Journal: Cancer Medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma

doi: 10.1002/cam4.70190

Figure Lengend Snippet: The effect of SUZ12 on metastasis related genes expression was tested by qRT‐PCR and western blotting. sh‐SUZ12 increased MMP1/2/3/9 mRNA expression (A) and TIMP1/2 protein expression (B), while decreased MMP14 mRNA expression (A), MMP1/9/14 (B), TIMP3 (B), and ITGB1/5(F) protein expression, without significantly effected ITGBs mRNA expression (E). oe‐SUZ12 increased MMP14 mRNA (C) and protein (D) expression, while decreased TIMP2 mRNA expression (C) and TIMP1/2 (D) protein expression.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122‐1‐AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103‐1‐AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052‐1‐AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939‐1‐AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554‐1‐AP), p18 (1:1000, BOSTER China, cat. no. M03299‐1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283‐2‐Ig), p‐p53 (1:2000; Proteintech, USA, cat. no. 28961‐1‐AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl‐2 (1:1000; Proteintech, USA, cat. no. 26593‐1‐AP), Bax (1:2000; Proteintech, USA, cat. no. 50599‐2‐lg), E‐cadherin (1:5000; Proteintech, USA, cat. no. 20874‐1‐AP), N‐cadherin (1:3000; Proteintech, USA, cat. no. 22018‐1‐AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366‐1‐AP), MMP1 (1:1000, BOSTER China, cat. no. A00733‐1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs‐0415R), TIMP2 (1:1000, Bioss China, cat. no. bs‐10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858‐1‐AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201‐1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD‐L1 (1:3000; Proteintech, USA, cat. no. 66248‐1‐Ig), and β‐actin (1:5000; Proteintech, USA, cat. no. 66009‐1‐Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) Schematic diagram depicting the principle of the PathHunter assay used to detect the βarr2 interaction with APH1A. The CHO-βarr2 cells, which stably express βarr2 covalently attached to a portion of β-galactosidase (enzyme acceptor [EA]), were transfected with ProLink (PK)-tagged APH1A (APH1A-PK). Upon βarr2 binding to APH1A, the EA and PK form an active β-galactosidase enzyme. Upon the addition of a β-galactosidase substrate, the interaction between βarr2 and APH1A is detected by a chemiluminescent signal. (B) βarr2 recruitment to APH1A WT in the CHO-βarr2 cell line following treatment with 10 μM CMPD101 relative to vehicle (0.1% DMSO) for 30 min as measured using the PathHunter assay. **p < 0.01 by unpaired t test. Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in quadruplicate. Individual technical replicates across all independent experiments are indicated (white circles). (C) Aβ 40 and Aβ 42 generation in human fAD neural progenitor cells (NPCs) that stably express APP Swe/Ind . *p < 0.05 by ANOVA with Sidak’s post-test. Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in triplicate. Individual technical replicates across all independent experiments are indicated (white circles).

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Stable Transfection, Transfection, Binding Assay

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) Heatmap of the fold change in APH1A phosphorylation following expression in each GRK KO cell line relative to control cells as measured by label-free LC-MS/MS in 3 independent experiments. The “x” in specific squares indicates that phosphorylation was not detected. (B) Snake diagram of APH1A. The serine residues that display increased (red) or decreased (blue) levels of phosphorylation in each GRK KO cell line are indicated. (C) βarr2 recruitment to APH1A in the CHO-βarr2 cell line following expression of APH1A WT or the APH1A ICL2 and C-terminal phosphorylation-deficient or phosphorylation-mimetic mutants. *p < 0.05, **p < 0.01, and ****p < 0.0001 by two-way ANOVA with Tukey’s post-test. (D) Aβ 40 generation in cells following expression of APP-C99 and APH1A WT , APH1A phosphorylation-deficient, or phosphorylation-mimetic mutants. *p < 0.05 and ****p < 0.0001 by two-way ANOVA with Tukey’s post-test. Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in triplicate. Individual technical replicates across all independent experiments are indicated (white circles). (E) Aβ 42 generation in cells following expression of APP-C99 and APH1A WT , APH1A phosphorylation-deficient, or phosphorylation-mimetic mutants. **p < 0.01 and ****p < 0.0001 by two-way ANOVA with Tukey’s post-test. Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in triplicate. Individual technical replicates across all independent experiments are indicated (white circles).

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Phospho-proteomics, Expressing, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) Structural model DOCK2 generated by docking simulations followed by MD refinement carried out at full atomic scale in explicit membrane (gray sticks) and water. The simulations were performed for the intact γ-secretase complex that displays tight interaction between APH1A (magenta) and βarr2 (salmon) and transient involvement of other γ-secretase complex subunits such as the PS1 (cyan) and NCT (green). (B) Detailed view of interfacial interactions robustly observed at the interface. Residues engaged in persistent interfacial contacts are shown as sticks and labeled. (C) Interactions between APH1A and βarr2 for two independent runs (40 ns each) were carried out for this model (the first two columns) and corresponding time evolution of interactions for both runs. The last column displays the cumulative fractional time during which those pairs made contacts. Here, interactions are defined when any pairs of heavy atoms belonging to the two respective proteins are separated by less than 5 Å. Equivalent results for two other models, termed Alignment and DOCK1, are presented in , supporting the robustness of the regions (ICL2 and ICL3 in APH1A and finger loop and C terminus in βarr2) and residues engaged in interfacial association, despite minor redistributions of the specific pairs of residues.

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Generated, Membrane, Labeling

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) Co-immunoprecipitation experiments in HEK293 cells expressing APH1A and βarr2 WT or the βarr2 finger loop mutants (βarr2 L72E , βarr2 S75R ). (B–D) Representative western blot expression of C99-FLAG and βarr2 WT , βarr2 L72E , or βarr2 S75R (B). (C) Aβ 40 and (D) Aβ 42 generation in HEK293 cells following expression of APP-C99 and empty-vector, βarr2 WT , or βarr2 finger loop mutants. **p < 0.01 by ANOVA with Tukey’s post-test (comparison with empty). # p < 0.05 and ### p < 0.001 by ANOVA with Tukey’s post-test (comparison with βarr2 WT ). Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in triplicate or quadruplicate. Individual technical replicates across all independent experiments are indicated (white circles). (E–G) Representative western blot expression of C99-FLAG and APH1A WT -PK or APH1A R184D -PK (E). (F) βarr2 recruitment to APH1A WT or APH1A R184D mutant in the CHO-βarr2 cell line. **p < 0.01 by unpaired t test. (G) Aβ 40 and Aβ 42 generation in the CHO-βarr2 cell line following expression of C99-FLAG and APH1A WT or APH1A R184D . ****p < 0.0001 by ANOVA with Sidak’s post-test. Bars denote mean ± SEM of 3 independent experiments (n = 3) performed in triplicate or quadruplicate. Individual technical replicates across all independent experiments are indicated (white circles). (H–J) Results for the βarr2 mutants (H) L72E and (I) S75R and (J) for the APH1A mutant R184D are presented in the respective panels using the structural model DOCK1. In each case, a representative snapshot for the complex with the mutant (top), and the histograms of the intermolecular C α -C α distances between the indicated residue pairs of APH1A and βarr2 are shown (bottom, solid curves) on the basis of duplicate runs (total of 80 ns) conducted for the indicated mutant. Dashed curves indicate the counterparts obtained with the βarr2 WT or APH1A WT proteins.

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Immunoprecipitation, Expressing, Western Blot, Plasmid Preparation, Comparison, Mutagenesis, Residue

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: (A) GRKs mediate APH1A phosphorylation within ICL2 and the C terminus of APH1A to regulate βarr2 recruitment via interactions between the βarr2 finger loop domain and the cytoplasmic TM core (ICL2/3) of APH1A. In turn, βarr2 recruitment to APH1A leads to γ-secretase cleavage of APP-C99 and the generation of Aβ peptides. (B) In GRK2 KO cells, APH1A ICL2 phosphorylation at both S105 and S110 is elevated relative to control cells. The γ-secretase complex exhibits increased activity and cleavage of APP-C99. (C) In GRK3 KO and GRK5 KO cells, APH1A displays reduced levels of ICL2 phosphorylation at S103 and S110 relative to control cells. The γ-secretase complex exhibits decreased activity and cleavage of APP-C99. (D) In GRK6 KO cells, APH1A phosphorylation at S110 alone is elevated relative to control cells. The γ-secretase activity and Aβ generation are unaffected relative to control cells. We hypothesize that changes in the phosphorylation barcode affect both the extent of βarr2 recruitment to APH1A and the APH1A-βarr2 conformation following binding. We predict the positioning of H171 and H197 in APH1A is altered by βarr2 engagement, which affects recognition and cleavage of APP-C99. The phosphorylation sites highlighted in red and blue indicate elevated or reduced levels of phosphorylation, respectively, relative to control (or basal) levels of phosphorylation at the sites highlighted in yellow. The figure was created with BioRender.com .

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Phospho-proteomics, Control, Activity Assay, Binding Assay

Journal: Cell reports

Article Title: GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-β generation

doi: 10.1016/j.celrep.2022.111110

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The CHO-KI β-arrestin 2 cell line (CHO-βarr2) was purchased from DiscoveRx.

Techniques: Recombinant, PathHunter β-Arrestin Assay, Mutagenesis, BIA-KA, Software, Membrane

Journal: Viruses

Article Title: Broad-Spectrum Host-Based Antivirals Targeting the Interferon and Lipogenesis Pathways as Potential Treatment Options for the Pandemic Coronavirus Disease 2019 (COVID-19)

doi: 10.3390/v12060628

Figure Lengend Snippet: Antiviral agents included in the primary screening in this study.

Article Snippet: At a concentration of 3000 IU/mL, Avonex (IFN-β1a), Rebif (IFN-β1a), and Betaferon (IFN-β1b) respectively achieved 3.1 log10 copies/mL, 2.8 log10 copies/mL, and 3.0 log10 copies/mL reduction in viral RNA load, whereas Pegasys (pegylated IFN-α2a) and Immukin (IFN-γ1b) achieved only 1.6 log10 copies/mL and 1.5 log10 copies/mL reduction in viral RNA load.

Techniques: Virus, Recombinant, Transplantation Assay, Protease Inhibitor, Infection

Journal: Viruses

Article Title: Broad-Spectrum Host-Based Antivirals Targeting the Interferon and Lipogenesis Pathways as Potential Treatment Options for the Pandemic Coronavirus Disease 2019 (COVID-19)

doi: 10.3390/v12060628

Figure Lengend Snippet: Antiviral activities and cytotoxicities of the anti-SARS-CoV-2 antiviral agents identified in the primary screening.

Article Snippet: At a concentration of 3000 IU/mL, Avonex (IFN-β1a), Rebif (IFN-β1a), and Betaferon (IFN-β1b) respectively achieved 3.1 log10 copies/mL, 2.8 log10 copies/mL, and 3.0 log10 copies/mL reduction in viral RNA load, whereas Pegasys (pegylated IFN-α2a) and Immukin (IFN-γ1b) achieved only 1.6 log10 copies/mL and 1.5 log10 copies/mL reduction in viral RNA load.

Techniques: