Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 2. [ 3H]Kainate binding and functional assessment by patch-clamp recording of GluR6-binding site mutants. A, Saturation analysis of [ 3H]kainate binding to GluR6-wt ex- pressedinHEK293cellsyieldedaKDof36nMandaBmaxof3.8pmol/mgprotein.Specificbinding wasdeterminedinthepresence(nonspecific)andabsence(total)of1mMglutamate.B,Specific binding of 100 nM [ 3H]kainate to GluR6-wt, R523G, T690G, E738G, and E738D (60 g of pro- tein). Western blots confirmed similar amounts of C-GluR6 immunoreactivity in 25 g of pro- tein used for the binding assays (inset). C, Outside-out patches were pulled from HEK293 cells expressing GluR6-wt, R523G, T690G, or E738G, voltage clamped at 70 mV, and tested by ultrafast application of 3 or 30 mM glutamate (GLU). For GluR6-wt, 3 mM glutamate was satu- rating and elicited large peak currents (2.8 0.4 nA). No currents were detected in patches from cells expressing R523G, T690G, or E738G. Application of 3 or 30 mM glutamate demon- stratesthatE738Dreceptorsarefunctional,whereas3mMglutamateisnolongersaturatingfor this mutant. D, Dose–response curve showing the rightward shift of the EC50 value ( 100- fold) for the mutation E738D compared with GluR6-wt in peak response to glutamate.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Binding Assay, Functional Assay, Patch Clamp, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 3. Cell-surface expression of the GluR6-binding site mutants. A, Confocal images of extracellular N-GluR6 (green) and intracellular C-GluR6 (red) immunofluorescence staining in HEK293 cells expressing GluR6-wt and GluR6-binding site mutants. N-GluR6 staining before cell membrane permeabilization demonstrates robust surface expression of GluR6-wt and E738D. In contrast, the absence of N-GluR6 staining indicates that R523G, T690G, and E738G are not present at the cell surface. C-GluR6 staining was evident for GluR6-wt and GluR6 mutants after permeabilization. Staining of nontransfected controls was negative. B,PrimaryculturesofratastrocytestransfectedwithGluR6-wtorT690GwerestainedforN-andC-GluR6asabove.C,Biotinylated cell-surfaceproteins(50g)wereprecipitatedwithNeutrAvidinbeads.Onlytraceamounts,ifany,ofR523G,T690G,andE738G werefoundatthecellsurface.GluR6-wtandE738Dwerebothisolatedinthebiotinylatedfraction.Thepercentageofcell-surface expressionofimmunoreactiveGluR6wasdeterminedbydensitometryusing10goftotalprotein.Theblotisrepresentativeof three separate experiments. IB, Immunoblot; IP, immunoprecipitate. D, Biotinylation studies, using GluR1-wt as a control, re- vealed that a homologous GluR1-binding site mutant, R499G, also fails to reach the cell surface.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Expressing, Binding Assay, Immunofluorescence, Staining, Membrane, Western Blot, Control, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure4. ERretentionandglycosylationstateofGluR6-bindingsitemutants.A,ConfocalimagesofintracellularC-GluR6(red) immunofluorescence staining in HEK293 cells coexpressing the EYFP-ER marker and GluR6-wt or GluR6-binding site mutants. GluR6-wt and E738D demonstrate robust surface staining that is not colocalized with EYFP-ER. In contrast, R523G, T690G, and E738G are not present at the cell surface and are colocalized entirely with EYFP-ER. The merged images show the overlapping localization of C-GluR6 and EYFP-ER markers. Identical results were obtained with antibodies to other ER markers, calnexin and calreticulin. B, Representative confocal images of the GluR6-wt (top) or the GluR6-binding-deficient mutant R523G (bottom) cotransfectedwiththeEYFP-Golgimarker.Themergedimageofimmunofluorescencestainingdemonstratesthelackofcolocal- ization of R523G (red) and the EYFP-Golgi marker (green). C, Triton X-100 (0.5%) solubilized lysates of HEK293 cells transfected withGluR6-wtorGluR6-bindingsitemutantsweresubjectedtoEndoHdigest.Westernblotanalysisofcontrol(Gly)andEndoH- digested(Cut)samples(15–30gofprotein)revealsthatR523G,T690G,andE738GareentirelyEndoHsensitive.Incontrast,the majorityofGluR6-wtandE738DareEndoHinsensitive.D,ParalleldigestsdemonstratePNGaseFandEndoHdeglycoslateR523G, T690G,andE738Gtothesameextent,confirmingtheirimmatureN-linkedglycosylationstate.Theblotsarerepresentativeofat least three separate experiments.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Immunofluorescence, Staining, Marker, Binding Assay, Mutagenesis, Transfection

Journal: bioRxiv

Article Title: Self-amplifying mRNA expression is governed by mitochondrial machinery and ACSL4

doi: 10.1101/2025.06.13.659366

Figure Lengend Snippet: A) Schematic for CRISPR-KO screen detailing the plan for each typical step of a CRISPR screen including model, perturbation, screen challenge, library generation, and analysis. Created in BioRender (biorender.com/u36hl45). B) 293T cells overnight co-transfected with saGFP and mod-mCherry at indicated ratios in 96 well format. mod-mCherry concentration was kept constant at 50 ng/well while saGFP varied from 4 to 25 ng/well. C) Volcano plots for each screen sample displaying log 2 (fold change) as calculated by MAGeCK. In the top left quadrant for each plot, genes highlighted in blue exhibit −log 10 (false discovery rate) > 1 and log 2 (fold change) < −0.5. Five named genes highlighted are common across all samples, appearing significant on all plots; the other five named genes highlighted are unique to Dual Expressors, only appearing significant in these samples. Essential genes integrated into each pool as an internal control performed as expected, indicating successful Cas9 editing efficiency (data not shown). D) Hits unique to Dual Expressor samples and suspected necessary factors for saRNA expression are primarily associated with mitochondrial function. General mitochondria (GO: mitochondrion) and oxidative phosphorylation (GOBP: OXPHOS) gene lists are taken from Gene Ontology and Gene Ontology Biological Process databases.

Article Snippet: 293T cells (ATCC) and fibroblasts (HFF-1, ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

Techniques: CRISPR, Transfection, Concentration Assay, Control, Expressing, Phospho-proteomics

Journal: bioRxiv

Article Title: Self-amplifying mRNA expression is governed by mitochondrial machinery and ACSL4

doi: 10.1101/2025.06.13.659366

Figure Lengend Snippet: A) 293T cells pre-treated with antimycin A overnight at the concentrations indicated before transfection with modGFP or saGFP. Each well received a standard dose of RNA (120 ng/well in 96 well format). MFI was read by HiBit assay after four hours and normalized to the negative control of each modality (DMSO). n=3 biological replicates. B) Comparison of fold change in MFI for each concentration of antimycin A pre-treatment. saRNA MFI is significantly more impacted at all concentrations. C) 293T cells pre-treated with ferrostatin-1 (Fer-1) overnight at indicated concentrations before transfection with modGFP or saGFP show Fer-1 only abrogates saRNA expression. Each well received a standard dose of RNA (120 ng/well in 96 well format). MFI was read by HiBit assay after four hours and normalized to the negative control of each modality (untreated/DMSO). n=3 biological replicates. D) 293T-Cas9 cells transfected with two unique ACSL4-targeting guide RNAs show ACSL4 dependency is unique to saRNA expression. KO cells were subcultured for 48 (left) or 72 (right) hours prior to transfection with modGFP or saGFP. Each well received a standard dose of RNA (120 ng/well in 96 well format). MFI was read by HiBit assay after four hours and normalized to the negative control of each modality (gRNA null/normal 293T-Cas9 cells). n=3 biological replicates. E) Jurkat cells co-transfected with saGFP and mod-mCherry overnight show dual expressors naturally exhibit higher levels of ACSL4 expression as measured by qPCR. Jurkat cells were nucleofected using Lonza 4D-Nucleofector™ 96-well Unit with a cell density of 5e5 cells per well; co-transfection wells received 1000 ng modRNA and 500 ng saRNA per well. Single modality and H 2 O nucleofection conditions were used as gating controls for FACS. 2 -ΔΔ CT was calculated using mCherry single expressors as the control.

Article Snippet: 293T cells (ATCC) and fibroblasts (HFF-1, ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

Techniques: Transfection, Negative Control, Comparison, Concentration Assay, Expressing, Cotransfection, Control

Journal: bioRxiv

Article Title: Self-amplifying mRNA expression is governed by mitochondrial machinery and ACSL4

doi: 10.1101/2025.06.13.659366

Figure Lengend Snippet: A) Schematic representing the mitochondrial-ferroptotic axis and the interaction between these biological processes. ACSL4 activity, electron transport chain (ETC) inhibitors, and ferroptosis inhibitors that have demonstrated a clear impact on saRNA expression are highlighted in blue, demonstrating their location upstream in this axis. Ferroptosis inducers and lipid peroxidation are highlighted in red and located at downstream points in this axis. Created in BioRender (biorender.com/5rmsile). B) 293T cells pre-treated with RSL3 for one hour at indicated concentrations show RNA expression is sensitive to high concentrations of RSL3 regardless of RNA modality. Each well received a standard dose of modGFP or saGFP (120 ng/well in 96 well format). MFI was read by HiBit assay after four hours and normalized to the negative control of each modality (untreated/DMSO). n=3 biological replicates. C) Lipid peroxidation detected by BODIPY dye oxidization shows no effect on lipid peroxidation post-transfection regardless of RNA modality. Each well received a standard dose of modGFP or saGFP (120 ng/well in 96 well format) for overnight transfection. Cells were analyzed by flow cytometry; debris, dead cells, doublets, and GFP− cells were gated out by FlowJo to observe oxidation in GFP+ cells only. n=3 biological replicates.

Article Snippet: 293T cells (ATCC) and fibroblasts (HFF-1, ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

Techniques: Activity Assay, Expressing, RNA Expression, Negative Control, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: Self-amplifying mRNA expression is governed by mitochondrial machinery and ACSL4

doi: 10.1101/2025.06.13.659366

Figure Lengend Snippet: A) ACSL4 expression as read by qPCR with two unique probes targeting different exons shows successful activation of ACSL4 via CRISPRa. Gene expression assay was conducted concurrently with RNA IVE. 2 -ΔΔ CT was calculated using normal 293T-dCas9 cells (no gRNA/normal 293T-dCas9 cells) as the control. B) ACSL4-activated cells show increased saRNA expression. CRISPRa cells were subcultured for 48 hours after gRNA transfection before being transfected with modGFP or saGFP. Each well received a standard dose of RNA (120 ng/well in 96 well format). MFI was read by HiBit assay after four hours and normalized to negative control of each modality (gRNA null/normal 293T-dCas9 cells). n=3 biological replicates. C) Transfection with modACSL4 successfully increases ACSL4 expression in 293T cells as read by qPCR. 293T cells were transfected with either modGFP, saGFP, modASCL4, or co-delivered modACSL4 with a GFP carrying RNA overnight in a 96 well format. Each single modality transfection received 100 ng/well RNA. Co-transfection with saRNA received 50 ng modRNA/well combined with 25 ng saRNA/well. Co-transfection of two modRNAs received 50 ng of each modRNA per well. All cells that received modACSL4 (either alone or co-delivered) exhibited significantly higher ACSL4 expression with both unique probes. 2 -ΔΔ CT was calculated using non-transfected 293T cells (H 2 O negative control) as the control. D) MFI of 293T cells with co-delivered modACSL4 and GFP carrying RNA show increased saRNA expression with no effect on modRNA expression. 293T cells were transfected with either modGFP, saGFP, modASCL4, or co-delivered modACSL4 with a GFP carrying RNA overnight in a 384 well format. Each single modality transfection received 50 ng/well RNA. Co-transfection with saRNA received 25 ng modRNA/well combined with 12.5 ng saRNA/well. Co-transfection of two modRNAs received 25 ng of each modRNA per well. MFI of each co-transfection was normalized to its single modality counterpart. n=4 biological replicates. E) Transfection with modACSL4 successfully increases ACSL4 expression in Jurkat cells as read by qPCR. Jurkat cells were nucleofected with either modGFP, saGFP, modASCL4, or co-delivered modACSL4 with a GFP carrying RNA overnight using Lonza 4D-Nucleofector™ 96-well Unit with a cell density of 5e5 cells per well. Each single modality transfection received 1000 ng/well RNA. Co-transfection with saRNA received 500 ng modRNA/well combined with 250 ng saRNA/well. Co-transfection of two modRNAs received 500 ng of each modRNA per well. All cells that received modACSL4 (either alone or co-delivered) exhibited significantly higher ACSL4 expression with both unique probes. 2 -ΔΔ CT was calculated using non-transfected Jurkat cells (H 2 O negative control) as the control. F) MFI of Jurkat cells with co-delivered modACSL4 and GFP RNA show increased saRNA expression with no effect on modRNA expression. Jurkat cells were nucleofected as described in 4E. Cells were analyzed by flow cytometry; debris, dead cells, doublets, and GFP− cells were gated out by FlowJo to observe MFI in GFP+ cells only. MFI of each co-transfection was normalized to its single modality counterpart. n=3 biological replicates.

Article Snippet: 293T cells (ATCC) and fibroblasts (HFF-1, ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

Techniques: Expressing, Activation Assay, Gene Expression, Control, Transfection, Negative Control, Cotransfection, Flow Cytometry