Journal: Science Advances

Article Title: NRF2 coordinates cystine import and glutathione use to sustain ferroptosis resistance and formaldehyde tolerance in cancer

doi: 10.1126/sciadv.aea1431

Figure Lengend Snippet: ( A ) Volcano plot showing dysregulated genes in lung adenocarcinoma tumors (TCGA-LUAD cohort) carrying driver mutations in NFE2L2 or KEAP1 [cutoffs: Log2(FC) = ±0.6 and P -adj ≤ 0.05]. ( B ) Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets in the TCGA-LUAD cohort with mutations in NFE2L2 or KEAP1 . ( C ) mRNA expression of selected genes involved in formaldehyde (FA) detoxification in KEAP1-NFE2L2 mutated and nonmutated (WT) LUAD tumors. ( D ) mRNA expression of selected genes in normal lung and whole blood obtained from GTEX. ( E ) Single-cell RNA-seq data from mouse hematopoietic and progenitor cells (HPC) plotted with kleintools ( https://kleintools.hms.harvard.edu/tools/spring.html ) . ( F ) Representative images of Western blots of selected factors from (A) performed in different cell lines. ( G ) Cystine uptake assay performed in selected cell lines (mean ± SEM, n = 3). ( H ) FA tolerance of A549 cells lacking SLC7A11 (sg SLC7A11 ) complemented with a wild-type copy of SLC7A11 (pSLC7A11) grown in the presence of 100 μM BSO (mean ± SEM, n = 3 and two-way ANOVA, **** P < 0.0001). ( I ) FA tolerance for H1299 cells harboring empty vector (pEV) or pSLC7A11 in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between untreated (UT) and BSO at each point); for H1299 pEV: **** P < 0.0001 and *** P = 0.0002]. ( J ) Viability of A549 cells carrying sg SLC7A11 and sgNTC (nontargeting control) under cystine deprivation [one-way ANOVA (Tukey test)]. ( K ) Glutathione (GSH) levels of the indicated cell lines maintained during 48 hours under cystine deprivation and exposing to FA during the last 24 hours [one-way ANOVA (Tukey test), ** P = 0.0058]. ( L ) FA tolerance of A549 cells lacking ADH5 in the presence of BSO (100 μM) or ERA (5 μM) [mean ± SEM, n = 6 and two-way ANOVA (Tukey test between UT and BSO or ERA at each point for multiple comparison); * P = 0.0300 and **** P < 0.0001].

Article Snippet: ADH5 -deficient cells were generated in A549 cells stably expressing Cas9 (Addgene, catalog no. 78546) infected with a lentiviral vector coding for an sgRNA against the exon 3 of ADH 5 previously published ( ).

Techniques: Analysis, Expressing, Single Cell, RNA Sequencing, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Cerebral Cortex (New York, NY)

Article Title: GABAergic subiculum–medial entorhinal projection in mice and rats

doi: 10.1093/cercor/bhag177

Figure Lengend Snippet: Anterograde tracing of subicular PV neurons in mice. a) Schematic of the experiment. AAV1-S5E2-GFP-fGFP was injected into the dorsal SUB to selectively express GFP in PV neurons. b) Sagittal section around the dorsal SUB near the AAV1-S5E2-GFP-fGFP injection site. c) PV expression in GFP-labeled SUB neurons. d) Sagittal section presenting GFP-labeled axons in the dorsal MEC ipsilateral to the injection site. Numbers, MEC layers; dotted lines, layer boundaries.

Article Snippet: The following AAVs were used: AAV1-mDlx-green fluorescent protein (GFP) (83900-AAV1, Addgene, Watertown, MA, United States; ≥7.0 × 10 12 vg/mL; 50 to 100 nL for mice; 250 to 300 nL for rats) , AAV1-CaMKIIα-mCherry (114469-AAV1, Addgene; ≥7.0 × 10 12 vg/mL; mixed at 1:1 volume ratio with AAV1-mDlx-GFP; 40 to 60 nL in total), AAV6-pgk-Cre (24593, Addgene; 2.4 × 10 12 vg/mL; 30 to 180 nL) , AAV1-hDlx-Flex-GFP (83895-AAV1, Addgene; ≥7.0 × 10 12 vg/mL; 40 to 100 nL) , AAV1-S5E2-GFP-fGFP (135631-AAV1, Addgene; 2.5 × 10 13 vg/mL; 50 nL) , and AAV1-mDlx-SynaptoTAG2 (7.4 × 10 12 vg/mL, 250 to 300 nL).

Techniques: Anterograde Tracing, Injection, Expressing, Labeling

Journal: Cerebral Cortex (New York, NY)

Article Title: GABAergic subiculum–medial entorhinal projection in mice and rats

doi: 10.1093/cercor/bhag177

Figure Lengend Snippet: Anterograde tracing of subicular PV neurons in rats. a) Low-magnification sagittal section showing GFP expression around the AAV1-S5E2-GFP-fGFP injection site in the rat dorsal SUB. b) High-magnification image of the dorsal SUB layer showing PV expression in GFP-labeled neurons. c) GFP-labeled axons in the dorsal MEC. Numbers, MEC layers; dotted lines, layer boundaries.

Article Snippet: The following AAVs were used: AAV1-mDlx-green fluorescent protein (GFP) (83900-AAV1, Addgene, Watertown, MA, United States; ≥7.0 × 10 12 vg/mL; 50 to 100 nL for mice; 250 to 300 nL for rats) , AAV1-CaMKIIα-mCherry (114469-AAV1, Addgene; ≥7.0 × 10 12 vg/mL; mixed at 1:1 volume ratio with AAV1-mDlx-GFP; 40 to 60 nL in total), AAV6-pgk-Cre (24593, Addgene; 2.4 × 10 12 vg/mL; 30 to 180 nL) , AAV1-hDlx-Flex-GFP (83895-AAV1, Addgene; ≥7.0 × 10 12 vg/mL; 40 to 100 nL) , AAV1-S5E2-GFP-fGFP (135631-AAV1, Addgene; 2.5 × 10 13 vg/mL; 50 nL) , and AAV1-mDlx-SynaptoTAG2 (7.4 × 10 12 vg/mL, 250 to 300 nL).

Techniques: Anterograde Tracing, Expressing, Injection, Labeling