-induced downstream ERK transcription in CAD cells. Data from 5–6 independent experiments with triplicate wells. ( H and I ) Sample traces of action potential firing in mouse DRG neurons evoked by injecting a 1-second ramp pulse from 0 to 250 pA ( G ), with the number of evoked action potentials ( H ). n = 7–10 cells. ( J – N ) NGF-induced pain. Effects of GIPC1 or Ctrl siRNA (i.t.) on NGF-induced (50 ng/10 μl, i.pl.) mechanical allodynia ( K and L ) and thermal hyperalgesia ( M and N ) in the ipsilateral paw. ( L and N ) AUC of time courses. ( O – S ) CFA-induced pain. Effects of GIPC1 or Ctrl siRNA (i.t.) on CFA-induced (i.pl.) mechanical allodynia ( P and Q ) and thermal hyperalgesia ( R and S ). ( Q and S ) AUC of time courses. n = 6–8 mice per group. B, basal. Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D and F ) One-way ANOVA, Sídák’s multiple comparisons. ( I ) Tukey’s multiple comparison. ( K , M , P , and R ) Two-way ANOVA, Sídák’s multiple comparisons. ( L , N , Q , and S ) Unpaired t test.

Article Snippet: Human DRG suspension cells were from AnaBios Corp. and were studied within 96 hours.

Techniques: RNAscope, Expressing, Clinical Proteomics, Membrane, Control, Marker, Comparison

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Images of the spheroids from static culture for morphological analysis and the determination of properties such as spheroid size and circularity ( n = 12; data are means ± STDV). (A) Morphological differences can be seen during the aggregation of the different β‐cell lines within 7 days. The images of INS‐1, MIN6, and MSCs were reduced by 60%, whereas the 1.1B4 images were reduced by 73% ( * ). In both cases the scale bar represents 100 μm. (B) The growth kinetics of the spheroids reveal the differences between cell lines. The INS‐1 cells (squares) showed fast aggregation and a continuous increase in spheroid size, whereas the MIN6 cells (circles) needed 3 days to form stable spheroids before slow growth was observed. The 1.1B4 cells (triangles) aggregated slowly over 5–7 days, represented by a peak on day 2 followed by a decline in size until a stable spheroid was formed and a volume increase was observed. The hMSC‐TERTs (crosses) and ad‐MSCs (stars) showed no growth and the spheroid size declined over time. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The viability of spheroids from static cultures determined by staining with calcein AM and ethidium after 7 days. (A) INS‐1 spheroids featured a dead core and a viable mantle, whereas MIN6 spheroids featured a heterogenous distribution of dead cells. The loose structure of the 1.1B4 spheroids promoted sufficient mass transfer resulting in a high viability. (B) Insulin secretion profiles of INS‐1 cells cultured as monolayers and spheroids cultured under static (96‐well plate) and dynamic (shaking flask) conditions ( n = 3; data are means ± STDV; significance intervals are * p < 0.05, ** p < 0.01, and *** p < 0.001). 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Cell Culture, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The insulin profiles of β‐cell spheroids in static culture was measured by GSIS ( n = 3; error = STDV)

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques:

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Aggregation of INS‐1 (upper row), MIN6 (middle row) and 1.1B4 (lower row) cells with hMSC‐TERTs at different ratios after 24 h. MSCs were stained blue (VPD) and β‐cells were stained with the green dye CFSE. Starting with monospheroids in the first (MSCs, blue) and second (β‐cells, green) columns, the cell ratios increase from left to right. Due to different scaling of the images, the MSC spheroids seem to have a different size in each setup, but the seeding density was always 1000 cells per well. In all cases the scale bar represents 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; CFSE, 5‐(and 6)‐carboxyfluorescein diacetate, succinimidyl ester; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Bright‐field and viability images (at day 7) of monospheroids (0–1 = MSC only; 1–0 = β‐cell only) and heterospheroids INS‐1 (upper row), MIN6 (middle row), and 1.1B4 (lower row) cocultured with hMSC‐TERTs at different cell ratios. The stated viabilities of the spheroids were assessed by measuring the red (core) and green (whole spheroid) diameter and the resulting volume to describe the real “3D viability,” but the displayed images only represent two dimensions of the spheroids, which could provide a deceptive impression. Scale bar = 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Molecules

Article Title: Structural Investigation of the Interaction Mechanism between Chlorogenic Acid and AMPA Receptor via In Silico Approaches

doi: 10.3390/molecules27113394

Figure Lengend Snippet: Effect of CGA on GluA1 in dorsal root ganglion (DRG) cells. The protein expression levels of GluA1 were assayed by Western blot analysis. n = 3, β-actin expression was used as loading control. * p < 0.05, ** p < 0.01, as compared with the control group (0 μM).

Article Snippet: The mouse dorsal root ganglion (DRG) cells purchased from Procell (CP-M166, Wuhan, China) were cultured in a medium consisting of Neurobasal-A medium (GIBCO, 21103049) with 2% B-27 (GIBCO, 17504044) at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Expressing, Western Blot, Control