Journal: BMC pulmonary medicine

Article Title: IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells.

doi: 10.1186/s12890-020-01319-z

Figure Lengend Snippet: Fig. 4 GRP78 expression characteristics induced by IL-32 or 4-PBA. a GRP78 mRNA expression levels. *P < 0.05 compared with the control group, **P < 0.05 compared with the rhIL-32 group. b and c GRP78 protein expression levels, measured by western blotting. *P < 0.05 compared with the control group, **P < 0.05 compared with the rhIL-32 group

Article Snippet: A sample containing 20 μg of protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Solarbio), transferred to a polyvinylidene fluoride membrane, and incubated with rabbit anti-mouse N-cadherin, GRP78, and α-SMA primary antibodies (Proteintech, Wuhan, China), or βactin antibody (Bioss, Beijing, China) as a loading control, at room temperature for 2 h. Subsequently, the membrane was washed, incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature, and exposed using the ECL kit.

Techniques: Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification and characterization of Nanobodies targeting the EphA4 receptor

doi: 10.1074/jbc.M116.774141

Figure Lengend Snippet: Nb 39 and Nb 53 do not completely inhibit ephrin-A5 binding to EphA7. With Alphascreen technology we determined whether Nb 39 and Nb 53 could inhibit ephrin-A5 binding to EphA7. These Nbs did not completely inhibit the interaction between ephrin-A5 and EphA7 at the highest concentrations tested. This assay was done in triplicate, and data are represented as the mean ± S.D.

Article Snippet: To test the inhibition of EphA7 and ephrin-A5 interaction, His-tagged human ephrin-A5 (Sino Biological, Beijing, China) was biotinylated with a five-times molar excess of EZ link NHS biotin (Thermo Fisher Scientific) as is described for the Nbs.

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Nature Communications

Article Title: CYCLIN-B1/2 and -D1 act in opposition to coordinate cortical progenitor self-renewal and lineage commitment

doi: 10.1038/s41467-020-16597-8

Figure Lengend Snippet: a t SNE-NN maps coloured by Hmmr or Slc1a5 expression levels in cortical cells. b Schematic showing dissection of E11.5, E13.5 and E15.5 Sox2 -GFP cortices, antibody labelling of cell-surface markers enriched in uncommitted progenitors and progenitors committed to upper-layer (HMMR and GPC6, enriched in upper-layer-trajectory gene set 1; EDNRB enriched in upper-layer-trajectory gene set 2), or deep-layer (SLC1A5, enriched in deep-layer-trajectory gene set 1 or EFNA5 enriched in deep-layer-trajectory gene set 2) differentiation trajectories, FACS-based isolation and in vitro differentiation or RNA-seq analysis. c , d Immunohistochemistry ( c ) and quantification ( d ) of BCL11B and SATB2 expression in TUJ1 + neurons derived from HMMR + and HMMR − progenitors after two days of differentiation. E11.5 ( n = 5 biological independent experiments; p-values SATB2 5.38e-4, BCL11B 0.023), E13.5 ( n = 4 biological independent experiments; P -values SATB2 0.015, BCL11B 0.048) and E15.5 ( n = 5 biological independent experiments). e , f Immunohistochemistry ( e ) and quantification ( f ) of BCL11B and SATB2 expression in TUJ1 + neurons derived from E11.5 SLC1A5 + and SLC1A5 − progenitors after 2 days of in vitro differentiation ( n = 5 biological independent experiments; P -values SATB2 1.48e-4, BCL11B 0.03). g Schematic of relationships between uncommitted cortical cells (green) and cortical cells committed to deep- (red) or upper-layer (blue) differentiation trajectories, labelled with markers analysed here. vz ventricular zone, svz subventricular zone, DL deep layer (layer V and VI), UL upper layer (layer II to IV). Scale bar in ( c , e ) represents 20 µm. Violin plots are inset by rings corresponding to the individual data points, a filled dot at the group mean and a vertical line showing standard error. Statistics based on two-tailed t tests; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Stainings were performed according to Hagey and Muhr using antibodies against SOX2 (Goat sc-17320, Santa Cruz, 1/200), Phospho-Histone H3 (mouse clone 3H10, Millipore, 1/1000), EOMES (Rabbit ab23345, Abcam, 1/1000), BCL11B (rat ab18465, Abcam, 1/1000), SATB2 (Rabbit ab92446, Abcam, 1/1000), SOX5 (Rabbit, Ludwig Institute for Cancer Research, Muhr laboratory, 1/500), POU3F2 (Goat sc-6029, Santa Cruz, 1/250), TUJ1 (Chicken ab41489, Abcam, 1/1000), GPC6 (Alexa Fluor-conjugated bs-2177R − A647, Bioss, 1/100), HMMR (Alexa Fluor-conjugated bs-4736R-A647, Bioss, 1/100), EDNRB (Alexa Fluor-conjugated bs-2363R-A647, Bioss, 1/100), EFNA5 (Alexa Fluor-conjugated bs-6048R-A647, Bioss, 1/100) and SLC1A5 (Alexa Fluor-conjugated bs-0473-A647, Bioss, 1/100).

Techniques: Expressing, Dissection, Isolation, In Vitro, RNA Sequencing Assay, Immunohistochemistry, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CYCLIN-B1/2 and -D1 act in opposition to coordinate cortical progenitor self-renewal and lineage commitment

doi: 10.1038/s41467-020-16597-8

Figure Lengend Snippet: a Hierarchical clustering on variable genes expressed >0.5 RPKM in sorted E11.5 HMMR + , EDNRB + , GPC6 + , SLC1A5 + or EFNA5 + cortical cell populations. b Bar graphs showing gene overlap enrichment of genes differentially expressed (according to DESeq2) between sorted E11.5 cortical HMMR + cells (blue bars) and SLC1A5 + cells (red bars) and genes differentially expressed between the other sorted E11.5 uncommitted and committed cell populations. c – e GSEA terms enriched in HMMR + ( c ), GPC6 + ( d ) and EDNRB + ( e ) E11.5 cortical cells when compared with EFNA5 + progenitors. Violin plots show expression of genes relevant to the enriched terms displayed. P -values, nominal (NOM) P -values and normalised enrichment scores (NES) are indicated. f – h FACS analysis of propidium iodide-treated HMMR + cells ( f ) ( P -values (vs. EFNA5 + cells); G1 phase 3.27e–4, S phase 0.016, G2/M phase 7.67e–4), GPC6 + cells ( g ) ( P -values (vs. EFNA5 + cells); G1 phase 1.8e–4, S phase 2.65e–3 and G2/M phase 5.15e–6), EDNRB + cells ( h ) and EFNA5 + ( f – h ) E11.5 cortical cells. N = 8797 HMMR + cells, 11309 GPC6 + cells, 3149 EDNRB + cells and 5341 EFNA5 + cells over three independent experiments. Violin plots show the proportion of analysed cells in G1, S and G2/M cell-cycle phases ( n = 3 biological independent experiments). Violin plots are inset by rings corresponding to the individual data points, a filled dot at the group mean and a vertical line showing standard error. Stars indicate significant differences between indicated groups based on two-tailed t tests; * P < 0.05, ** P < 0.01 and *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Stainings were performed according to Hagey and Muhr using antibodies against SOX2 (Goat sc-17320, Santa Cruz, 1/200), Phospho-Histone H3 (mouse clone 3H10, Millipore, 1/1000), EOMES (Rabbit ab23345, Abcam, 1/1000), BCL11B (rat ab18465, Abcam, 1/1000), SATB2 (Rabbit ab92446, Abcam, 1/1000), SOX5 (Rabbit, Ludwig Institute for Cancer Research, Muhr laboratory, 1/500), POU3F2 (Goat sc-6029, Santa Cruz, 1/250), TUJ1 (Chicken ab41489, Abcam, 1/1000), GPC6 (Alexa Fluor-conjugated bs-2177R − A647, Bioss, 1/100), HMMR (Alexa Fluor-conjugated bs-4736R-A647, Bioss, 1/100), EDNRB (Alexa Fluor-conjugated bs-2363R-A647, Bioss, 1/100), EFNA5 (Alexa Fluor-conjugated bs-6048R-A647, Bioss, 1/100) and SLC1A5 (Alexa Fluor-conjugated bs-0473-A647, Bioss, 1/100).

Techniques: Expressing, Two Tailed Test