Journal: Molecular Neurobiology

Article Title: Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

doi: 10.1007/s12035-020-02276-8

Figure Lengend Snippet: Cav2.2 protein expression is downregulated in DRGs after SNI. a Total Cav2.2 protein expression deceases after SNI. Sample bands of Cav2.2 expression were demonstrated at different time after sham surgery or SNI from rat DRGs. α-Tubulin served as internal control. Summary data showed that Cav2.2 protein expression but not mRNA level decreases after SNI on day 3, 14, or 28. b Cav2.2 protein level decreases at the plasma membrane after SNI. Cav2.2 at plasma membrane decreases after day 14 of SNI. N-Cadherin expression from streptavidin beads pulled down served as internal control. Data are means ± SEM; two-way ANOVA, or Student’s t test; * P < 0.05, ** P < 0.01

Article Snippet: Plasmid vectors used were pCMV6-CACNA1B-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC217170), pCMV6-EIF4EBP1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC201348), pCMV6-FOS-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC202597), pCMV6-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. PS100001), pCMV6-MZF1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC220791), pCMV6-OCT1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC208599), pCMV3-GFP control vector (Sino Biological, Wayne, PA; Cat. CV026), pCMV3-HA-Sec61β□ (Sino Biological, Wayne, PA; Cat. MG51753-NY), pCMV3-HA-SP3 (Sino Biological, Wayne, PA; Cat. HG13964-CY), pCMV3-Sigma-1R-GFP (Sino Biological, Wayne, PA; Cat. MG57873-ACG), pCMV3-HA control vector (Sino Biological, Wayne, PA; Cat. CV017), pcDNA3-HA-CEBPD (self-construct), pcDNA3-HA-SOX2 (self-construct), and Sigma-1R-EYFP (self-construct).

Techniques: Expressing

Journal: Molecular Neurobiology

Article Title: Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

doi: 10.1007/s12035-020-02276-8

Figure Lengend Snippet: Sigma-1 receptor transcription, protein expression, and surface level do not change after SNI in rat DRGs. a Sample blots of sigma-1 receptor (Sig-1R) expression at different time points after SNI. α-Tubulin served as internal control. b Summary data show that Sig-1R protein expression decreases slightly at day 14 (nonsignificant per ANOVA) but not at days 3 or 28 after SNI (left panel). The Sig-1R mRNA does not change after SNI at days 3, 14, or 28 (right panel). c Surface level of Sig-1Rs remains unchanged after SNI. Streptavidin beads were used to pull down biotinylated lysate (200 μg) from DRGs 14 days after SNI or sham surgery. Na + -K + ATPase served as internal control and GAPDH served as negative control. d Sig-1R KO increases Cav2.2 expression in HEK cells. Western blotting shows an increase of Cav2.2 protein level in total cellular lysates of Sig-1R KO HEK cells when compared to wild-type cells. Data represent means ± SEM; two-way ANOVA or Student’s t test

Article Snippet: Plasmid vectors used were pCMV6-CACNA1B-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC217170), pCMV6-EIF4EBP1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC201348), pCMV6-FOS-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC202597), pCMV6-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. PS100001), pCMV6-MZF1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC220791), pCMV6-OCT1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC208599), pCMV3-GFP control vector (Sino Biological, Wayne, PA; Cat. CV026), pCMV3-HA-Sec61β□ (Sino Biological, Wayne, PA; Cat. MG51753-NY), pCMV3-HA-SP3 (Sino Biological, Wayne, PA; Cat. HG13964-CY), pCMV3-Sigma-1R-GFP (Sino Biological, Wayne, PA; Cat. MG57873-ACG), pCMV3-HA control vector (Sino Biological, Wayne, PA; Cat. CV017), pcDNA3-HA-CEBPD (self-construct), pcDNA3-HA-SOX2 (self-construct), and Sigma-1R-EYFP (self-construct).

Techniques: Expressing, Negative Control, Western Blot

Journal: Molecular Neurobiology

Article Title: Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

doi: 10.1007/s12035-020-02276-8

Figure Lengend Snippet: cFOS increases 4E-BP1 transcription via its interaction with Sig-1R. a Overexpression of 4E-BP1 decreases Cav2.2 in HEK cells. b Sig-1R interacts with cFOS in HEK cells. In cFOS-Myc/DDK and Sig-1R-GFP HEK co-expressing HEK cells, sample blot demonstrates the co-immunoprecipitation of those two proteins (lane 6). c Co-overexpressed Sig-1R and cFOS bind to the promoter of 4E-BP1. DNAs obtained from chromatin immunoprecipitation assays testing the binding of GFP-tagged Sig-1R (upper panel) or Myc-tagged cFOS (lower panel) to the 4E-BP1 promoter in HEK cells were examined. The precipitated DNA was amplified using specific primers for the 4E-BP1 promoter. Quantified results from the PCR products were normalized to input. Summary data are presented as means ± SEM; Student’s t test; * P < 0.05, *** P < 0.001; N = 3

Article Snippet: Plasmid vectors used were pCMV6-CACNA1B-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC217170), pCMV6-EIF4EBP1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC201348), pCMV6-FOS-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC202597), pCMV6-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. PS100001), pCMV6-MZF1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC220791), pCMV6-OCT1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC208599), pCMV3-GFP control vector (Sino Biological, Wayne, PA; Cat. CV026), pCMV3-HA-Sec61β□ (Sino Biological, Wayne, PA; Cat. MG51753-NY), pCMV3-HA-SP3 (Sino Biological, Wayne, PA; Cat. HG13964-CY), pCMV3-Sigma-1R-GFP (Sino Biological, Wayne, PA; Cat. MG57873-ACG), pCMV3-HA control vector (Sino Biological, Wayne, PA; Cat. CV017), pcDNA3-HA-CEBPD (self-construct), pcDNA3-HA-SOX2 (self-construct), and Sigma-1R-EYFP (self-construct).

Techniques: Over Expression, Expressing, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay, Amplification

Journal: Molecular Neurobiology

Article Title: Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

doi: 10.1007/s12035-020-02276-8

Figure Lengend Snippet: Sig-1R regulates 4E-BP1 and its effect on eIF4E. a Sig-1R KO decreases the transcription and translation of 4E-BP1 in HEK cells. Sample blot demonstrates a decrease of 4E-BP1 protein in Sig-1R KO HEK cells. Summary data reveal an elevated level of protein and mRNA of 4E-BP1 in Sig-1R KO HEK cells. b Sig-1R knockout (KO) increases eIF4E and Cav2.2 mRNA binding. In the RNA-IP test, eIF4E binding to Cav2.2 5′cap region increases in Sig-1R KO HEK cells. Data represent means ± SEM; Student’s t test; Mann-Whitney test for b ; * P < 0.05, *** P < 0.001

Article Snippet: Plasmid vectors used were pCMV6-CACNA1B-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC217170), pCMV6-EIF4EBP1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC201348), pCMV6-FOS-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC202597), pCMV6-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. PS100001), pCMV6-MZF1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC220791), pCMV6-OCT1-myc/DDK (Origene Technologies Inc., Rockville, MD; Cat. RC208599), pCMV3-GFP control vector (Sino Biological, Wayne, PA; Cat. CV026), pCMV3-HA-Sec61β□ (Sino Biological, Wayne, PA; Cat. MG51753-NY), pCMV3-HA-SP3 (Sino Biological, Wayne, PA; Cat. HG13964-CY), pCMV3-Sigma-1R-GFP (Sino Biological, Wayne, PA; Cat. MG57873-ACG), pCMV3-HA control vector (Sino Biological, Wayne, PA; Cat. CV017), pcDNA3-HA-CEBPD (self-construct), pcDNA3-HA-SOX2 (self-construct), and Sigma-1R-EYFP (self-construct).

Techniques: Knock-Out, Binding Assay, MANN-WHITNEY

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