Journal: bioRxiv

Article Title: A binary effector module secreted by a type VI secretion system

doi: 10.1101/2021.07.29.453783

Figure Lengend Snippet: A) Toxicity of periplasm-targeted proteins in E. coli . E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide ( peri sfGFP, peri VP1388, and peri VP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.1% arabinose, to induce protein expression. B) VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δ vp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. C) VP1390 induces cell lysis in E. coli . Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 ( peri sfGFP, peri VP1388, and peri VP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 μm.

Article Snippet: Next, 500 μL of supernatant were transferred to tubes containing 25 μL of prewashed magnetic α-Myc beads (Myc-tag [9B11] mouse mAb magnetic beads conjugated #5698; Cell Signaling Technology) and incubated at 4°C for 2 hours.

Techniques: Expressing, Control, Immunoprecipitation, Lysis, Time-lapse Microscopy, Plasmid Preparation

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: SARS-CoV-2 spike-transfected cells mimic viral envelope for fusion with ACE2 + target cell membrane. ( A ) Schematic representation of the fusion process for SARS-CoV-2 and a potential target for fusion inhibitors. See text for detailed description of the fusion process. FP, fusion peptide; HR, heptad repeat domain; 6-HB, 6 helix bundle; FI, fusion inhibitor. ( B ) Schematic representation of a split neongreen fusion assay (figure adapted from ). A549.ACE2 + cells (transfected to express the first 10 betasheets of neongreen) were overlayed with HEK293T cells co-transfected with a plasmid encoding the SARS-CoV-2 spike protein and a plasmid encoding the 11 th betasheet of neongreen. Only cell-cell fusion of an A549 cell with a HEK293T cell will result in the assembly of a functional neongreen protein and give a green fluorescence signal as the former expresses spike and the latter human ACE2. Light microscopic picture shows fused cells with neongeen expression (20x magnification). ( C ) Same as in (B). A549.ACE2 + cells were overlayed with HEK293T cells either transfected (TF) with an empty vector (left panels; mock-TF), or with Wuhan-Hu-1 S protein and left untreated (middle) or treated with the fusion inhibitor EK1 (2 μM; right panels). Light microscopic pictures were taken at 3 and 12 hours post overlay (20x magnification). Note that cell-cell fusion in the untreated spike-transfected condition is already visible at 3h post overlay but that neongreen fluorescence is still absent. Cartoons were created with BioRender ( www.biorender.com ).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Single Vesicle Fusion Assay, Plasmid Preparation, Functional Assay, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Comparison of impedance signal of A549.ACE2 + cells overlayed with mock-transfected versus Wuhan-Hu-1 SARS-CoV-2 spike-transfected HEK293T cells. At time point 0, A549.ACE2 + cells were seeded and impedance was recorded of the proliferating cell monolayer. At 24h post plating (phase #1), empty vector- (grey) and spike-transfected (blue) HEK293T cells were added. The graph depicts the raw impedance signal (expressed as cell index) over time of 4 technical replicates (mean ± SD). Vertical dotted lines 1 to 3 indicate important phases, which are further explained in the text. Note the bigger variation in CI response between the replicates during the disruption of the cell monolayer (starting at phase #2).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: SARS-CoV-2 spike expression correlates with the intensity and kinetics of impedance signal in CEI quantified cell-cell fusion assay. ( A ) Different ratios of A549.ACE2 + acceptor (A) and trypsinized Wuhan-Hu-1 spike-transfected HEK293T donor (D) cells. In the 1:1 cell ratio, 15,000 cells of acceptor and donor were used. The graph depicts the impedance signal (expressed as cell index) over time, starting at the moment of cell overlay, of 4 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). ( B ) Different amounts of Wuhan-Hu-1 SARS-CoV-2 S expressing plasmid DNA (as indicated) were added to 200 μl transfection mixture for the transfection of 400,000 HEK293T donor cells. The next day, cells were trypsinized and added to an A549.ACE2 + acceptor cell monolayer. The graph depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms at the right show the background-substracted mean fluorescence intensity (MFI) values (on a logarithmic scale) for cell surface S staining (Ab R001) of the transfected cells by flow cytometry. See also Supplementary Figure 1D for corresponding flow cytometric histogram plots. ( C ) Comparison of impedance signal of A549.ACE2 + cells either mock-transfected (dark blue) versus TMPRSS2-transfected (light blue) and overlayed by trypsinized Wuhan-Hu-1 SARS-CoV-2 S transfected HEK293T cells. The graph depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the corresponding mock-transfected HEK293T condition (grey horizontal curve).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Expressing, Cell-Cell Fusion Assay, Transfection, Plasmid Preparation, Fluorescence, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Optimization of CEI-measured S-induced cell-cell fusion. ( A ) Cell-cell fusion depends both on the expression of ACE2 on A549 cells and SARS-CoV-2 S protein on transfected HEK293T cells. Different combinations of acceptor and donor cells were tested as indicated. The graph depicts the impedance signal of 4 technical replicates (mean ± SD). Bar histograms at the right represent the cell index value at 8:43h post overlay, when the maximum was reached in the positive control. Note that no increase in impedance signal (CI max ∼ 0) was obtained in the conditions in which ACE2 and/or spike were not (over)-expressed. ( B ) Comparison of impedance signal of A549.ACE2 + cells overlayed with SARS-CoV-2 Wuhan Hu-1 S-transfected HEK293T cells, either trypsinized or collected by resuspending. The graph depicts the impedance signal of 2 technical replicates (mean ± SD). ( C ) HEK293T cells were transfected with SARS-CoV-2 Wuhan Hu-1 S. After 6h, transfection reagent was removed and cells were incubated either at 34°C or 37°C for 18h. S-expressing cells were then trypsinized, collected and administered to a A549.ACE2 + cell monolayer, and further incubated at 37°C for the CEI measurement. The graph depicts the impedance signal of 2 technical replicates (mean ± SD). ( D ) Flow cytometric histogram plots of the samples presented in (see figure legend to for experimental details). HEK293T cells were collected 24h post transfection, stained with anti-S Ab (R001) and an AF647-labeled secondary Ab. Of each sample 10,000 cells were analyzed on a FACSCelesta to calculate the mean fluorescence intensity (MFI) value. The grey histogram represents the stained mock-transfected background control sample, whereas the S-transfected cells are indicated in blue. The values in color refer to the respective MFI value.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Expressing, Transfection, Positive Control, Incubation, Staining, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Validation of CEI cell-cell fusion assay with entry inhibitors of SARS-CoV-2. ( A ) The fusion inhibitor EK1 inhibits cell-cell fusion of A549.ACE2 + acceptor with S-transfected (20A.EU2 strain) donor cells. Inhibitor and donor cells were added simultaneously to the A549.ACE2 + acceptor cells. ( B ) Same as in (A) but for the attachment inhibitor R001, an RBD binding antibody that neutralizes viral entry of authentic SARS-CoV-2 virus, and with SARS-CoV-2 Wuhan-Hu-1 S. ( C ) Same as in (A) but for the entry inhibitor UDA, a carbohydrate-binding small monomeric plant lectin from stinging nettle rhizomes. The graphs on the left depict the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms on the right show the inhibition of impedance response relative to the untreated control sample, calculated from the maximum CI values obtained for each treated sample.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Cell-Cell Fusion Assay, Transfection, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Validation of CEI cell-cell fusion assay with entry inhibitors of SARS-CoV-2. ( A ) Fusion inhibitor EK1 inhibits concentration-dependently the cell-cell fusion of S-transfected (Wuhan-Hu-1 strain) HEK293T donor with A549.ACE2 + acceptor cells. Inhibitor and donor cells were added simultaneously to the A549.ACE2 + acceptor cells. The graph on the left shows the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition. Concentration-response curve on the right shows the inhibition of impedance response relative to the untreated control sample, calculated from the CI values obtained at the time point when maximum CI was reached in the positive control. The calculated 50% inhibitory concentration (IC 50 ) is given in the boxed insert. ( B ) The S-binding attachment inhibitor Ab R001 delays the the cell-cell fusion of S-transfected (Wuhan-Hu-1 strain) HEK293T donor with A549.ACE2 + acceptor cells transiently transfected with TMPRSS2. R001 (10 μg/ml) and donor cells were added simultaneously to the A549.ACE2 + .TMPRSS2 + acceptor cells. The graph shows the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition. Bar histograms on the right show the maximum CI values (mean ± SD; n=2). ( C ) RBD peptide from SARS-CoV-2 Wuhan-Hu-1 S delays the cell-cell fusion of S-transfected (Wuhan-Hu-1) HEK293T donor with A549.ACE2 + acceptor cells. RBD (81 nM) and donor cells were added simultaneously to the A549.ACE2 + acceptor cells (red curve). In parallel, RBD (81 nM) was administered to a monolayer of A549.ACE2 + cells in the absence of spike-expressing cells to measure the (small) morphological changes induced by RBD binding to the ACE2 receptor (green curve). The graph shows the impedance signal of 4 technical replicates (mean ± SD), normalized to the respective mock-transfected or untreated condition. Bar histograms on the right show the maximum CI values (mean ± SD; n=4).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Cell-Cell Fusion Assay, Concentration Assay, Transfection, Inhibition, Positive Control, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Plant lectin UDA inhibits CEI quantified cell-cell fusion through binding to SARS-CoV-2 S. ( A ) A monolayer of A549.ACE2 + cells were pretreated with UDA (2μM) for 1h at 37°C, washed and overlaid with S-transfected (Wuhan-Hu-1) HEK293T cells without additional compound administration. ( B ) At 24h post transfection, S-transfected (Wuhan-Hu-1) HEK293T cells were first pretreated with UDA (2μM) for 1h at 4°C, trypsinized, collected and washed. Cells were resuspended in culture medium and overlaid on a monolayer of A549.ACE2 + cells without additional compound administration. Graph show the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Binding Assay, Transfection

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: CEI measures the alteration in fusogenic potential of SARS-CoV-2 S variants. ( A ) Comparison of impedance signal of A549.ACE2 + cells overlayed with HEK293T cells transfected with plasmid DNA coding for SARS-CoV-2 S either from Wuhan-Hu-1, carrying D614 (blue) or a mutant with G614 (red) as found in the Nextstrain clade 20A and its descendants. In both conditions, 2.5 μg S expressing plasmid DNA was added to 200 μl transfection mixture for the transfection of 400,000 HEK293T donor cells. Graph on the left depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms on the right show the maximum CI values (mean ± SD). ( B ) Same as in (A) but for the comparison between Wuhan-Hu-1 and Omicron. ( C ) Fusion-inhibitory effect of UDA (2 μM) on different N-glycosylation deletion mutants. Mutants of Wuhan-Hu-1 S that contained two deletions of adjacent N-glycosylation sites in the S2 subunit were generated (by N into Q conversion) and analyzed in a CEI-based cell-cell fusion assay for their sensitivity to UDA. Graphs show the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Generated, Cell-Cell Fusion Assay

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: CEI measures the alteration in fusogenic potential of SARS-CoV-2 S variants. ( A ) Same as in but with transfection of Omicron S (BA.1 variant). ( B ) Transfected HEK293T samples (each with 2.5 μg plasmid DNA) from were collected 24h post transfection, stained with anti-S Ab (R001) and an AF647-labeled secondary Ab. Bar histograms represent the background-corrected mean fluorescence intensity (MFI) values (on a logarithmic scale), calculated from 10,000 cells analyzed by a FACSCelesta flow cytometer. ( C ) Untreated control samples from were plotted together in one graph to compare the fusion efficiency of N-glycosylation mutants of S. ( D ) Transfected HEK293T samples from (C) were collected 24h post transfection, stained with anti-S Ab (MM57) and an PE-labeled secondary Ab. Bar histograms represent the background-corrected MFI values (on a logarithmic scale), calculated from 10,000 cells analyzed by a FACSCelesta flow cytometer.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Variant Assay, Plasmid Preparation, Staining, Labeling, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: (A) Immunoblot of Nek2 at various timepoints between days 0-17 of RA treatment. (B) Densitometry of immunoblot in A. N=3. Bars represent mean values ±s.e.m. P-values were determined by One-way ANOVA using Tukey’s post-hoc analysis.

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: (A) Immunoblots of Nek2 in wildtype (WT), knockout (KO), knockdown (KD), pcDNA and hNek2-Myc transfected cells in the undifferentiated state. (B) Densitometry of immunoblots in A. Bars represent mean values ±s.e.m. P-values were determined by One-way ANOVA using Tukey’s post-hoc analysis. (C) Total cell counts of WT, KO, KD, pcDNA and hNek2-Myc transfected cells between 0-96 hours after plating in the undifferentiated state. Dots represent mean values ±s.e.m. P-values were determined by Two-way ANOVA using Sidak’s post-hoc analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Western Blot, Knock-Out, Transfection

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: (A) Area, and (B) Aspect ratio (major axis/minor axis of EB from images in D formed after 4 days of RA treatment in wildtype (WT), Nek2 knockout (KO) and knockdown (KD) cells, and pcDNA and pNek2-Myc transfected cells. Points represent individual EB. (C) Number of EB per image area from images in D. Points represent mean number of EB across all images per replicate. Lines across points represent mean values ±s.e.m. (D) Phase contrast images of EB. White dotted lines outline perimeter. Scale bar = 200 um. N=3. P-values were determined by One-way ANOVA with Tukey’s post-hoc analysis. *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Knock-Out, Transfection

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: RT-qPCR of pluripotency markers Oct4 and Nanog in untreated, undifferentiated (A) wildtype (WT), Nek2 knockout (KO) and knockdown (KD) cells, and (B) pcDNA and pNek2-Myc transfected cells. P-values were determined by One-way ANOVA with Tukey’s post-hoc analysis. Expression of Nanog in (C) WT and KO cells and (D) pcDNA and pNek2-Myc transfected cells during 0-17 days of RA treatment. Immunoblotting of ESRRB in (E) WT and KO cells during days 0-17 of RA treatment and (F) untreated pcDNA and pNek2-Myc transfected cells. P-values were determined by Two-way ANOVA with Sidak’s post-hoc analysis or by Student’s t-test. N=3. Bars represent mean values ±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Quantitative RT-PCR, Knock-Out, Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: (A) Immunoblot of neuron marker, β-III-tubulin, and astrocyte marker, GFAP, in wildtype (WT), Nek2 knockout (KO), pcDNA and pNek2-Myc transfected cells at various times during RA treatment. Densitometry of (B) β-III-tubulin and (C) GFAP from immunoblots in A). P-values were determined by Student’s t-test. (D) Immunofluorescence of GFAP (red), β-III-tubulin (green), and DAPI (blue) staining in WT and KO cells at day 17 of RA treatment. (E) RT-qPCR of neural stem cell marker Nestin in WT and KO cells during days 0-17 of RA treatment. P-values were determined by Two-way ANOVA with Sidak’s post-hoc analysis. N=3. Bars represent mean values ±s.e.m. *P<0.05, **P<0.01, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Western Blot, Marker, Knock-Out, Transfection, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: RT-qPCR of Hh target gene Gli1 in (A) wildtype (WT) and Nek2 knockout (KO) cells, and (B) in pcDNA and pNek2-Myc transfected cells during days 0-17 of RA treatment. RT-qPCR of Hh target gene Ptch1 in (C) WT and KO, and (D) pcDNA and pNek2-Myc transfected cells during days 0-17 of RA treatment. P-values were determined by Two-way ANOVA with Sidak’s post-hoc analysis. N=3. Bars represent mean values ±s.e.m. **P<0.01, ****P<0.0001.

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Quantitative RT-PCR, Knock-Out, Transfection

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: RT-qPCR of Wnt target gene Dkk1 in (A) wildtype (WT) and Nek2 knockout (KO) cells, and (B) in pcDNA and pNek2-Myc transfected cells during days 0-17 of RA treatment. RT-qPCR of Hh target gene Dab2 in (C) WT and KO, and (D) pcDNA and pNek2-Myc transfected cells during days 0-17 of RA treatment. P-values were determined by Twoway ANOVA with Sidak’s post-hoc analysis. N=3. Bars represent mean values ±s.e.m. **P<0.01, ***P<0.001, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Quantitative RT-PCR, Knock-Out, Transfection

Journal: bioRxiv

Article Title: Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

doi: 10.1101/2022.08.23.504995

Figure Lengend Snippet: Proteins identified through LC-MS that are (A) upregulated and (B) downregulated in KO cells compared to WT cells at days 0, 4 and 10 of RA treatment. GSEA enrichment analysis of LC-MS results identifying pathways that are (C) enriched in KO cells compared to WT cells and (D) in WT cells compared to KO cells at day 10 of RA treatment. (E) Immunoblot of electron transport chain components, SDHB, MTCO1, UQCRC2 and ATP5A corresponding to subunits in complexes II-V, respectively, in WT and KO cells at days 0 and 4 of RA treatment. (F) Densitometry analysis of immunoblots in E. P-values were determined by One-way ANOVA with Tukey’s post-hoc analysis. Phase contrast images and viability of cells determined through trypan blue exclusion in cells treated with (G) media only or 50 mM 2-DG, and (H) DMSO or 2.5 μM oligomycin A for 24 hours. Scale bar = 200 μm. P-values were determined by Two-way ANOVA with Sidak’s post-hoc analysis. (I) Immunoblot of HIF1α in untreated WT and KO cells. (J) Proposed mechanism of Nek2 action. Image created using BioRender. Bars represent mean values ±s.e.m. **P<0.01, ***P<0.001, ****P<0.0001 .

Article Snippet: Plasmids used include: 1436 pcDNA3 Flag HA (Addgene plasmid #10792) and pCMV3-human NEK2-Myc (HG10054-CM, Sino Biological).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot