Journal: iScience

Article Title: Aryl hydrocarbon receptor is a tumor promoter in MYCN -amplified neuroblastoma cells through suppression of differentiation

doi: 10.1016/j.isci.2023.108303

Figure Lengend Snippet:

Article Snippet: siRNA pools against MYCN , Santa Cruz Biotechnology , Cat#sc-36003.

Techniques: Recombinant, Staining, Amplification, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: PLOS Pathogens

Article Title: Contrasting effects of filamin A and B proteins in modulating filovirus entry

doi: 10.1371/journal.ppat.1011595

Figure Lengend Snippet: Analysis of FLN KD cells (A) Representative Western blot showing stable shRNA knockdown of FLNa in FLNaKD cells (lane 2) and FLNb in FLNbKD cells (lane 3). Both FLNa and FLNb are expressed in WT HT-1080 cells (lane 1). (B) MTT assay showing no significant growth rate or proliferation differences between WT and the filamin KD cells at 24, 48, and 72 hours post-seeding. Statistical analysis of 4 independent experiments using 2-sample student t-test is shown.

Article Snippet: FLNa-specific siRNA, FLNb-specific siRNA and control siRNA pools were purchased from Santa Cruz and Origene.

Techniques: Western Blot, shRNA, MTT Assay

Journal: PLOS Pathogens

Article Title: Contrasting effects of filamin A and B proteins in modulating filovirus entry

doi: 10.1371/journal.ppat.1011595

Figure Lengend Snippet: (A) Representative Western blot showing detection of the indicated proteins in cells receiving no siRNA, control siRNA, FLNa-specific siRNA, or FLNb-specific siRNA. Cells were non-transduced, or transduced with psVSV-RFP-eGP pseudotypes as shown. (B) Bar graph showing relative psVSV-RFP-eGP particle entry into FLNKD cells as compared to WT cells in 4 independent experiments. Statistical analysis was performed using 2-sample student t test; ** = p value < .01.

Article Snippet: FLNa-specific siRNA, FLNb-specific siRNA and control siRNA pools were purchased from Santa Cruz and Origene.

Techniques: Western Blot, Transduction

Journal: PLOS Pathogens

Article Title: Contrasting effects of filamin A and B proteins in modulating filovirus entry

doi: 10.1371/journal.ppat.1011595

Figure Lengend Snippet: (A) Representative Western blot showing detection of the indicated proteins in macrophages receiving control siRNA or three individual FLNa-specific siRNAs (1, 2, and 3). (B) Bar graph showing the relative number of infected cells to non-specific siRNA controls after infection with EBOV-GFP (MOI = 0.2) at 24 hours post infection in 3 independent experiments. (C) Bar graph showing relative number of infected cells to non-specific siRNA controls after infection with MARV (MOI = 0.2) at 24 hours post infection in 3 independent experiments. A one-way ANOVA followed by Dunnett’s multiple comparison test for one variable was used to assess a statistical difference between infection efficiencies in cells transfected with non-specific siRNA vs. FLNa-specific siRNAs. **** = p value <0.0001 was determined for each sample pair analyzed.

Article Snippet: FLNa-specific siRNA, FLNb-specific siRNA and control siRNA pools were purchased from Santa Cruz and Origene.

Techniques: Western Blot, Infection, Comparison, Transfection

Journal: Science translational medicine

Article Title: Inhibition of VEGF binding to neuropilin-2 enhances chemosensitivity and inhibits metastasis in triple-negative breast cancer

doi: 10.1126/scitranslmed.adf1128

Figure Lengend Snippet: (A) Ability of aNRP2-10 alone or in combination with cisplatin (at either C1 = 6.1 or C2 = 12.1 μM within IC10 to IC30 based on the cisplatin monotherapy tests) to reduce the viability of MDA-MB-231 cells grown in methylcellulose is shown. hIgG4 was used as an isotype control. Consistent results were obtained in three independent experiments, and representative data with n = 3 biological replicates are shown. Statistical significance was determined by two-sided, unpaired t test. **P < 0.01 and ****P < 0.0001. (B) Breast cancer (BC) cell lines covering Luminal, Her2+, and TNBC subtypes were used in methylcellulose 3D viability assays. Cells responding to aNRP2-10 treatment in the combination therapy with chemo drug cisplatin (responders) showed higher EMT scores and enriched gene expression of NRP2 and ZEB1 compared with nonresponder cells. EMT color scheme is based on EMT scores by Le et al. (30) (dark orange: 0.5 to 1, indicating mesenchymal cells; light orange: 0 to 0.5, and light blue: −0.5 to 0, indicating intermediate cells; dark blue: −1 to −0.5, indicating epithelial cells). Cell line gene expression data were from CCLE [expressed as Log2(TPM + 1) in the file: OmicsExpressionProteinCoding-Genes TPMLogp1.csv downloaded from the CCLE website]. TPM, transcript per million. (C) NRP2 gene expression in chemo-resistant organoids (R) and chemosensitive organoids (S) derived from two TNBC patient tumors (TPDO-1 and TPDO-2) is shown. The chemoresistant organoids were generated by culturing them in the presence of either 5-FU or cisplatin for 2 weeks. Organoids were pooled from 24 wells, and qPCR was performed with technical triplicates. Statistical significance was determined by two-sided, unpaired t test comparing R and S organoids from the same tumors. ****P < 0.0001. (D) TPDO-1 TNBC organoids were treated with aNRP2-10 or aNRP2-14 and either 5-FU or cisplatin, and viability was compared with either treatment alone. n = 4 biological replicates. Statistical significance was determined by two-sided, unpaired t test. ****P < 0.0001. (E) Effect of NRP2 siRNA and aNRP2-10, either alone or in combination, on the sensitivity of TNBC organoids to 5-FU was measured and plotted as relative percentage viability. n = 3 biological replicates. Statistical significance was determined by two-sided, unpaired t test. ***P < 0.001; ns, not significant. KD, knockdown. (F) CSC and EMT markers were analyzed after aNRP2-10 treatment of the TPDO-1 organoids using Fluidigm 84-gene panels. Organoids were pooled from 24 wells, and the Fluidigm experiment was performed with technical triplicates. Green indicates >2-fold down-regulation, and orange indicates >2-fold up-regulation of gene expression by aNRP2-10 versus vehicle treatment. (G) Expression of CSC pluripotency, EMT, and epithelial markers was measured in the second patient-derived organoids, TPDO-2, by qPCR analysis after aNRP2-10 treatment. hIgG4 was used as an isotype control. Organoids were pooled from 24 wells, and qPCR was performed with technical triplicates. Statistical significance was determined by two-sided, unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. (H) Effect of overexpression of ZEB1 on TPDO-1 viability was measured for the indicated treatment groups. n = 3 biological replicates. Statistical significance was determined by two-sided, unpaired t test. **P < 0.01. Data are presented as means ± SEM.

Article Snippet: For knockdown experiments using VEGF or NRP2 siRNA, the harvested organoids were incubated with the siRNA pool (Santa Cruz Biotechnology) or nontargeting control for 8 hours in ultralow attachment plates.

Techniques: Expressing, Derivative Assay, Generated, Over Expression

Journal: bioRxiv

Article Title: EGR1 regulates oral epithelial cell responses to Candida albicans via the EGFR- ERK1/2 pathway

doi: 10.1101/2023.03.31.535186

Figure Lengend Snippet: TR146 cells were treated with a single siRNA or a pool of siRNAs targeting EGR1 and then infected with C. albicans . (A) Western blot analysis to confirm knockdown of EGR1 in OECs. (B) LDH quantification and (C) Luminex analysis of culture supernatant at 24 h post-infection. All data is shown as mean fold change ± S.E.M. Significance relative to non-targeting siRNA control calculated using one-way ANOVA: * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Knockdown of gene expression was performed using previously reported siRNAs for EGR1 , or commercially available siRNA pools for EGR1 and EphA2 (Santa-Cruz Biotech).

Techniques: Infection, Western Blot, Luminex

Journal: Cellular microbiology

Article Title: Subtilase cytotoxin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli induces stress granule formation

doi: 10.1111/cmi.12565

Figure Lengend Snippet: Protein kinase D1-knockdown enhances SubAB-induced SG formation.

Article Snippet: To suppress PKD1 expression, we transfected PKD1 siRNA mixture as follows: PKD1-a, 5′-GUCGAGAGAAGAGGUCAAATT-3′( Fuchs et al., 2009 ), PKD1-b, 5′-CAGGAAGAGAUGUAGCUAU-3′( Yin et al., 2008 ) and PKD1 siRNA pool (Santa Cruz Biotechnology, Inc.).

Techniques: