Journal: The Journal of Biological Chemistry

Article Title: Hypoxia-induced Expression of Carbonic Anhydrase 9 Is Dependent on the Unfolded Protein Response

doi: 10.1074/jbc.M109.006510

Figure Lengend Snippet: ATF4 is required for CA9 mRNA expression during hypoxia. U373 cells were transiently transfected with siRNA duplexes directed against HIF1α, ATF4, CHOP, and ATF6. 24 h after transfection cells were exposed to 16 h of hypoxia. A, the knockdown efficiency was determined for gene-specific siRNAs using Q-PCR. Expression was normalized to mRNA levels in cells transfected with scrambled control (n = 3). B, relative expression of CA9 mRNA post transfection with siRNA after culturing under normoxia or 16 h of hypoxia (n = 3, Student's t test; *, p < 0.05, scrambled versus knock down). C, exogenous ATF4 was overexpressed in U373 cells and exposed to 0, 8, and 16 h of hypoxia. Relative levels of CA9 mRNA were determined using Q-PCR compared with empty vector control under aerobic conditions (n = 5, Student's t test; *, p < 0.05, pcDNA versus ATF4).

Article Snippet: For transient knockdown U373 cells were transfected with 100 n m siRNA duplexes using Oligofectamine according to the manufacturer's instructions. siRNA duplex targeting ATF4 122372, CHOP 146321, ATF6 115889, and HIF1α 42840 was obtained from Ambion, and scramble control siRNA was from Dharmacon.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Synergistic immunotherapy of glioblastoma by dual targeting of IL-6 and CD40

doi: 10.1038/s41467-021-23832-3

Figure Lengend Snippet: a BM-derived Mϕs were isolated from mice and treated with 50 ng/ml IL-4 and IL-6 for 2 days, followed by RNA-seq analysis ( n = 3 mice). Shown are top upregulated transcriptional factors induced by IL-6. Left, heatmap. Right, means of fold expression of control. b Human monocytes were transfected with siRNA targeting NF-κB2, Stat3, or control sequence and treated with IL-6 or control medium. Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. c – e Human monocytes were treated with IL-6 or control medium under d normoxia or e hypoxia. Nuclei protein was immunoprecipitated with d anti-Stat3 or e anti-HIF-1α antibody, or IgG, and subjected to ChIP analysis with different primers. c Results shown are from quantitative real-time polymerase chain reaction (RT-PCR) analysis ( n = 3 human samples, means ± SEM). Statistical analysis by two-way ANOVA with Tukey’s test. f Human monocytes were treated with IL-6 or control medium under normoxia or hypoxia, followed by immunoblot analysis. This experiment was repeated independently twice with similar results. g Human monocytes were pretreated with siRNA targeting HIF-1α or control sequence, and treated with IL-6 or control medium under hypoxia. Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. Source data are provided as a Source data file.

Article Snippet: Human monocytes were transfected with siRNAs targeting HIF-1α (Thermo Fisher, 42840), Stat3 (Life Technologies, 4390824), nuclear factor-κB (NF-κB) (Thermo Fisher, 106835), or control siRNA (Qiagen, 1027280) using Amaxa 4D-Nucleofector (Lonza) with program EA-100.

Techniques: Derivative Assay, Isolation, RNA Sequencing Assay, Expressing, Transfection, Sequencing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot