Journal: bioRxiv

Article Title: Lineage transcription factors co-regulate subtype-specific genes providing a roadmap for systematic identification of small cell lung cancer vulnerabilities

doi: 10.1101/2020.08.13.249029

Figure Lengend Snippet: (A) RNA-seq (log2 FPKM) showing expression of SCN3A and KCNB2 , in SCLC tumors (T) or cell lines (C) in the specified SCLC subtype or NSCLC. The line indicates the mean, and the asterisks indicate p-values for each sample compared to SCLC-A tumors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (B) UCSC Genome Browser tracks showing ASCL1, NKX2-1, PROX1 and H3K27ac ChIP-seq signals in NCI-H2107 cells around the genes encoding SCN3A and KCNB2 . Black bars above the tracks indicate computationally called binding sites. (C-E) Immunoblots showing SCN3A and KCNB2 protein in NCI-H2107 cells, 72 hours post-transfection with control siRNAs or siRNA targeted against either ASCL1, NKX2-1 , or PROX1 (C), or SCN3A (D) or KCNB2 (E). (F) WST-1 assay for cell viability from cells from (D,E). Each data point represents a biological replicate, and error bars indicate SEM. ANOVA with Bonferroni’s multiple comparisons test was used to determine significant differences relative to control. ns; not significant. (G) Quantification of colony formation assays in soft agar using varying doses of KCNB2 inhibitor Quinine shows increased sensitivity of SCLC-A NCI-H889, compared to SCLC-N NCI-H524, and SCLC-P NCI-H526. (H) Histograms showing SCN3A+ cells are detected in live SCLC-A but not SCLC-N or SCLC-P cell cultures using an SCN3A extracellular domain specific antibody (red). The background fluorescence with the secondary antibody only is shown (gray). (I,J) RT-qPCR for SCN3A (I) and ASCL1 (J) mRNA from FACs isolated SCN3A+ (red) or SCN3A-(black) cells from mixtures of SCLC-A with SCLC-P or – N. Each data point represents a biological sample, error bars = SD around mean, unpaired t-test, * p<0.05, ** p<0.01.

Article Snippet: Cells were incubated with SCN3A antibody (Alomone Labs, Anti-SCN3A (Nav1.3) (extracellular), Cat #: ASC-023, 4.25 µg/ml) for 30 min on ice, washed 3 times with ice cold FACs buffer, incubated with Alexa Fluor 568-conjugated secondary antibody (Thermofisher, Catalog # A10042, 1:400) for 30 min on ice, washed 3 times as above, and resuspended in ice cold FACs buffer.

Techniques: RNA Sequencing Assay, Expressing, ChIP-sequencing, Binding Assay, Western Blot, Transfection, WST-1 Assay, Fluorescence, Quantitative RT-PCR, Isolation

Journal: The Journal of Headache and Pain

Article Title: ROS/TRPA1/CGRP signaling mediates cortical spreading depression

doi: 10.1186/s10194-019-0978-z

Figure Lengend Snippet: Both ROS and the TRPA1 activation reversed the inhibitory effects of the anti-CGRP antibody on CSD in the mouse brain slice. CSD was induced by 260 mM KCl. There were four groups: anti-IgG antibody at 0.025 μM (i, n = 6) as the control, anti-CGRP antibody at 0.4 μM in the absence (ii, n = 6) or presence of 50 μM of the TRPA1 agonist, AITC (iii, n = 6) or the ROS activator, H 2 O 2 (vi, n = 6). In order to minimize the animal use, data in anti-IgG antibody control group and the anti-CGRP antibody were adopted and transformed from that in Fig. in our recent paper . Representative trace of the 2nd CSD episode in each group are shown in the panel a . The data showed that both ROS and the TRPA1 activation reversed the prolonged CSD latency ( b ), but not magnitude ( c ) under the perfusion of the anti-CGRP antibody. Data were plotted as percentage of their initial levels (1st CSD episode) and indicated as median (range). Mann-Whitney U test, one-tailed, was used for significant analysis between two independent groups. * p < 0.05, ** p < 0.01. Abbreviation: Ab indicates antibody

Article Snippet: Three groups were designed: (i) pretreatment of the anti-TRPA1 antibody with a total 0.8 μg (Alomone Labs, n = 10) 4 days before CSD induction. (ii, iii) Unconjugated rabbit IgG (H + L) (Sangon, D110502, n = 8) with a total 0.8 μg being applied for both the CSD group and the sham group as controls ( n = 7 in each group).

Techniques: Activation Assay, Slice Preparation, Transformation Assay, MANN-WHITNEY, One-tailed Test

Journal: The Journal of Headache and Pain

Article Title: ROS/TRPA1/CGRP signaling mediates cortical spreading depression

doi: 10.1186/s10194-019-0978-z

Figure Lengend Snippet: Effects of the anti-TRPA1 antibody on MDA level (μmol/mg protein) induced by CSD in the ipsilateral cerebral cortex of rat and correlation analysis of each CSD characteristic with levels of cortical ipsilateral MDA between the anti-TRPA1 antibody or anti-IgG antibody groups. a CSD promoted ipsilateral cortical MDA level, which was inhibited by the pretreatment of anti-TRPA1 antibody perfused into the contralateral i.c.v in rats. Mann-Whitney U test, one-tailed, for significance between each group (* p < 0.05, *** p < 0.001). The reduced CSD magnitude ( d ), but not CSD number ( b ) and latency ( c ) positively correlated with a lower MDA level after the anti-TRPA1 antibody perfusion. Red dotted lines indicated positive correlation between CSD magnitude and MDA level

Article Snippet: Three groups were designed: (i) pretreatment of the anti-TRPA1 antibody with a total 0.8 μg (Alomone Labs, n = 10) 4 days before CSD induction. (ii, iii) Unconjugated rabbit IgG (H + L) (Sangon, D110502, n = 8) with a total 0.8 μg being applied for both the CSD group and the sham group as controls ( n = 7 in each group).

Techniques: MANN-WHITNEY, One-tailed Test

Journal: The Journal of Headache and Pain

Article Title: ROS/TRPA1/CGRP signaling mediates cortical spreading depression

doi: 10.1186/s10194-019-0978-z

Figure Lengend Snippet: Effects of the anti-TRPA1 antibody, perfused into the contralateral ventricle, on cortical susceptibility to CSD in rats. a CSD was induced by topical application of 2 M KCl for 30 min onto cerebral cortex with dura intact via the posterior burr hole on the right parietal bone. The ipsilateral anterior hole was used for CSD recording. The anti-TRPA1 antibody (i, n = 10) or anti-IgG antibody (ii, n = 8) was perfused through a cannula implanted in the contralateral ventricle ( i.c.v ) at 4 days prior to CSD induction. In the sham group (iii), the anti-IgG antibody was i.c.v perfused in the absence of KCl application as the control ( n = 7). The whole ipsilateral cerebral cortical tissue was subsequently used for detecting MDA level immediately after the in vivo experiment. b A representative trace showing CSD propagation wave after i.c.v perfusion of the anti-IgG antibody. CSD number, latency (minute) and magnitude (area under the curve of each CSD wave, mV × minute) were used for quantifying the excitation phase of CSD. The effects of the anti-TRPA1 antibody at 0.8 μg on CSD number are shown in panel ( c ), latency in panel ( d ) and magnitude in panel ( e ). All the values shown are median (range). * p < 0.05, Mann-Whitney U test with one-tailed calculation was used for comparison of the anti-IgG antibody and the anti-TRPA1 antibody group

Article Snippet: Three groups were designed: (i) pretreatment of the anti-TRPA1 antibody with a total 0.8 μg (Alomone Labs, n = 10) 4 days before CSD induction. (ii, iii) Unconjugated rabbit IgG (H + L) (Sangon, D110502, n = 8) with a total 0.8 μg being applied for both the CSD group and the sham group as controls ( n = 7 in each group).

Techniques: In Vivo, MANN-WHITNEY, One-tailed Test