Journal: bioRxiv

Article Title: The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models

doi: 10.1101/2021.01.10.426114

Figure Lengend Snippet: A. Schematic showing ACE2 tagged with LgBIT and SmBIT. B. HEK-293 cells were transfected with combinations of plasmids encoding LgBIT or SmBIT fused to either protein kinase A regulatory subunit (PRKAR2) or catalytic subunit (PRKACA), ATG5 or ACE2. The results (mean ± S.D., n = 5) were normalized to the luminescence measured in cells transfected with protein kinase A reporters (positive control). C. HEK-293 cells were transfected with plasmids encoding ACE2 nanoBIT reporters under the control of the HSV TK promoter and ACE2 or prolactin (PRL) under the control of the CMV promoter. The results (mean ± S.D., n = 4) were normalized to the luminescence measured in cells transfected with protein kinase A reporters and prolactin. D. HEK-293 cells were transfected with NanoBIT-tagged ACE2 reporters and incubated with sodium valproate or clofibrate at a concentration equal to 1x, 2x or 3x the reported C max of the drug. After 1 hour, luminescence was measured and normalized (mean ± S.D., n =4) to that measured in cells treated with DMSO. E. A series of other fibrates were similarly evaluated in the assay. The luminescence measured (mean ± S.D., n = 5-11, solid bars) was significantly different to that measured in cells treated with solvent where shown. When these fibrates were incubated with purified LgBIT and HiBIT-RBD to create a constitutively active nanoluc, each of these fibrates were found to inhibit nanoluciferase (bezafibrate 35 ± 7 %, ciprofibrate 55 ± 6 %, fenofibric acid 46 ± 3 %, fenofibrate 69 ± 5 %, gemfibrozil 61 ± 2 % of the activity measured in the presence of DMSO). To correct for this, the luminescence measurements from cells treated with fibrates in cells were divided by these latter values to estimate the effect of the drugs on dimerization (hatched bars). Significant difference from control is shown as *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Article Snippet: The plasmid pcDNA3 encoding ACE2 was obtained from GenScript (OHu20260); the plasmid encoding prolactin (PRL) was obtained from Sino Biological (HG10275-CY).

Techniques: Transfection, Positive Control, Incubation, Concentration Assay, Purification, Activity 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: 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

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. Cadherin-11 protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Staining, Invasion Assay, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Negative Control, Invasion Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: shRNA, Quantitative RT-PCR, In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Labeling, Invasion Assay, Quantitation Assay, In Vitro, Micro-MRI, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: 1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing, Luciferase, Over Expression, In Vivo, In Vitro