Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 1. Differentiation of 3D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 3D-iMACs. All cell culture was performed under hypoxic (5% O2) conditions. B. Bright-filed images at day 4, 10, 17, and 24. Scale bar, 200 μm. C. Surface marker expressions of 3D-iMACs at day 24. Most cells express both of CD163 and CD206. D. Comparison of cytokine production profiles between 3D-iMACs and primary M2-like macrophages. They were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS). n = 3 biological replicates. iMACs and primary MACs showed similar cytokine profiles with or without LPS stimulation. E. Phagocytic activities of 3D-iMACs were confirmed under a fluorescent microscope. E. coli BioParticles conjugated with pHrodo Green appear bright green when they are taken up by macrophages. Scale bar, 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Cell Culture, Marker, Comparison, Microscopy

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 2. Differentiation of 2D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 2D-iMACs. B. Bright-field images at days 12 and 19. Scale bar, 200 μm. C. Surface marker expression of 2D- iMACs at days 12 and 19 (WT1323). They are positive for CD45, CD14, and CD11b. D. Polarization protocol and their subsequent surface marker expression. While M2-like iMACs showed high CD163 and low CD80 positivity, the expression patterns were opposite in M1-like iMACs. E. mRNA expression levels of macrophage- related genes in M1-like and M2-like 2D-iMACs. M1-related and M2-related genes are shown in the upper and lower portions respectively. They showed the expected expression patterns except for TGF-β. Expression levels are normalized to the housekeeping gene β-actin. Student's t-test was used for comparison of two groups. **p < 0.01. Data represent mean ± SEM of four independent experiments with technical triplicates. F. Phagocytic activities of M1-like and M2-like 2D-iMACs were confirmed (WTC11). E. coli BioParticles taken up by macrophages appear bright green. Scale bar, 200 μm. G. Comparison of cytokine production profiles between 2D- iMACs and primary macrophages. M1-like and M2-like 2D-iMACs showed similarities to primary M1- and M2-like macrophages respectively. n = 4–6 biological replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Marker, Expressing, Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 4. Response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) stimulation in 2D-iMACs. A. Protocol for testing the response to PAMPs and DMAPs. B. Comparison of cytokine production profiles in response to a PAMP (LPS) and DAMPs (HMGB1, S100A8/ A9) between primary M1-like macrophages and M1-like 2D iMACs. Primary cells and iMACs showed similarities regarding their responses to LPS. NT, HMGB, and S100 labels refer to untreated, stimulated with HMGB1, and stimulated with S100A8/A9, respectively. Primary cells and iMACs showed strong similarities regarding their responses to LPS. C. Comparison of cytokine production profiles in response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) between primary M2-like macrophages and M2-like 2D iMACs. n = 2–3 biological replicates for each condition.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 5. Comparison between WT-iMACs and FOP-iMACs. A. Comparison of cytokine production profiles between WT and FOP 2D-iMACs. Cells were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS) based on the protocol shown in Fig. 4A. n = 6 biological replicates. Differences between WT- and FOP-iMACs were more apparent in M1-like iMACs. B. Cytokines that showed higher production in FOP-M1-like iMACs compared with WT-M1-like iMACs at their baseline state (nontreated, NT). Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. C. Cytokine concentrations showing significant differences between WT- and FOP-M1-like iMACs stimulated with 10 ng/ml LPS. Key pro-inflammatory cytokine concentrations were higher in WT-M1-like iMACs when stimulated with 10 ng/ml LPS. Student's t-test was used for comparison of two groups. **p < 0.01Data represent mean ± SEM of six independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 6. Activin A concentrations in iMACs and cytokine concentrations in FOP-M1-like iMACs treated with inhibitors of Activin A pathways. A. Activin A concentrations in NT and LPS-stimulated group were analyzed using Human/Mouse/Rat Activin A Quantikine ELISA Kit. Analyzed samples were the same as those used in Fig. 5A. The concentrations of FOP-M1-like iMACs were significantly higher than WT-M1-like iMACs in NT group, while their response to LPS was downregulated. Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. B. FOP-M1-like iMACs were treated with 100 ng/ml anti-human/mouse/rat Activin A antibody (Anti) or 10 mM SB431542 (SB) after M1 polarization. Each inhibitor was added every 24 h. After 3 days culture, supernatants were collected and analyzed. There were no significant differences in key pro- inflammatory cytokines that were found elevated in FOP-M1-like iMACs as shown in Fig. 5B. Dunnett's test was used to compare each group to control (CTRL). Data represent mean ± SEM of five independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: The 51 genes within the functional annotation cluster were input into the STRING database. A network of how each gene interacts with others was produced. A sub-group of genes which interact closely, primarily through binding to each other, was highlighted (inset image). The genes of interest were CD247; LCK: Lymphocyte-specific protein tyrosine kinase; LAT: Linker of activated T cells; SYK: spleen tyrosine kinase; VAV2: VAV2 guanine nucleotide exchange factor; VAV3: VAV3 guanine nucleotide exchange factor.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Functional Assay, Produced, Binding Assay

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Gene expression levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by both human U133 plus 2 affymetrix gene assay and by quantitative reverse transcription polymerase chain reaction (PCR). Fold change from blood to lung is charted. Statistically significant differences between blood and pulmonary levels measured by PCR are indicated by *p<0.05, **p<0.001. Statistically significant differences between blood and pulmonary levels measured by microarray are indicated by † Q<0.01.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Gene Expression, Gene Assay, Reverse Transcription, Polymerase Chain Reaction, Microarray

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Pulmonary CD8 cells were isolated from chronic obstructive pulmonary disease (COPD, grey n = 6) and smokers with normal lung function (S, black, n = 6). Transcript levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by quantitative real time polymerase chain reaction. Statistically significant differences between COPD and S are represented by *p<0.05.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: The MT3-Zn 2+ axis suppresses TRIF signaling resulting in decreased IRF3 phosphorylation. When MT3 is absent, TRIF-IRF3-STAT1 signaling and non-canonical inflammasome activation are exaggerated. A lack of MT3 augments immunity to gram-negative bacteria, an effect, that is further enhanced by the combined absence of MT3 and caspase-11 in vivo . Thus, while MT3 curtails caspase-11 activation, the two molecules act together in compromising antibacterial immunity.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Phospho-proteomics, Activation Assay, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Table S1 and Files S1 , S2 |Protein interaction network of Mus musculus MT3 to determine functionally enriched GO BP categories using the STRING database.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Transduction, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S1 MT3 suppresses caspase-11 inflammasome activation in BMDMϕ. qRT-PCR analysis of Mt3 expression in WT BMDMϕ stimulated with (A) iLPS (2 μg/ml) or vehicle control, 3-5 independent experiments and (B) exLPS (10 μg/ml) for 48h, 3 independent experiments, two-tailed t-test. (C) Western Blots of pro- and active-caspase-11, pro-caspase-1, pro-IL1β and β-actin in cell lysates and active-caspase-1 and active-IL-1β in supernatants of WT and Mt3 -/- BMDMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin, 3-4 independent experiments, one-way ANOVA, data are mean ± SEM. (D) Western Blots of pro- and active-caspase-11 and β-actin in lysate + supernatant samples from WT and Mt3 -/- BMDMϕ stimulated with iLPS (2 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S2 . MT3 curtails CASPASE-4 and caspase-11 signaling and antibacterial immunity in hMϕ and in vivo . (A) MT3 and MT2A expression analyzed by qRT-PCR in hMϕ transfected with scramble siRNA or MT3 siRNA for 24h, 3 independent experiments, two-tailed t-test. (B) Scramble siRNA or MT3 siRNA treated hMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of pro-CASPASE-4 and active-CASPASE-4 in cell extracts, 3 independent experiments, one-way ANOVA. (C) Active-IL-1β measured by ELISA in supernatants of hMϕ treated as above, 3 independent experiments, one-way ANOVA. (D) E . coli growth inhibition in hMϕ transfected with MT3 siRNA and infected with 25 E . coli (K12): 1 hMϕ for 24h compared to scramble siRNA treated hMϕ, 3 independent experiments, two-tailed t-test. (E) E . coli growth inhibition in WT and Mt3 -/- BMDMϕ infected with 25 E . coli (K12):1 hMϕ for 24h, 4 independent experiments, two-tailed t-test. (F) WT and Mt3 -/- mice infected i.p. with 1X10 9 E . coli for 6h, log CFUs of E . coli in blood, kidney and peritoneal lavage samples, n = 12-15 per group, two-tailed t-test. (G) Western blots of inflammasome mediators in kidney homogenates of WT and Mt3 -/- mice infected as above, n = 6 per group, two-tailed t-test. (H) WT and Mt3 -/- mice infected i.p. with 1 X10 9 E . coli for 1h and IL-1β measured in peritoneal lavage and serum by ELISA. n = 3 per group, two-tailed t-test. (I) WT and Mt3 -/- mice primed i.p. with poly(I:C) (10 mg/kg) for 6h and challenged with LPS (2 mg/kg) i.p. After 18h, IL-1β was measured in peritoneal lavage and serum by ELISA, n = 3/group, two-tailed t-test. (J) Bacterial growth in spleen, lung and kidney of WT and Mt3 -/- mice infected i.n. with K. pneumoniae (4 X10 4 CFUs/mouse) for 48h, n = 8-12 per group, two-tailed t-test, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Infection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, C asp-11 -/- , Mt3 -/- and Casp-11 -/- Mt3 -/- mice were infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3 -/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. IL1β was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S4 Myeloid-MT3 suppresses non-canonical inflammasome activation and blunts gram-negative bacterial clearance in vivo . (A) Generation of Mt3 fl/fl mice by inserting loxp sites flanking exon 3 of the Mt3 gene using the CRISPR-Cas9 gene targeting approach. Mt3 fl/fl mice crossed with Lys2Cre mice to obtain Lys2Cre Mt3 fl/fl mice. (B) Efficacy of myeloid Mt3 deletion assessed by genotyping peritoneal Mϕ (PMϕ) and BMDMϕ from Lys2Cre , Mt3 fl/fl and Lys2Cre Mt3 fl/fl mice. Gel electrophoresis analysis demonstrating efficient deletion of the Mt3 gene from BMDMϕ and PMϕ of Lys2Cre Mt3 fl/fl mice. (C) Western blots of pro-caspase-11, active-caspase-11, pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in whole kidney homogenates of mice infected as above, n = 3-5 per group, two-tailed t-test. (D) Bacterial CFUs in kidney and whole blood of Lys2Cre and Lys2Cre Mt3 fl/fl mice infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h, n = 3-5 per group, two-tailed t-test, data are mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, In Vivo, CRISPR, Nucleic Acid Electrophoresis, Western Blot, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S5 MT3 thwarts TRIF-IRF3-STAT1 signaling to suppress non-canonical inflammasome activation. (A) Functional enrichment analysis of differentially expressed genes using RNA-seq data from resting WT and Mt3 -/- BMDMϕ (NCBI SRA: PRJNA533616) FDR, false detection rates. (B, C) Heat map (left) and table (right) show differentially expressed IFN-related genes in resting Mt3 -/- BMDMϕ compared to resting WT BMDMϕ obtained from RNA-seq analysis. (D) Western blots of pIRF3, pSTAT1, STAT1, GBP2 and GBP5 in vehicle or iLPS (10 μg/ml)-treated WT and Mt3 -/- BMDMϕ lysates, 3-4 independent experiments, one-way ANOVA. (E) Western blots of TRIF in lysates from WT and Mt3 -/- BMDMϕ stimulated as above, 3 independent experiments, one-way ANOVA. (F) Scramble and Ticam1 siRNA treated WT and Mt3 -/- BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of TRIF (2 independent experiments), pro-caspase-11, and active-caspase-11 in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Functional Assay, RNA Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figures S6 , 7 MT3-Zn 2+ axis drives negative regulation of the non-canonical inflammasome. (A) SEC-ICP-MS of WT and Mt3 -/- BMDMϕ exposed to vehicle or iLPS (10 ug/ml) for the indicated time points, chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (B) Bar graphs of total Zn 2+ and MT-Zn 2+ in WT and Mt3 -/- BMDMϕ post iLPS (10 μg/ml) or vehicle exposure. Two-way t-test against respective BMDMϕ controls at each time point, 3 independent experiments, data are mean ± SD. (C) WT BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ sufficient or Zn 2+ deficient Opti-MEM media, immunoblots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL-1β in lysates and active-IL-1β in media supernatants, one-way ANOVA, data are mean ± SEM. (D, E) Mt3 -/- BMDMϕ transfected with Pro-Ject™ or Pro-Ject™ complexed with apo-MT3, 4Zn 2+ MT3 or 6Zn 2+ MT3 and treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ deficient Opti-MEM media. (D) Chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ signal associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (E) Western blots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL1β in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Comparison, Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: Reagents and resources.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Expressing, Plasmid Preparation, Control, Extraction, Blocking Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Filtration, Transfection, Reverse Transcription, Cytotoxicity Assay, Isolation, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner

doi: 10.1152/ajplung.00362.2021

Figure Lengend Snippet: Genes assayed, grouped by functional categories

Article Snippet: , IFITM3 , Interferon induced transmembrane protein 3 , Hs03057129_s1.

Techniques: Functional Assay, TaqMan Probe Assay, Recognition Signal

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner

doi: 10.1152/ajplung.00362.2021

Figure Lengend Snippet: Statistically significant virus-induced changes in gene expression in hNECs from males and females at 0, 24, and 72 h p.i. with SARS-CoV-2

Article Snippet: , IFITM3 , Interferon induced transmembrane protein 3 , Hs03057129_s1.

Techniques: Virus, Gene Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner

doi: 10.1152/ajplung.00362.2021

Figure Lengend Snippet: Statistically significant effects of particle exposure on virus-induced gene expression in hNECs at 0, 24, and 72 h p.i.

Article Snippet: , IFITM3 , Interferon induced transmembrane protein 3 , Hs03057129_s1.

Techniques: Virus, Gene Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner

doi: 10.1152/ajplung.00362.2021

Figure Lengend Snippet: Statistically significant, sex-disaggregated effects of particle exposure on virus-induced gene expression in infected hNECs at 0, 24, and 72 h p.i.

Article Snippet: , IFITM3 , Interferon induced transmembrane protein 3 , Hs03057129_s1.

Techniques: Virus, Gene Expression, Infection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner

doi: 10.1152/ajplung.00362.2021

Figure Lengend Snippet: Genes assayed, grouped by functional categories

Article Snippet: , IL6 , Interleukin 6 , Hs00265051_s19.

Techniques: Functional Assay, TaqMan Probe Assay, Recognition Signal

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Mmu-miR-151-5p cleaves E2f6 in the absence of a seed match. (a) Genomic locus of mmu-miR-151 encoded by a LINE2 repeat element. (b) Schematic of the binding site of mmu-miR-151-5p to E2f6 3′UTR. (c) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (per 5p and mut 5p) in presence of miR-151-5p overexpression (sh-151-5p). (d) Western blot for E2f6in presence of sh-151-5p or a scrambled control (sh-scr). Actin serves as a loading control. Uncropped blot in . (e) E2f6 qPCR on miR-151-5p overexpression. Error bars, s.e.m. (n = 3 replicates). (f) Dual-luciferase reporter assay for E2f6 3′UTR or control ( Sox4 3′UTR) in presence of an increasing dosage of miR-151-5p inhibitor. (g) Dual-luciferase reporter assay in Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-5p and a functional copy of Ago2 or cleavage deficient Ago2 (D597A) or Ago1. (h) 5′-RACE of E2f6 . Arrowhead in the E2f6 3′UTR sequence (schematic) indicates the 5′ end of majority of E2f6 cleavage products in mouse lung tissue. Agarose gel showing E2f6 cleaved products (shown by an asterisk) is shown in the top gel (uncropped gel in ). The bottom gel serves as a RACE reaction control to detect the presence of E2f6 and ARHGDIA cDNAs. (c,f, g) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates), ns denotes not significant,*** P = 0.001 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Luciferase, Reporter Assay, Over Expression, Western Blot, Control, Functional Assay, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: miR-151-3p suppresses E2f6 expression by binding to E2f6 3′UTR adjacent to where miR-151-5p binds. (a) Schematic of a putative binding site of the miR-151-3p to the E2f6 3′UTR region adjacent to where 5p strand binds, in both mice and humans. The seed regions (nucleotides 2-8) are indicated for both the 5p and 3p arms. (b) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (mut 3p and seed 3p as shown in the schematic) in presence of a miR-151-3p overexpression (sh-151-3p) or a scrambled control(sh-scr). A reporter construct with deletion of the entire miR-151-3p binding site in E2f6 3′UTR was also included. (c) Dual-luciferase reporter assay in wildtype MEF and Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-3p. For comparision, dual-luciferase reporter assay for E2f6 3′UTR in presence of sh-151-5p is also shown. (b,c) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Control, Construct

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Precursor miR-151 competes with the mature miR-151-5p for binding to E2f6 3′UTR. (a) Thermodynamics of pre-miR-151 binding to E2f6 . (b) Schematic of the stem-loop structure of the pre-miR-151 with the 5p arm (blue), 3p arm (purple) and two adenosines (green) substituted to guanosines (orange). (c) Northern analysis of miR-151 processing from pre-miR-151 overexpression plasmid (pEZX-151) or the double mutant form of pre-miR-151 (pEZX-DM). Let-7a serves as a loading control. (d) Dual-luciferase reporter assay for E2f6 3′UTR in presence of only the mature miR-151-5p (sh-miR-151-5p), or both the pre-miR-151 and mature miR-151-5p (pEZX-151 and pEZX-DM). (e) Schematic of the binding site of pre-miR-151 in E2f6 3′UTR and its modifications ( E2f6 3p del and E2f6 3p-5p swap). (f) In-vitro gel shift assay with radiolabed (denoted by an asterisk) synthetic pre-miR-151(I) or a control pre-miR-122 and increasing molar concentrations of wildtype E2f6 3′UTR (1, 10 and 100 nM) or its modified forms. (g) Dual-luciferase analysis for E2f6 3′UTR (wt), 3p del or 3p-5p swap reporters with pEZX-151 or pEZX-DM. (h) In-vitro gel shift assay of E2f6 3′UTR bound to miR-151-5p with increasing molar concentrations of a synthetic pre-miR-151 or a control pre-miR-122. Bands below the blue star and orange star represent radiolabeled pre-miR-151 and pre-miR-122 respectively. “*” denotes radiolabeled oligos. (d, g) For reporter assays, normalization was done with respect to a scrambled control (sh-scr). Error bars, s.e.m. (n = 3 biological replicates, each with 3 technical replicates). * P = 0.05, ** P = 0.01 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Northern Blot, Over Expression, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, In Vitro, Gel Shift, Modification, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues. (a) Schematic of the ChIRP method used to pull-down E2f6 mRNA from mouse brain. Quantitative PCR of (b) E2f6 mRNA and a control Ctdnep1 mRNA, (c) pre-miR-151 and a control pre-miR-124, (d) mature miR-151-5p and a control miR-124, pulled down by the biotinylated tiling oligonucleotides against the E2f6 3′UTR or a control lacZ mRNA. Quantitative PCR of (e) E2f6 mRNA, (f) pri-miR-151, (g) mature miR-151-5p in quiescent and non-quiescent tissues. In each case ( e — g ), the data are presented as fold induction after normalization to the liver sample (value = 1). (h) Northern analysis of miR-151 processing in various tissues. The blot on the left was probed with a LNA probe against mature miR-151-5p as shown by the schematic above the blot. The primary or intermediate product in the miR-151 biogenesis pathway is indicated by an arrowhead (→) and the mature miR-151-5p is indicated by a circle (○). U6 serves as a loading control. The blot on the right was probed (sequence is provided in ) for a region just outside the annotated stem loop structure of mmu-miR-151 (as shown by the schematic above the blot). (i) Quantitative PCR analyses of E2f6 , pri-miR-151 and miR-151-5pduring differentiation of muscle cells (C2C12) ( b — g, i ) For qPCR data, error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Control, Northern Blot, Sequencing

Journal: PLoS Biology

Article Title: Machine Learning Helps Identify CHRONO as a Circadian Clock Component

doi: 10.1371/journal.pbio.1001840

Figure Lengend Snippet: (A) Overexpression of either BMAL1 or BMAL2, along with CLOCK, activates Per1 :Luciferase reporter activity. Both are repressed by overexpression of CRY1. CHRONO specifically represses BMAL1-induced reporter activity. (B) BMAL1 and BMAL2 have similar structures with conserved bHLH DNA binding domains and PAS A and B interaction domains. BMAL1 contains a unique C-terminal region. Chimeric proteins were constructed by swapping corresponding domains from each protein as shown. Two-hybrid screening in HEK 293T cells demonstrates that BMAL1 truncation mutants (C) and chimeric proteins (D) that contain the 487–586 region of BMAL1 bind CHRONO and induce UAS:Luc reporter expression. This region is adjacent to but distinct from the annotated CRY1 binding site. (E) All BMAL1–BMAL2 constructs induce Per1 -luc reporter activity in HEK 293T cells. In all constructs, reporter signal is repressed by the addition of CRY1. Functional repression by CHRONO is limited to BMAL constructs containing the implicated binding domain. (F) In cells overexpressing MYC–CHRONO along with BMAL1, BMAL2, or a chimeric BMAL2–BMAL1 construct, co-IP confirms complex formation between CHRONO and proteins containing the implicated BMAL1 C-terminal region.

Article Snippet: The Taqman probe identifiers were as follows: For Mus musculus : Arntl , Mm00500226_m1; Arntl2 , Mm00549497_m1; Per1 , Mm00501813_m1; Per2 , Mm00478113_m1; Per3 , Mm00478120_m1; Nr1d1 , Mm00520708_m1; Chrono (Gm129), Mm01255906_g1; Importin 8 , Mm01255158_m1.

Techniques: Over Expression, Luciferase, Activity Assay, Binding Assay, Construct, Two Hybrid Screening, Expressing, Functional Assay, Co-Immunoprecipitation Assay

Journal: Thyroid

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

doi: 10.1089/thy.2012.0457

Figure Lengend Snippet: Change in hepatic LRP1 expression in a hypothyroidism animal model. C57BL/6 mice were fed a normal diet (control group, n=6) or a low-iodine diet supplemented with 0.15% propylthiouracil (PTU/LI group, n=4) for 4 weeks. (A, B) Western blot analysis of LRP1 (β-chain) in liver samples. (C) RT-PCR quantification of LRP1 mRNA in liver samples. Results are normalized with β-actin protein or mRNA levels and are expressed as ratios relative to the control group. Data are mean±SE. *p<0.05 vs. control group. RT-PCR, real-time polymerase chain reaction; LRP1, low-density lipoprotein receptor–related protein 1; SE, standard error.

Article Snippet: Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction analysis was performed using a TaqMan assay kit for LRP1 (Hs00233856_m1, Mm00464608_m1) and an ABI 7500 instrument (Applied Biosystems, Foster City, CA). β-Actin (Hs99999903_m1, Mm00607939_s1) was used as an invariant control.

Techniques: Expressing, Animal Model, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Thyroid

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

doi: 10.1089/thy.2012.0457

Figure Lengend Snippet: Immunohistochemistry of LRP1 in the liver samples. L C57BL/6 mice were fed a normal diet (control group, n=6) or a low-iodine diet supplemented with 0.15% propylthiouracil (PTU/LI group, n=4) for 4 weeks. Three samples of each group are presented and LRP1 expression is shown by brown color. Bars indicate 100 μm. Color images are available online at www.liebertpub.com/thy

Article Snippet: Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction analysis was performed using a TaqMan assay kit for LRP1 (Hs00233856_m1, Mm00464608_m1) and an ABI 7500 instrument (Applied Biosystems, Foster City, CA). β-Actin (Hs99999903_m1, Mm00607939_s1) was used as an invariant control.

Techniques: Immunohistochemistry, Control, Expressing

Journal: Thyroid

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

doi: 10.1089/thy.2012.0457

Figure Lengend Snippet: Change in hepatic LRP1 expression after T3 treatment in animals. C57BL/6 mice were fed a low-iodine diet supplemented with 0.15% propylthiouracil (n=11). Various doses of T3 (0, 30, and 150 μg/kg of body weight) were administered daily to the mice through intraperitoneal injection for 7 days. (A, B) Western blot analysis of LRP1 (β-chain) in liver samples. (C) RT-PCR quantification of LRP1 mRNA in liver samples. Results are normalized with β-actin protein or mRNA levels and are expressed as ratios relative to 0 μg/kg. Data are mean±SE. *p<0.05 vs. 0 hour. T3, triiodothyronine.

Article Snippet: Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction analysis was performed using a TaqMan assay kit for LRP1 (Hs00233856_m1, Mm00464608_m1) and an ABI 7500 instrument (Applied Biosystems, Foster City, CA). β-Actin (Hs99999903_m1, Mm00607939_s1) was used as an invariant control.

Techniques: Expressing, Injection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Thyroid

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

doi: 10.1089/thy.2012.0457

Figure Lengend Snippet: The effect of T3 on LRP1 expression in HepG2 cells. HepG2 cells incubated in CSS or unstripped FBS for 24 hours were treated with the indicated concentrations of T3 for 48 hours. (A) Western blot analysis of LRP1 (β-chain) in HepG2 cells (n=5 in each group). (B) Western blot analysis of LRP1 (β-chain) in HepG2 cells incubated in CSS (n=5 in each group). (C) RT-PCR quantification of LRP1 mRNA in HepG2 cells incubated in CSS (n=5 in each group). Results are normalized with β-actin protein or mRNA levels and are expressed as ratios relative to T3 0 nM in CSS. Data are mean±SE. *p<0.05 vs. T3 0 nM in CSS; †p<0.05 vs. T3 0.5 nM in CSS. CSS, charcoal-stripped fetal bovine serum; FBS, fetal bovine serum.

Article Snippet: Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction analysis was performed using a TaqMan assay kit for LRP1 (Hs00233856_m1, Mm00464608_m1) and an ABI 7500 instrument (Applied Biosystems, Foster City, CA). β-Actin (Hs99999903_m1, Mm00607939_s1) was used as an invariant control.

Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Thyroid

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

doi: 10.1089/thy.2012.0457

Figure Lengend Snippet: Uptake of lipid-conjugated ApoE3 by T3 in HepG2 cells. HepG2 cells incubated in CSS for 24 hours were treated with the indicated concentrations of T3 for 48 hours. Human recombinant ApoE3 conjugated with lipid was added to the culture medium and cells were incubated for 1 hour. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells transfected with nontargeting negative siRNA (siCTRL) and siRNA targeting LRP1 (siLRP1). (C) Western blot analysis of ApoE3 in HepG2 cells transfected with siCTRL and siRNA targeting the LDL receptor (siLDLR). The human recombinant receptor–associated protein was used as a functional blocker of LRP1 and LDLR. The human recombinant receptor–associated protein was added to the culture medium before adding ApoE3. Three independent experiments were performed for the representative figures. siRNA, small interfering RNA; LDL, low-density lipoprotein; LDLR, LDL receptor.

Article Snippet: Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction analysis was performed using a TaqMan assay kit for LRP1 (Hs00233856_m1, Mm00464608_m1) and an ABI 7500 instrument (Applied Biosystems, Foster City, CA). β-Actin (Hs99999903_m1, Mm00607939_s1) was used as an invariant control.

Techniques: Incubation, Recombinant, Western Blot, Transfection, Functional Assay, Small Interfering RNA

Journal: Arthritis Research & Therapy

Article Title: Low-intensity pulsed ultrasound rescues insufficient salivary secretion in autoimmune sialadenitis

doi: 10.1186/s13075-015-0798-8

Figure Lengend Snippet: Anti-inflammatory mechanism of LIPUS on NS-SV-AC and NS-SV-DC cellular function. a Western blot analysis showed IκBα phosphorylation was obviously induced by TNF-α stimulation; however, when LIPUS treatment was administered following TNF-α treatment, IκBα, NF-κB, and IKKβ phosphorylation were inhibited. b Similar inhibitory effects of LIPUS were observed in IL-1β stimulation. c In Western blot analysis, IRAK1 was degraded after IL-1β stimulation, and LIPUS exposure failed to inhibit it. d The stimulation of both NS-SV-AC and NS-SV-DC cells with TNF-α or IL-1β resulted in a significant increase in levels of A20 mRNA, and A20 mRNA expression was further increased following treatment with LIPUS after TNF-α or IL-1β stimulation. * p < 0.05; ** p < 0.01 (n = 6). A20 tumor necrosis factor-α-induced protein 3 ( TNFAIP3 ), IKKβ inhibitor of nuclear factor κB kinase subunit β, IL-1β interleukin 1β, IRAK1 interleukin 1 receptor-associated kinase 1, IκBα inhibitor of nuclear factor of κ light polypeptide gene enhancer in B cells, α subunit, LIPUS low-intensity pulsed ultrasound, NF-κB nuclear factor κB, NS-SV-AC salivary gland acinar cells, NS-SV-DC salivary gland ductal cells, TNF-α tumor necrosis factor α

Article Snippet: The following TaqMan probe mixtures were used: TaqMan gene expression assays; AQP5 , Hs00387048_m1; TNF-α , Hs01113624_g1; A20 , Hs00234713_m1; and β-actin , Hs01060665_g1 (Applied Biosystems).

Techniques: Cell Function Assay, Western Blot, Phospho-proteomics, Expressing