Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. HEK-293 cells overexpressing distinct α-syn constructs were lysed 48 h after transfection, and the cell lysates were analysed by immunoblotting for α-syn (Syn-1), without boiling. GAPDH was used as the loading control. A representative blot of n=3 experiments is shown B. Densitometry of data shown in (A.); a significant difference was indicated (p=0.045) by one-way ANOVA followed by a Tukey test for BiFC (Vn-α-syn) vs. BiFC (α-syn-Vc). C. WB of lysates from HEK-293 cells transfected with various amounts of vector α-syn DNA (depicted above the blot). α-syn was detected by anti-α-syn (Syn-1). The samples were boiled to disrupt complex formation to allow accurate determination of the levels of the two fragments. D. Relative protein levels (α-syn/β-tubulin) of BiFC proteins determined by quantification of the respective bands in (D) using Image Studio Lite. Data are shown as the mean of three independent experiments, each performed as technical duplicates (error bars represent the S.D.). E. Schematic of bidirectional inducible plasmid for expression of Vn-α-syn & α-syn-Vc under the control of a single tetracycline promoter. Immunoblot showing expression of both Vn-α-syn & α-syn-Vc upon addition of 1 μg /mL doxycycline. Samples were boiled prior to SDS-PAGE. No relevant significant difference was observed between the tested conditions using one-way ANOVA followed by a Tukey test. Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length)

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Construct, Transfection, Western Blot, Plasmid Preparation, Expressing, SDS Page

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. Size exclusion chromatography of a HEK-293 cell lysate 48 h post-transfection with the indicated α-syn constructs. The SEC fractions were analyzed by WB using anti-α-syn (Syn-1) antibodies. B. Calculated apparent MW depicted with a log scale plotted against the Kav value of each α-syn construct (large colored dots). Mixed circles represent either Vn-α-syn (red) or α-syn-Vc (blue) when being co-expressed. C. Standard curve generated using the indicated six reference proteins. D. Working model suggesting that the high propensity of Vn-α-syn to oligomerize and how such oligomers could interact with or entrap α-syn-Vc. Note, the UV280 signal in A does not correspond to the α-syn signal, but mostly proteins in the remaining cell lysate. Representative data shown from n=3 experiments. Vo: dead volume; Ve: elution volume; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length).

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Size-exclusion Chromatography, Transfection, Construct, Generated

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. Living HEK-293 cells were crosslinked by the addition of DSG 48 h post transfection with the indicated α-syn constructs, and the resulting cell lysate was analysed by Western blotting against α-syn using the Syn-1 antibody. All analysed α-syn proteins displayed higher MWs upon addition of the DSG crosslinker, except for α-syn-Vc. B. The physiological dimer DJ-1 was used as an internal control so that the correct crosslinker concentration was applied. DJ-1 immunoblotting confirms that dimers are only detected upon crosslinking. C. Immunoblot of DSG crosslinked to live SH-SY5Y cells following 24 h BacMam delivery of either Vn-α-syn or α-Syn-Vc or both constructs. All displayed samples treated with DSG. Cells treated with only Vn-α-syn displayed higher order crosslinked oligomeric species in addition to monomeric Vn-α-syn. Cells treated with only α-syn-Vc displayed only monomeric α-syn-Vc. Treatment with both Vn-α-syn and α-Syn-Vc displayed higher order crosslinked oligomeric species in addition to both monomeric proteins. To determine if the higher order crosslinked oligomeric species in the sample treated with both Vn-α-syn and α-Syn-Vc were comprised of both proteins a separate WB for the FLAG epitope present only in the α-Syn-Vc protein was performed, this revealed the higher order crosslinked oligomeric species were predominantly comprised of Vn-α-syn. EV: empty vector; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length). Blots are representative of n=3 experiments.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Transfection, Construct, Western Blot, Concentration Assay, FLAG-tag, Plasmid Preparation

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: A. HEK-293 cells overexpressing distinct α-syn constructs were lysed 48 h after transfection, and the cell lysates were separated into a soluble and insoluble fraction by centrifugation. The resulting fractions were analysed by immunoblotting of α-syn (Syn-1). GAPDH was used as the loading control. Shown is a representative blot of n=3 experiments. B. Resulting soluble and insoluble band intensities of were quantified and normalized to soluble GAPDH. Significant differences were indicated (p<0.005) by one-way ANOVA followed by a Tukey test. C. Ratio of insoluble to soluble α-syn of Vn-α-syn and α-syn. Vc EV: empty vector; Vn-α-syn: (amino terminal fraction of Venus)-α-syn; α-syn-Vc: α-syn-(carboxy terminal fraction of Venus); α-syn-V (α-synuclein-Venus full length).

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Construct, Transfection, Centrifugation, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: HEK-293 cells expressing the indicated α-syn constructs were fixed 48 h post-transfection and stained with the Syn-1 antibody. Upon co-transfection of the Vn-α-syn and α-syn-Vc constructs, Venus was successfully reconstituted and emitted a fluorescent signal that colocalized with the signal for α-syn. Mouse primary neurons were treated by PFF α-syn for 14 days and are positive for pS129. The nucleus was counterstained with DAPI. The scale bar is 20 μm in all the non-zoomed images and 10 μm for the zoomed images. The microscopy settings were kept the same among each construct. Shown are representative images from three independent experiments.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Expressing, Construct, Transfection, Staining, Cotransfection, Microscopy

Journal: bioRxiv

Article Title: Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

doi: 10.1101/2020.05.02.074161

Figure Lengend Snippet: HEK-293 cells expressing the indicated α-syn constructs were fixed 48 h post-transfection and stained with the Syn-1 antibody. Upon co-transfection of the Vn-α-syn and α-syn-Vc constructs, Venus was successfully reconstituted and emitted a fluorescent signal that colocalized with the signal for α-syn. Mouse primary neurons were treated by recombinant pre-formed-fibrils α-syn for 14 days and are positive for the amytracker. The nucleus was counterstained with DAPI. MAP2 was used as a neuronal marker for the primary neurons and also colocalize with the amytracker. Both α-syn-V and the reconstituted BiFC fragments showed a nuclear localization and signal that did not colocalize with that of α-syn (Syn-1) The scale bar is 20 μm in all the non-zoomed images and 10 μm for the zoomed ones. Shown are representative images from two independent experiments. The microscopy settings were kept the same among each construct.

Article Snippet: Induced expression of the bidirectional plasmid required the use of HEK 293 Tet-On ® 3G cells (Takara Clontech Cat# 631182).

Techniques: Expressing, Construct, Transfection, Staining, Cotransfection, Recombinant, Marker, Microscopy

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Steady-state mRNA levels of firefly luciferase variants. Shown is a northern blot, probing for the common 3’UTR within different firefly luciferase reporter mRNAs, which vary by optimal codon content, and U6 snRNA loading control. Quantification of Firefly luciferase mRNA (relative to 0% optimal construct) is shown at bottom (error bars denote standard deviation; n=3). (B) More optimal luciferase mRNA variants are more stable. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and firefly luciferase mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline, which shut-offs transcription of the reporter. t½ corresponds to the half-life (min) ± standard deviation (n=3). See for loading control. (C) More optimal MECP2 mRNA variants are more stable. As in B, expect for MECP2 . See for loading control. (D) More optimal CFTR ΔF508 mRNA variants are more stable. As in B, except for CFTR ΔF508. See for loading control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Luciferase, Northern Blot, Construct, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Schematic of the ORFeome workflow. The ORFeome collection contains ~16,000 full-length coding sequences corresponding to 14,000 genes in a lentiviral background. Each ORF derived from the ORFeome is flanked by invariant UTRs, and also contains a C-terminal V5 tag. Lentiviral pools containing ~3000 ORFeome clones were used to infect HEK293T and generate stable cell lines. Metabolic labeling was used to determine stabilities of endogenous and ORFeome-derived mRNAs in these stable lines. (B) Changing coding regions changes mRNA stability. Plotted are boxplots of half-lives for endogenous HEK293T (End.) and ORFeome-derived mRNAs (in blue and gray, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) ORFeome mRNAs show as much variation in stability as endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T and ORFeome-derived mRNAs (in blue and gray, respectively). (D) Treatment with 4EGI-1 inhibits translation. Shown are A 254 traces from sucrose density gradients of cell lysates from HEK293T cells treated with the translation inhibitor 4EGI-1 (orange) or DMSO (grey). (E) Transaltion inhibition destabilizes endogenous mRNAs. Plotted are boxplots of half-lives for endogenous HEK293T mRNAs with DMSO or 4EGI-1 treatment (in grey and orange, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (F) Translation inhibition has a minor effect on the variation in stability for endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T in cells treated with DMSO or 4EGI-1 (in gray and orange, respectively). (G) Inhibition of translation destabilizes ORFeome-derived mRNAs and reduces the variation in stability. As in E, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. (H) Translation inhibition reduces the variation in stability for ORFeome-derived mRNAs. As in F, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. See also and Table S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Clone Assay, Stable Transfection, Labeling, Inhibition

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) ORFeome complexity was maintained through stable cell line generation. Shown is a western blot probing lysates from the pooled ORFeome stable lines with V5 (the common C-terminal tag in the ORFeome collection). WT, parental HEK293T line; S, supernatant; P, pellet. (B) ORFeome-derived mRNAs are expressed in the stable cell lines. Shown is a scatter plot comparing steady-state RNA-seq reads (with a +1 pseudocount) for each gene between the two pooled lines used in this study. In black, genes in neither pool; in blue, genes in pool 1; in orange, genes in pool 4; in green, genes in both pools. Red dashed lines represents y = 3X and y = X/3, which were used as cut-offs to classify genes as ORFeome-expressed. ORFeome genes that did not pass threshold were not used for subsequent analysis (see Methods for more details). Numbers refer to the total number of genes in each pool and the number passing the 3-fold threshold. (C) ORFeome mRNAs are expressed in a pool-dependent fashion. Shown are boxplots of normalized read counts (with a +1 pseudocount) for ORFeome-derived mRNAs (split into pool 1 and pool 4) in the two pooled stable cell lines. Abundance in cell line 1 is shown in blue; in cell line 4, in orange. Note that the ORFeome pools are expressed in the appropriate cell line. (D) 4EGI-1 substantially reduces translation. Cells were treated with DMSO, 4EGI-1, or cyclohexamide (as a positive control) for the indicated times and then briefly treated with puromycin, which is incorporated into nascent peptides. Lysates were separated by gel electrophoresiss and probed for puromycin (top) or tubulin (bottom). (E) DMSO treatment does not substantially affect mRNA stability. Shown are scatterplots comparing half-lives for endogenous genes (averaged from both pools) from the original experiment and DMSO-treated cells. Red dashed line represents x = y. (F) 4EGI-1 treatment affects mRNA stability. As in E, except comparing half-lives from the original experiment and 4EGI-1-treated cells.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Stable Transfection, Western Blot, Derivative Assay, RNA Sequencing Assay, Positive Control

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Endogenous mRNA stability negatively correlates with length. Shown are boxplots for half-lives of endogenous HEK293T mRNAs binned into quartiles by ORF length. (B) ORFeome mRNA stability does not correlate with length. As in A, except for ORFeome mRNAs. (C) Endogenous mRNA stability weakly correlates with local secondary structure. For each ORF, the folding energy in 100 bp sliding windows was calculated, and the minimum value taken. Shown are boxplots for half-lives of endogenous HEK293T binned into quartiles by folding energy (with increased secondary structure on the right). (D) ORFeome mRNA stability does not correlate with local secondary structure. As in B, except for ORFeome mRNAs. (E) microRNA-mediated regulation cannot explain the variation in ORFeome stability. ORFs were classified as containing or lacking seed-matched sites for the top five expressed mRNAs (site ORFs [orange] and no site ORFs [blue], respectively). Shown are boxplots for their half-lives. Significance was calculated by the Kolmogorov-Smirnov test. (F) AU-rich elements cannot explain the variation in ORFeome stability. As in E, except for AU-rich elements. (G) AU-rich elements in ORFs destabilize mRNAs upon translational repression. As in F, except for half-lives determined in the presence of 4EGI-1.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codons are differentially associated with stability. Shown are spearman correlations, for each codon, of their frequency with mRNA stability (codon stability coefficient; CSC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. The 15 codons most associated with stability (as defined by the ORFeome collection) were designated as “optimal” (blue), while 15 codons most associated with instability were designated as “non-optimal” (orange). (B) HeLa and HEK293T cells have similar codon stability coefficients (CSCs). Plotted are the CSC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (C) As in B, except comparing endogenous HEK293T and ORFeome-derived CSC values. (D) ORFeome mRNAs with more optimal codons are more stable. Shown are boxplots of mRNA half-lives for ORFeome mRNAs, binned into quartiles by the frequency of optimal codons. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (E) As in D, except for endogenous HEK293T mRNAs. (F) Endogenous HEK293T CSCs weakly correspond with pause scores. Using HeLa ribosome profiling, pause scores were calculated for each codon in the A site, and then codons were divided into three groups (slow in orange; neutral in green; fast in blue). Shown are boxplots for the corresponding CSC values as determined by endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived CSCs. See also .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codon stability coefficients (CSCs) have little relationship to tRNA abundance. Plotted are the normalized tRNA abundance in comparison to CSC values for endogenous HEK293T and and ORFeome mRNAs (left and right panels, respectively). (B) tRNA abundance has little impact on elongation speed. Codons were divided into thirds by their A-site pause scores (slow in orange; neutral, green; fast, blue). Shown are boxplots for the abundance of corresponding tRNAs. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) Amino acids are differentially associated with stability. Shown are spearman correlations, for each amino acid, of their frequency with mRNA stability (amino acid stability coefficient or AASC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. Polar amino acids (in pink) have charged or highly electronegative side chains; nonpolar amino acids (dark gray) have aliphatic and weakly electronegative side chains. (D) HeLa and HEK293T have similar AASCs. Plotted are the AASC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (E) As in B, except comparing endogenous HEK293T and ORFeome-derived AASC values. (F) Hydrophobic amino acids are associated with stability. Plotted are the hydrophobicity scores for each amino acid compared to their stability coefficient for endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived AASC values. (H) Schematic diagram of LSM8 reporter constructs. In the middle of the LSM8 coding region, five repeats of instability-associated amino acids (S, H and E) or stability-associated amino acids (V, I, and L) were inserted. (I) Insertion of instability-associated amino acids destabilizes the LSM8 reporter mRNA. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and LSM8 mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline. t½ corresponds to the half-life (min) ± standard deviation (n=4). See for loading control. (J) The destabilized LSM8 reporter mRNA has shorter poly(A) tails. High resolution northern blotting was performed to measure poly(A)-tail lengths on the SHE and VIL LSM8 mRNAs. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. (K) LSM8 reporter mRNAs are deadenylated. Transcription of the SHE and VIL LSM8 reporters was shut-off, as in I, and poly(A)-tail lengths were measured by high-resolution northern blotting. Timepoints represent time elapsed after transcription shutoff with 2 μg/mL doxycycline. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Construct, Northern Blot, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Valine frequency correlates with mRNA stability. Shown are boxplots of mRNA stabilities for HeLa, endogenous HEK293T, and ORFeome mRNAs binned into quartiles by valine frequency. (B) Serine frequency negatively correlates with mRNA stability. As in A, except for serine. (C) U6 snRNA northern analysis for transcription shut-off experiments for the LSM8 variants shown in . (D) U6 snRNA Northern analysis for LSM8 variable amino acid content transcription shutoff/Northern mRNA decay analyses shown in .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Northern Blot

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Amino acids, when in the A-site, are translated at different rates. Plotted are the pause scores for each amino acid when in the predicted A-site (see methods for details). Nonpolar amino acids, grey; polar amino acids, pink. (B) Amino acid stability coefficients correlate with A-site pause scores. Shown are plots comparing A-site pause scores for each amino acid with its stability coefficient, as defined by endogenous HeLa, endogenous HEK293T, and ORFeome mRNAs (left, middle, and right, respectively). (C) As in A, except for the P site. (D) As in A, except for the E site. (E) A-site pause scores correlate poorly with P- and E-site pause scores. Plotted are the pairwise comparisons for A-, P-, and E-site pause scores. (F) ORFeome amino acid stability coefficients poorly correlate with P-site pause scores. Shown are plots comparing P-site pause scores for each amino acid with its stability coefficient, as defined by ORFeome mRNAs. (G) As in F, except for E-site pause scores.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: For each pair of codons, the absolute difference in the corresponding CSC values was calculated and then normalized to the maximal difference (to correct for differences in overall variance between organisms). Pairs of codons were binned into these encoding the same or different amino acid (n = 87, in grey, and n = 1742, in green, respectively). Shown are boxplots of those differences for S. pombe , trypanosomes, zebrafish, and endogenous HEK293T mRNAs. Significance determined by Wilcoxon rank sum test.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a Representative images of hMC4R GFP+ HEK 293 cells (green) 30 min after application of DyLight 550®-labeled (red) α-MSH affinity-purified IgG from eating disorder (anorexia nervosa, n = 9; bulimia nervosa, n = 7; binge eating disorder, n = 7), obese ( n = 10), and Ctrl ( n = 9) preincubated or not with α-MSH. Quantification of DyLight 550®-positive spots in hMC4R+ HEK 293 cells ( n = 50/group): b at the membrane; c intracellularly (cytosolic), and d ratios of cytosolic/membrane staining. Affinity kinetics properties of α-MSH/IgG IC for hMC4R + HEK 293 cells including e dissociation equilibrium constant (KD); f association rate (ka), and g dissociation rate (kd). Data are means ± s.e.m. Kruskal–Wallis test with Dunns’ post-tests ( b – d , f , g ) or analysis of variance with Tukey’s post-test ( e ), *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Labeling, Affinity Purification, Staining

Journal: Translational Psychiatry

Article Title: Immunoglobulin G modulation of the melanocortin 4 receptor signaling in obesity and eating disorders

doi: 10.1038/s41398-019-0422-9

Figure Lengend Snippet: a cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC formed by IgG pooled in patents and control groups and adjusted to α-MSH-reactive IgG plasma levels of controls. cAMP dose–response curves to α-MSH preincubated or not with individual total IgG and corresponding EC50 ( b ) and maximal cAMP production ( c ). d Control experiments including cAMP dose–response curves to α-MSH by MC4R-expressing and non-expressing control HEK 293 cells and to α-MSH 1–4 peptide by MC4R-expressing cells ( n = 4). e cAMP dose-response curves to α-MSH and IgG from patients and controls without their overnight pre-incubation. f , cAMP dose–response curves to α-MSH alone or α-MSH/IgG IC co-administered (solid line) with agouti-related protein (AgRP; 100 nM) or added after AgRP preincubation (dotted line— n = 2/group) as well as in g cAMP maximal response. h , i cAMP dose–response curves of α-MSH preincubated with h purified total IgG from patients and controls depleted for α-MSH-reactive IgG ( n = 3/group) and i affinity-purified α-MSH-reactive IgG ( n = 6/group); j EC 50 and k maximal cAMP production at the plateau. Data are means ± s.e.m. Analysis of variance with Tukey’s post-test ( b , c , g , j ) or Kruskal–Wallis test with Dunns’ post-tests ( m ), *** p < 0.001, ** p < 0.01, * p < 0.05; Mann–Whitney test, $ p < 0.05. a α-MSH ( n = 9), Ctrl, anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED; n = 6), obese (OB; n = 4); d HEK 293-hMC4R+ ( n = 5), HEK 293-CTRL ( n = 6); e α-MSH ( n = 3), Ctrl, BN, and BED ( n = 2), AN and OB ( n = 3)

Article Snippet: MC4R-expressing HEK 293 cells (AMS Biotechnology, Abingdon, UK) were cultured in glass bottom Petri dishes (MatTek, ≈250,000 cells/dish).

Techniques: Expressing, Incubation, Purification, Affinity Purification, MANN-WHITNEY

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of the preparatory and analytical workflows. (A) POMC derived ligands and their downstream signaling cascades. (B) Schematic overview of the thermal proteome profiling (TPP) workflow. MC3R-expressing HEK293 cells were treated with ACTH, α-MSH, or γ-MSH at concentrations of 20, 100, and 500 nM or with DMSO as a vehicle-only negative control. (C) Schematic overview of the TPP data analysis workflow. Protein identification and relative quantification were achieved by direct analysis of the raw LC–MS data, after which various bioinformatics tools were used to infer changes in transcription factor (TF) activity, perform enriched pathway analysis, and identify thermally affected proteins.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Derivative Assay, Expressing, Negative Control, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Overview of identified proteins and thermally stabilized or destabilized proteins. (A) Venn diagrams showing the numbers of proteins exhibiting altered melting points, associations with enriched pathways, and phosphorylation in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH. (B) Venn diagrams showing the numbers of stabilized, destabilized, and phosphorylated proteins after incubation with ACTH, α-MSH, and γ-MSH. (C) Upset plot representing individual numbers of stabilized and destabilized proteins for each ligand and those common between various combinations of ligands.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Phospho-proteomics, Expressing, Incubation

Journal: Analytical Chemistry

Article Title: Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

doi: 10.1021/acs.analchem.3c03643

Figure Lengend Snippet: Characterization of transcription factors. (A) Heat map showing the relative abundance (compared to vehicle-only controls) of the transcription factors CCAR2, HMGB2, DDX21, SRSF7, and TET2 in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, and γ-MSH at different ligand concentrations and temperatures. (B) Phosphorylation of tryptic peptides derived from the thermally stabilized and destabilized transcription factors shown in panel A whose activity was inferred to change following stimulation with ACTH, α-MSH, or γ-MSH. Phosphorylation sites are indicated by asterisks next to the modified amino acid (shown in parentheses when the exact amino acid is unknown). (C) Transcription factor activities and relational networks inferred from differential expression data using BITFAM. The heatmap shows fold changes in transcription factor activities (relative to vehicle-only treatments) in MC3R-expressing HEK293 cells incubated with ACTH, α-MSH, or γ-MSH. (D) Network showing the interconnectivity of the transcription factors identified within our experimental LC–MS data set.

Article Snippet: A human embryonic kidney 293 cell line transfected with a tetracycline-regulated expression system to overexpress MC3R (HEK-TREx-MC3R) was provided by Astra Zeneca.

Techniques: Expressing, Incubation, Phospho-proteomics, Derivative Assay, Activity Assay, Modification, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number in gene reporter assays. pGL3-Promoter-derived constructs containing PTCH1b 5'UTRs (188 or 300 nucleotides) fused to the Firefly luciferase (Fluc) were transiently transfected in HCT116 p53+/+ (A), MCF7 (B) and HEK 293T cells (C). Presented are the average fold inductions relative to the empty vector and standard deviations of at least 3 biological replicates. The luciferase values are expressed relative to the value obtained with an empty control vector. The same constructs were used to measure relative Fluc mRNA transcript levels in transiently transfected HCT116 p53+/+ (D), MCF7 (E) and HEK 293T cells (F). Presented is the average Fluc mRNA expression normalized for the average plasmid copy number and for relative cDNA synthesis efficiency as revealed by the amplification of GAPDH and B2M mRNAs. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Control, Expressing, cDNA Synthesis, Amplification

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number on cap-independent translation. Bicistronic pRuF-derived constructs were used to study the potential of the PTCH1b 5'UTR to drive cap-independent translation of the Firefly luciferase gene in transiently transfected MCF7 (A) or HEK 293T cells (B). Presented are the average ratios and standard deviations between Firefly (Fluc) and Renilla luciferase (Rluc) relative light units, normalized with the results obtained for empty pRuF vector, and standard deviation of at least 3 replicates. The PTCH1b 5'UTR size and CGG-repeat number are indicated. (C, D) The bicistronic nature of the transcript expressed by pRuF constructs was estimated measuring the Fluc/Rluc mRNA ratio by a qPCR approach. A ratio equal to 1.0 would strongly argue against the presence of a cryptic promoter within cloned PTCH1b 5'UTR resulting in a monocistronic Fluc mRNA transcript. A pRuF-type plasmid with cloned c-MYC 5'UTR was used as a positive control. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Standard Deviation, Clone Assay, Positive Control

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: An internal ribosome entry site (IRES) motif maps in the 3' end of the PTCH1b 5'UTR. (A) Putative PTCH1b IRES motif cloned into pRuF-type plasmid is not sufficient to obtain Firefly luciferase (Fluc) activity observed with the complete PTCH1b 5'UTR in MCF7 cells, while the remaining part of PTCH1b 5'UTRs (188ΔIRES and 300ΔIRES) retains certain levels of IRES activity. (B) Similar results were obtained in HEK 293T cells. (C) The ratios between Fluc and Renilla luciferase (Rluc) mRNA for remodeled pRuF PTCH1b 5'UTR plasmids in transfected MCF7 cells didn't deviate from 1.0. (D) The same results were obtained in HEK 293T cells. Results are presented as described in Figure 4.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Hypoxia enhances PTCH1b 5'UTR mediated translation. MCF7 and HEK 293T cells transiently transfected with the different pRuF reporter constructs were grown in normoxic (dark gray) or severe hypoxic (light gray) conditions for 16 hours. Luciferase assays were performed as described in Materials and Methods and shown as those presented in Figure 5.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Construct, Luciferase

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Higher PTCH1b mRNA relative translation efficiency during hypoxia in HEK 293T cells overexpressing GLI1. HEK 293T cells were transfected with an empty vector or a construct overexpressing GLI1 and then cultured in normoxia or hypoxia for 16 hours (48 hours post transfection). (A). Total RNA levels of the indicated mRNAs, quantified by qPCR as described in the method section and presented as relative expression compared to the reference genes (ΔCq). c-MYC mRNA was included as an example of mRNA whose 5'UTR is considered to possess IRES activity, while PCNA was included as negative control for IRES function. Error bars plot the average and standard deviations of 3 replicates (B). Cytoplasmic lysates of HEK 293T cells transfected and cultured as for panel A, were separated by sucrose-gradient equilibrium density; subpolysomal (SUB) and polysome-associated (POL) mRNAs were identified and collected by UVC scanning and the indicated transcripts were quantified by qPCR, as for panel A. (C). The results from panel B, are also plotted as ratio between polysome-associated and subpolysomal mRNAs as a function of the different treatment. This ratio is considered an estimate of relative mRNA translation efficiency. (D). Global relative mRNA translation rates in HEK 293T cells cultures in normoxia or hypoxia for 16 hours were assessed by incorporation of an immune-detectable methionine analog, as described in Materials and Methods. (E). Western blot analysis of PTC1-L protein and PCNA. Transfection of the GLI1 overexpression plasmid and treatment are indicated. Beta-tubulin was used as reference protein.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Plasmid Preparation, Construct, Cell Culture, Expressing, Activity Assay, Negative Control, Western Blot, Over Expression