Journal: Molecules

Article Title: Oleoylethanolamide and Palmitoylethanolamide Enhance IFNβ-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

doi: 10.3390/molecules29071592

Figure Lengend Snippet: Involvement of PD-L1 and p38 MAPK in the effects of OEA, PEA, and IFNβ treatment on SH-SY5Y cells. Cells were treated with IFNβ for different lengths of times ( A ) and at different concentrations ( B ) to assess the effect of time and dose exposure to IFNβ on the levels of the programmed death-ligand 1 (PD-L1). * p < 0.05 versus vehicle. The values are expressed as percentage of IFNβ and are represented as the mean ± SEM of four independent experiments ( A , B ). Cells were treated as mentioned in A and cell lysates were analyzed for PD-L1. The values are expressed as percentage of IFNβ. ** p < 0.01 and *** p < 0.001 versus vehicle; # p < 0.05 and ### p < 0.001 versus IFNβ. The results are represented as the mean ± SEM of four independent experiments ( C ). Cells were treated as reported in A. The total cell extract (cell lysate) and biotinylated proteins (surface protein) were analyzed for PD-L1 by Western blot. The levels of PD-L1 in whole cell lysate and cell surface were normalized to pan cadherin (pan cadh), a plasma membrane marker. * p < 0.05 and ** p < 0.01 in surface protein versus IFNβ; ## p < 0.01 and ### p < 0.001 in the cell lysate versus IFNβ. Values are represented as the mean ± SEM of three independent experiments ( D ). Cells were treated as in A, and cell lysates were analyzed for the phosphorylated and total p38 protein. * p < 0.05 versus vehicle; # p < 0.05 versus IFNβ ( E ).

Article Snippet: Membranes were blocked, washed, and incubated overnight at 4 °C with one of the following primary antibodies: PD-L1 (cat. no. 13684, Cell Signaling Technology, Danvers, MA, USA) (1:1000); IKBα (cat. no. 4814, Cell Signaling Technology) (1:1000); cleaved caspase 3 (Asp175) (cat. no. 9664, Cell Signaling Technology) (1:1000); caspase 3 (cat no. 9665, Cell Signaling Technology) (1:1000); cleaved-poly (ADP-ribose) polymerase (PARP) (Asp214) (cat. no. 5625, Cell Signaling Technology) (1:1000); PARP (cat. no. 9542, Cell Signaling Technology) (1:1000); phospho-Tyr701-STAT1 (1:1000) (cat no. ST1P-11A5, Thermo Fisher Scientific, Rockford, IL, USA); anti-STAT1 (1:500) (cat no. sc-592, Santa Cruz Biotechnology, Paso Robles, CA, USA); PKR (1:1000) (cat no. 3072, Cell Signaling Technology); survivin (cat. no. 2808, Cell Signaling Technology); Mcl-1 (1:1000) (sc-819, Santa Cruz Biotechnology); pan cadherin (cat. no. 4073, Cell Signaling Technology) (1:2000); actin (1:3000) (cat no. A2066, Sigma-Aldrich); GAPDH (1:5000) (cat no. 247-002, Synaptic Systems, Gottingen, Germany).

Techniques: Western Blot, Clinical Proteomics, Membrane, Marker

Journal: Cellular and Molecular Life Sciences

Article Title: Downregulation of p53 drives autophagy during human trophoblast differentiation

doi: 10.1007/s00018-017-2695-6

Figure Lengend Snippet: Transcriptome analyses of BeWo cell differentiation. Immunofluorescence staining of E-cadherin in BeWo cells incubated either with vehicle control ( a , DMSO, 0.1%) or forskolin ( b , 20 µM). Stimulation with forskolin induced formation of multinucleated syncytia (outlined by dotted lines) after 48 h culture. Scale bar in ( b ) represents 100 µm. c qPCR analysis showed significantly upregulated mRNA expression of β subunit of human chorionic gonadotropin (CGB) in forskolin-treated BeWo cells after 48 h. Data are presented as mean ± SEM * p ≤ 0.05. d Bioinformatic pipeline applied to twofold regulated genes is depicted. e Top five positive (positive z -score) and negative (negative z -score) upstream regulators derived from ingenuity pathway analysis (IPA), sorted by adjusted p values given next to bars. f Heatmap of genes comprising the gene ontology (GO) term “autophagy” that is significantly enriched (adjusted p value = 7.9E−3) in a DAVID functional annotation analysis focused on GO biological processes. Data are from three independent experiments, using different cell passages

Article Snippet: Monoclonal anti-E-Cadherin antibody (Acris Antibodies GmbH, OriGene EU, Herford, Germany) was diluted 1:15 in antibody diluent (DAKO, Carpintera, CA, USA) and incubated on cells for 30 min. PBS washing steps were followed by incubation with secondary antibody, Alexa Fluor 555 goat anti-mouse (1:200; Invitrogen, Lifetechnologies, Carlsbad, CA, USA) for 30 min.

Techniques: Cell Differentiation, Immunofluorescence, Staining, Incubation, Control, Expressing, Derivative Assay, Functional Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Downregulation of p53 drives autophagy during human trophoblast differentiation

doi: 10.1007/s00018-017-2695-6

Figure Lengend Snippet: Effect of forskolin on p53 and autophagy in non-fusing JAR trophoblasts. Immunofluorescence staining of E-cadherin in JAR cells incubated either with vehicle control ( a , DMSO, 0.1%) or forskolin ( b , 20 µM) did not show formation of multinucleated syncytia after 48 h culture. qPCR analysis of p53 ( c ) and LC3B ( d ) mRNA as well as immunoblotting ( e ) with subsequent band densitometry for p53 ( f ), LC3B-I, and LC3B-II ( g – i ) protein levels did not show significant differences between forskolin (20 µM) treated JAR cells and vehicle control (DMSO, 0.1%) after 48 h. Scale bar in b represents 100 µm. Data in c , d and f – i are presented as mean ± SEM from three independent experiments, using different cell passages

Article Snippet: Monoclonal anti-E-Cadherin antibody (Acris Antibodies GmbH, OriGene EU, Herford, Germany) was diluted 1:15 in antibody diluent (DAKO, Carpintera, CA, USA) and incubated on cells for 30 min. PBS washing steps were followed by incubation with secondary antibody, Alexa Fluor 555 goat anti-mouse (1:200; Invitrogen, Lifetechnologies, Carlsbad, CA, USA) for 30 min.

Techniques: Immunofluorescence, Staining, Incubation, Control, Western Blot

Journal: Dalton transactions (Cambridge, England : 2003)

Article Title: Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

doi: 10.1039/c4dt02884h

Figure Lengend Snippet: Fig. 1 Schematic presentations of EMT (A, cells in epithelial (I), inter- mediate (II) and mesenchymal (III) state) and the distinction between E-cadherin and N-cadherin coexpression (B and D) and E/N-cadherin clus- tering (C and E) by TG FRET using Tb-donor antibody (green) excitation (magenta arrow) and dye-acceptor antibody (orange) emission (orange arrow). (D) When E- and N-cadherins are not in a close distance (>20 nm) UV-excitation of the immunostained cells leads to long-life- time Tb PL (green), a strong nano- to microsecond autofluorescence (brown) and a weak short-lived (nanoseconds) acceptor PL. (E) Cluster- ing of E- and N-cadherins brings the Tb-donor and dye-acceptor in close proximity, which causes Tb-to-dye FRET. As the FRET efficiency ηFRET depends on the PL decay times of the pure Tb-donor (τD) and the one of the Tb-dye donor–acceptor pair (τDA) by ηFRET = 1 −(τDA/τD),23

Article Snippet: Primary antibodies: Anti-E cadherin goat polyclonal (ref. AF648, R&D Systems, Minneapolis, USA), anti-N Cadherin [8C11] antibody (ref. ab19348, Abcam, Cambridge, UK), pan Cadherin Antibody (3F4) (ref. H00000999-M01, Novus Biologicals).

Techniques:

Journal: Dalton transactions (Cambridge, England : 2003)

Article Title: Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

doi: 10.1039/c4dt02884h

Figure Lengend Snippet: Fig. 2 Confocal images of A549 cells, which coexpress E-cadherin (A, FITC dye) and N-cadherin (B, AlexaFluor 594 dye). The overlay image (C) shows colocalization (indicated by the white pixels) with Mander’s overlap coefficients of M1 = 0.95 (fraction of AF594 pixels overlapping FITC pixels) and M2 = 0.48 (fraction of FITC pixels overlapping AF594 pixels) but does not contain any information about the E/N-cadherin distances (E/N-cadherin clusters). Scale bars correspond to 20 µm.

Article Snippet: Primary antibodies: Anti-E cadherin goat polyclonal (ref. AF648, R&D Systems, Minneapolis, USA), anti-N Cadherin [8C11] antibody (ref. ab19348, Abcam, Cambridge, UK), pan Cadherin Antibody (3F4) (ref. H00000999-M01, Novus Biologicals).

Techniques:

Journal: Dalton transactions (Cambridge, England : 2003)

Article Title: Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

doi: 10.1039/c4dt02884h

Figure Lengend Snippet: Fig. 5 Investigation of E/E-cadherin clustering in MCF-7 cells by Tb-to- dye FRET. For better clarity, positive signal images have green frames whereas negative signal images have red frames. (A) Time-gated (TG, 0.01–2.51 ms after excitation pulse) and steady-state (SS, excited at 520 ± 14 nm) images in the Tb detection channel (Tb, 542 ± 10 nm) and the AF568 detection channel (AF568, 607 ± 5 nm) using polyclonal primary Tb and AF568 antibodies resulted in positive TG Tb, SS AF568 and TG AF568 (FRET) PL signals, which could be caused by antibody-protein recognition on the same or different E-cadherins (top or bottom scheme, respectively). (B) Similar results as in A were found when using unlabelled polyclonal antibodies against E-cadherin and Tb and AF568 (left) or FITC (right) secondary antibodies, which offers again two possi- ble binding scenarios (top and bottom scheme, respectively). (C) Using monoclonal Tb and AF568 primary antibodies led to efficient costaining but no FRET signal (TG AF568) due to too large distances (>ca. 12 nm) between Tb and AF568 antibodies (scheme). (D) For a verification of efficient TG Tb-to-dye FRET Tb primary antibodies and AF568 secondary antibodies (against the Tb primaries) were used for immunostaining. Efficient costaining as well as FRET due to antibody–antibody recog- nition (scheme) are clearly visible in the TG Tb, SS AF568, and TG AF568 PL images, respectively. Control experiments using only primary Tb anti- bodies showed that the TG AF568 signal is not caused by spectral cross- talk from the Tb PL (Fig. S7 in the ESI†).

Article Snippet: Primary antibodies: Anti-E cadherin goat polyclonal (ref. AF648, R&D Systems, Minneapolis, USA), anti-N Cadherin [8C11] antibody (ref. ab19348, Abcam, Cambridge, UK), pan Cadherin Antibody (3F4) (ref. H00000999-M01, Novus Biologicals).

Techniques: Binding Assay, Immunostaining, Control

Journal: Dalton transactions (Cambridge, England : 2003)

Article Title: Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

doi: 10.1039/c4dt02884h

Figure Lengend Snippet: Fig. 6 TG (0.01–2.51 ms after excitation pulse) and SS (excited at 520 ± 14 nm) images in the Tb detection channel (Tb, 542 ± 10 nm) and the AF568 detection channel (AF568, 607 ± 5 nm) of different FRET-pair antibody combinations to detect a possible E/N-cadherin clustering. Costaining of Tb-antibodies (against E-cadherin) and AF568-antibodies (against N-cadherin) is clearly visible in the TG Tb and SS AF568 PL images. However, no FRET signal (TG AF568 signal) was detected, which shows that Tb-donor and dye-acceptor are not in close (

Article Snippet: Primary antibodies: Anti-E cadherin goat polyclonal (ref. AF648, R&D Systems, Minneapolis, USA), anti-N Cadherin [8C11] antibody (ref. ab19348, Abcam, Cambridge, UK), pan Cadherin Antibody (3F4) (ref. H00000999-M01, Novus Biologicals).

Techniques: Control

Journal: Dalton transactions (Cambridge, England : 2003)

Article Title: Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

doi: 10.1039/c4dt02884h

Figure Lengend Snippet: Fig. 7 (A) SS images of different combinations of AF488 donor antibodies with AF568 acceptor antibodies on M4-T cells were recorded in the AF568 PL emission channel upon excitation of AF488 (438 ± 12 nm, no significant direct excitation of AF568). Although the two possible AF488– AF568 FRET combinations (II and V) led to positive PL signals, the dye–dye FRET pair could not provide clear evidence for FRET because direct exci- tation of AF568 (I and IV), spectral crosstalk of AF488 PL in the AF568 detection channel (VI), and autofluorescence of immunostained cells without any dyes (III) also led to positive PL signals. Control experiments using specific excitation of only AF488 antibodies and AF568 antibodies confirmed that both dye-labelled antibodies were bound to the cell membranes (Fig. S12 in the ESI†). (B) Strong photobleaching (three serial image acquisitions from top to bottom) of both AF488 (excitation via 438 ± 12 nm and detection via 522 ± 6 nm transmission filters, 100 ms acquisition per image) and AF568 (excitation via 542 ± 10 nm and detection via 607 ± 5 nm transmission filters, 350 ms acquisition per image) with both dye-labelled antibodies against N-cadherin or with only one type of each dye-labelled antibody (Fig. S11 in the ESI†).

Article Snippet: Primary antibodies: Anti-E cadherin goat polyclonal (ref. AF648, R&D Systems, Minneapolis, USA), anti-N Cadherin [8C11] antibody (ref. ab19348, Abcam, Cambridge, UK), pan Cadherin Antibody (3F4) (ref. H00000999-M01, Novus Biologicals).

Techniques: Control, Transmission Assay

Journal: Cell reports

Article Title: Single-cell and spatial analyses reveal a tradeoff between murine mammary proliferation and lineage programs associated with endocrine cues

doi: 10.1016/j.celrep.2023.113293

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pan-Cadherin (CycIF) , Novus , Cat# NBP2–73237AF488; Clone OTI4F1.

Techniques: Software, Microscopy