Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 1 DHX9 expression significantly increased in oxLDL or interferon-γ-treated macrophages and peripheral blood mononuclear cells from patients with coronary artery disease. a Single-cell RNA analysis of DHX9 mRNA expressions in mononuclear phagocytes (MNPs) a public MNP single-cell RNA compendium. b Western blot analysis of DHX9 expressions PBMCs from health volunteers stimulated with 50 ng/ml of recombi- nant human GM-CSF for different hours. c Western blot analysis of DHX9 expressions in the THP-1-derived macrophages stimulated with oxLDL for 24 h. d Western blot analysis of DHX9 expressions in the THP-1-derived macrophages stimulated with 40 μg/ml oxLDL for different hours. e Western blot analysis of DHX9, STAT1, p-STAT1 expressions in the THP-1-derived macrophages stimulated with 20 ng/mL IFN-γ for different hours. GAPDH was used as a loading control. f Immunofluorescence analysis of DHX9 cellular distribution in macrophages treated with or without 40 μg/mL oxLDL for 24 h. Scale bars, 10 μm. g Western blot analysis of DHX9 expressions in PBMCs isolated from health volunteers (HV) or patients with (CAD). GAPDH was used as a loading control.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Derivative Assay, Control, Immunofluorescence, Isolation

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 2 Knockdown of DHX9 inhibits lipid uptake and pro-inflammatory factor expressions of macrophages, and ameliorates TNF-α-mediated monocyte adhesion capacity. a qPCR detection of IL-6 and TNF mRNAs in THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. b Flow cytometry analysis of cell cycle of THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. c Flow cytometry analysis of cell apoptosis of THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. d Immunofluorescence analysis of Dil-oxLDL (red) uptake of THP-1-derived macrophages transfected si-NC or si-DHX9 for 48 h. Scale bars, 5 μm. e qPCR detection of IL-1β, IL-6 and TNF-α mRNAs in THP-1-derived mac- rophages transfected with si-NC or si-DHX9 for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. Data are represented as means ± SD (n = 3; * P < 0.05 vs. si-nc + oxLDL group). f and g Correlation analysis of DXH9 and IL-6 or TNFα gene expressions in monocytes. h Representative images of the attachment of THP-1 cells transfected si-NC or si-DHX9 to HUVECs. Scale bars, 160 μm. Data are represented as means ± SD. n = 3; Statistical differences were calculated using unpaired two-tailed Student’s t test. *P < 0.05.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, Derivative Assay, Transfection, Control, Flow Cytometry, Immunofluorescence, Incubation, Two Tailed Test

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 3 oxLDL stimulation promotes DHX9 interaction with p65 in macrophages. a Western blotting analysis of p38, JNK, ERK signaling in mac- rophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. b Western blotting analysis of NF-κB signaling in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. c Co-IP detection of the interaction between DHX9 and p65 in macrophages treated with or without 40 μg/mL oxLDL for 24 h by using DHX9 antibody. d Immunofluores- cence analysis of DHX9 and P65 expression in macrophages treated with or without 40 μg/mL oxLDL. Scale bars, 10 μm. e Co-IP detection of the interaction between DHX9 and p65 in the nuclear fractions of macrophages by using DHX9 antibody. f P65 dimer formation was detected using naïve PAGE when macrophages were transfected with or without FLAG-DHX9 and treated with or without oxLDL (-, 0 μg/mL oxLDL; +, 40 μg/ mL oxLDL; ++, 80 μg/mL oxLDL).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Western Blot, Transfection, Incubation, Co-Immunoprecipitation Assay, Expressing

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 4 oxLDL stimulation promotes the transcriptional activity of DHX9-p65-RNA Polymerase II complex. a ChIP analysis of the binding of DHX9 to IL-6 promoter in macrophages treated with oxLDL (-, 0 μg/mL oxLDL; +, 40 μg/mL oxLDL; ++, 80 μg/mL oxLDL). IgG was used as the control of anti-DHX9. Data are represented as means ± SD (n = 3; *P < 0.05). b and c ChIP-re-ChIP analysis of the binding of DHX9-p65 com- plex to IL-6 promoter in macrophages treated with oxLDL. d ChIP analysis of the binding of RNA Polymerase II to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. e ChIP analysis of the binding of p65 to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. f and g ChIP-re-ChIP analysis of the binding of RNA Polymerase II-p65 complex to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Activity Assay, Binding Assay, Control, Transfection, Incubation

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 5 Knockdown of DHX9 alleviates AS progression in vivo. a Western blot analysis of DHX9 protein expression in the arterial tissues of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. b Representative images and quantification of the aorta en face lesion stained with oil red O (n = 6 for each group). Data are represented as means ± SD (n = 6; *P < 0.05). c Representative images and quantification of the aortic root lesion area stained with oil red O (n = 6 for each group). Data are represented as means ± SD (n = 6; *P < 0.05). d Immunofluorescence analysis of F4/80 and p-p65 in the plaques of arteries of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. Scale bars, 20 μm. e qPCR detection of IL-6 and TNF-α mRNA expressions in the plaques of arteries of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. g and f ELISA detection of IL-6 and TNF-α expressions in the plasma of mice. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 6 Knockdown of DHX9 inhibits p65 activation, inflammatory factor expressions, and the transcriptional activity of p65-RNA Polymerase II complex in PBMCs from CAD patients. a Western blot analysis of DHX-9 and p-p65 protein expressions in the PBMCs from CAD patients trans- fected with si-DHX9 or si-NC. b qPCR detection of IL-6 and TNF-α mRNA expressions in the PBMCs from CAD patients transfected with si- DHX9 or si-NC. c ChIP analysis of the binding of DHX9 to IL-6 promoter in PBMCs. d ChIP analysis of the binding of RNA Polymerase II to IL-6 promoter in the PBMCs from CAD patients transfected with si-DHX9 or si-NC. e ChIP analysis of the binding of p65 to IL-6 promoter in the PBMCs from CAD patients transfected with si-DHX9 or si-NC. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, Activation Assay, Activity Assay, Western Blot, Transfection, Binding Assay

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 7 DHX9 promotes ox-LDL-induced inflammation in macrophages via interacting with p65. DHX9 interacts with p65 in ox-LDL-stimulated macrophages to enhance the transcriptional activity of DHX9-p65-RNA Polymerase II complex to produce inflammatory factors.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Activity Assay

Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Schematic shows the morphology of sarcomere disassembly. B. Schematic of Co-IP/MS by TNNT2 pulldown. C. Table lists selected TNNT2 associated proteins identified from the Co-IP/MS. * PSM: Peptide Spectrum Matches. Number of spectra assigned to peptides that contributed to the inference of the protein. **MIC Sin: The normalized spectral index statistic for the protein for the specific group. Calculated from the intensity of fragment ions in each spectrum assigned to a protein. ***Ratio: Quantitative ratio for protein between groups derived from the MIC Sin value. D. Coimmunoprecipitation of ADD1 from total heart extracts at P1 and P7, probed for TNNT2 and α-actinin. E. (Left) Representative WB to show endogenous expression profile of ADD1 and ADD3 at different ages. (Right) Quantification of adducin expression level relative to internal control GAPDH. (n=3 per group). F. Expression of four selected cytoskeletal proteins from MS results, α-, γ-adducin, spectrin, and filamin were stained in neonatal hearts, showing profile of proliferation (left) and non-proliferation (right) cardiomyocytes. Arrows point to representative cells. Arrow heads in filamin staining images refer to nucleus location. Scale bar=10 μm.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Expressing, Control, Staining

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. (Top) Schematic of exons and introns of Add1 gene. Red rectangle represents exon 15 which is the target of alternative splicing. (Bottom) Schematic shows inclusion or exclusion of exon 15 in mRNA alternative splicing events. The corresponding translated protein schematic is shown next to the mRNA. Inclusion of exon 15 has an in frame stop code which generates ADD1 isoform 2, composing 636 amino acids (a.a.). Exclusion of exon 15 translates into ADD1 isoform 1, composing 732 a.a. B. Western blot shows endogenous profile of ADD1 isoform 2 at different ages. GAPDH is used as internal control. C. Representative images of NRVM transduced with AAV6-Add1i1 (i, ii), AAV6-Add1i2 (iii, iv), and AAV6-GFP (v) at day 3. Schematics of AAV expression cassettes are shown with each group images. Cells were fixed and immunostained with α-actinin (ACTN2, red) to reveal sarcomere organization. Green fluorescence represents either Adducin isoform 1 and 2, or GFP. High magnification images of squared regions are shown on the right. Dotted lines indicated the boarder of cardiomyocytes. (i) and (iii) represents partial disassembled sarcomeres. (ii) and (iv) represents fully disassembled sarcomeres. (v) represents assembled sarcomeres. Percentage of sarcomere assembly changes by AAV are shown in the graph. D. (Top) Schematic to show the strategy of generating Add1 isoform 2 single transgenic line. (Bottom left) H&E staining of WT and cardiac-specific Add1 i2 transgenic. Scale bar = 1 mm. (Bottom right) Heart weight to body weight ratio in control and cardiac-specific Add1i2 transgenic. E. (Top) Images to show expression pattern of ADD1 i2 transgenic at P14 and P28. (Bottom) High magnification images of squared region from the top panels. Scale bar = 10 μm. F. (Top) Representative images to show sarcomere pattern from ADD1 i2 transgenic at P14 and P28. Insets are high magnification images of single cardiomyocyte (squared regions). Dotted lines are drawn around outer and inner borders of sarcomeres. (Bottom) Quantification graph to show the extent of sarcomere disassembly. Disassembly scores were defined as: (Outer Area – Inner Area) / Outer Area. Age matched litter maters of WT mice were used as controls. At P14, ADD1 i2 transgenic mice have significantly thinner sarcomeric zone with central clearance compared to controls. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Alternative Splicing, Western Blot, Control, Transduction, Expressing, Fluorescence, Transgenic Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Endogenous phospho-adducin expression pattern in neonatal mouse heart. Schematics of phosphosite locations is shown on the left. Right: immunostaining images show co-staining images of anti-phospho-site antibody with sarcomeric protein TNNT2 or α-actinin. From top to bottom are Phospho-sites T445/T480, S12/S355, S714/S724, and S408/S436/S481. Arrows indicate phospho-adducin colocalized with disassembled sarcomeres. Arrow heads indicate colocalization with assembled sarcomeres. B. Endogenous expression of phospho-adducin T445 in NRVM as stained with α-actinin (red), anti-phospho-adducin pT445 (green) and DAPI. Phospho-adducin translocates from the nucleus to the cytoplasm during mitosis. C. (Top): WB examination of endogenous phospho-T445 expression level. (Bottom) Quantification of pT445 expression level in relative to ADD1 expression level. D. Overexpression of adducin phospho- or non-phospho mimic T445/T480 in NRVM as induced by AAV6. (Top left) Schematic diagram of AAV shuttle vector shows that adducin mutants are expressed with 3 tandem FLAG epitopes in frame at the N-terminus. (Bottom left) Immunofluorescent staining with α-actinin antibody to show sarcomeric structure. Adducin mutants are detected by Flag antibody in green. High magnification images of representative region (white box) were shown on the right. Dotted lines were drawn around the cell boarder. (Right) Quantitative analysis of disassembled sarcomeres induced by adducin overexpression. Scale Bar = 10 μm. E. (Top) Schematics to show strategy of generating cardiac specific inducible Add1 i1 phospho single transgenic (p-TRE T445E/T480E, α-MHC tTA). (Bottom left) H&E staining of control and cardiac-specific Add1 i1 phospho transgenic. Scale bar = 1 mm. (Bottom right) Heart weight to body weight ratio in control and cardiac-specific phospho-single transgenic. F. (Top) Images to show expression pattern of phospho-Add1i2 transgenic at P14 and P28. (Bottom) High magnification images of selective phospho-adducin mimic overexpression cardiomyocytes. At P14, we saw phospho-mimic expresses both in cytoplasm and on membrane (Bottom left). At P28, phospho-mimic adducin expresses on membrane and in nucleus (Bottom right). Scale bar = 10 μm. G. (Left) Representative images to show sarcomere pattern from phospho-ADD1 i1 transgenic at P14 and P28. Insets are high magnification images of single cardiomyocyte. Dotted lines are drawn around outer and inner edges of cardiomyocytes. (Right) Quantitative graph to show sarcomere disassembly. Disassembly scores were defined the same way as that of Add1i2 transgenic in . Scale bar = 10 μm. Scale bar= 10 μm. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Expressing, Phospho-proteomics, Immunostaining, Staining, Over Expression, Plasmid Preparation, Transgenic Assay, Control, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Phospho-adducin expression profile in regenerating neonatal heart at P4 by anti-pT445 staining. Note that the expression of pT445 translocates from nucleus to cytoplasm during cardiomyocyte mitosis. Scale bar= 10 μm. B & C. Overexpression of adducin mutants S12/S355 ( B ), and S714/724 ( C ) in NRVM induced by AAV6. (Top) Schematic diagram of AAV shuttle vectors for adducin expression. Mutants are expressed with 3 tandem FLAG epitopes in frame at the N-terminus. Middle. Immunofluorescent staining with α-actinin antibody (red) to show sarcomeric structure. Adducin mutants are detected by Flag antibody in green. Insets show high magnification images of boxed region. Bottom. Quantitative analysis of disassembled sarcomeres induced by adducin overexpression. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Expressing, Staining, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. HEK293T cells were transfection of variant 1(p-3xFlag-Add1i1) and Add1 variant 2 (p-3xFlag-Add1i2) in the presence or absence of Add3 (p-3xTy1-Add3). 24 hours after the transfection, the cells were treated with 80 μM cycloheximide (CHX) to inhibit protein translation. Cells were collected 0, 24, and 48 hours after treatment. (Left) WB to show protein level. (Right) Measurement of relative ADD1i1 or Add1i2 protein level (calculated as FLAG vs GAPDH) in the absence or presence of ADD3. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a one-tailed t -test. B. Schematic diagram to show the constructs of generating Add1 i2 and Add3 double transgenic. Both expression constructs were driven under an α-MHC promoter. C. QPCR results to show the mRNA level of Add1i2 in 2-month-old double transgenic with low expression. D. (Left) H&E staining of control and cardiac-specific Add1 i2/Add3 double transgenic with low expression. Samples are of two months old mice. Scale bar = 1 mm. (Right) Heart weight to body weight ratio in WT and low expression double transgenic. E. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in 2-month old ADD1 i2/ADD3 double transgenic (Low expression). n=3 for each group. F. Longitudinal views to show sarcomere pattern from 2 months old WT and Adducin double transgenic (Low expression). The high magnification images of boxed region in low magnification image are shown on the right. Dotted lines the border where adducin is expressed. Inset shows partial disassembled sarcomeres (disorganized but not absent sarcomeres). The corresponding adducin expression are indicated by arrows. Scale bar= 10 μm. G. QPCR results to show the mRNA level of Add1i2 in 2-month-old double transgenic with high expression. H. (Left) H&E staining of control and cardiac-specific Add1 i2/Add3 double transgenic with low expression. Samples are of 2-month-old mice. Scale bar = 1 mm. (Right) Heart weight to body weight ratio in WT and high expression double transgenic. I. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in 1-month-old ADD1 i2/ADD3 double transgenic (high expression). (n=3 for each group). J. (Top) Representative images to show sarcomere pattern from 1-months old ADD1 i2/ADD3 double transgenic (high expression). (Bottom) Enlarged images of Insets from above panels. K. (Left) ADD1 i2/ADD3 double transgenic with high expression has evidence of cardiomyocytes fragmentation. (Right) High magnification of boxed region on the left. Arrows point to sarcomere fragments where ADD1 and TNNT2 are colocalized. Scale bar = 10 μm. Scale bar= 10 μm. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Transfection, Variant Assay, One-tailed Test, Construct, Transgenic Assay, Expressing, Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. (Top) Schematic diagram of the constructs of generating phospho-mimic Add1 i2 and Add3 double transgenic. Glu substitution on T445 and T480 mimic phosphorylation form of Add1 i2. Both expression constructs were driven under an α-MHC promoter. (Bottom) H&E staining of control and cardiac-specific phospho-Add1 i2/Add3 double transgenic. Samples are of two-month-old mice. Scale bar = 1 mm. B. Heart weight to body weight ratio in WT and phospho double transgenic at P21 days and 2 months. (n=3 for P21, and n=5 for 2 months). C. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in P21 and 2 months old ADD1 i2/ADD3 double transgenic (Low expression). (n=3 for P21, and n=5 for 2 months). D. QPCR of mRNA level of Add1i2 in 2-month-old phospho-double transgenic. E. Sarcomere pattern of phospho-double transgenic at P7 (i), P14 (ii), P21 (iii) and 2-month-old (iv). The high magnification images of boxed region are shown on the right side of zoom out image. Dotted lines circle out the boarder where adducin expresses outside sarcomeres. Arrows refer to regions with high level of adducin expression. Note the clearance of sarcomeres where adducin is expressed. Scale bar= 10 μm. F. Large scale kinase screening assay identified 7 candidates that potentially phosphoorylate T445 (left) and T480 (right) sites. Corrected kinase activity values (raw value minus sample peptide background, in cpm) with sample peptide at one concentration in 245 Ser/Thr peptide kinase assays. Blue: Kinase with sample peptide; yellow: Kinase activity w/o sample peptide. G. Serine/Threonine Kinases Hit Assay to verify 3 kinases, NEK1, DCAM1 and IRAK4, selected from initial kinase screening assay. Each kinase was tested at three concentrations in triplicates. These results suggested that the kinases NEK1, DCAMKL2, IRAK4 all show a concentration-dependent ability to phosphorylate the sample peptide “T445” and “T480”. H. Validation of endogenous expression pattern of IRAK4 (Left) and NEK1 (Right) in the regenerating neonatal heart. Positive signals are indicated by arrow. I. (Top) Schematic of IRAK4 expression cassettes in AAV6 constructs. Overexpress IRAK4 (green) in NRVM causes sarcomere (stained with α-actinin, red) disassembly (i) and pT445 adducin (stained with pT445, red) translocated from nucleus to cytoplasm (ii). Arrowheads indicate adducin is expressed in the nuclei in IRAK4 negative cells. Scale bar = 10 μm. J. (Top left) Schematic of IRAK4 expression cassettes in AAV9 constructs. (Top right) Schematic drawing of experimental design. (Bottom) AAV9-IRAK4-GFP was injected into CD1 pups at P1 and hearts were collected at P15 for sectioning and staining. IRAK4 overexpression caused strong sarcomere disassembly (i) in cardiomyocyte compared to littermate controls (ii).

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Construct, Transgenic Assay, Phospho-proteomics, Expressing, Staining, Control, Screening Assay, Activity Assay, Concentration Assay, Biomarker Discovery, Injection, Over Expression

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Table lists selected ADD1 associated proteins identified from the Co-IP/MS. * PSM: Peptide Spectrum Matches. Number of spectra assigned to peptides that contributed to the inference of the protein. **MIC Sin: The normalized spectral index statistic for the protein for the specific group. Calculated from the intensity of fragment ions in each spectrum assigned to a particular protein. ***Ratio: Quantitative ratio for protein between groups derived from the MIC Sin value. Scale bar=10 μm. B . Schematic of the purification process of short and long adducin complexes. C. Schematic drawing shows the flowchart of Co-IP/MS by purified adducin complex pulldown D. Bar graph for Canonical Pathways enriched in each sample. X-axis shows transformed adjusted p value (Benjamini-Hochberg Procedure) of overlap between sample proteins and pathways. FDR refers to false discovery rate. *P<0.05, **P<0.01, ***P<0.001; Statistical significance was calculated using a two-tailed t -test. E. Network plot of cytoskeleton proteins from the pull down assay interact with adducin_long or adducin_short at different time points (P1 and P21). Different color labeling highlighting common and unique proteins among sample groups. Red color represents the four sample groups in the pulldown assay. Yellow color represents proteins common to all groups. Green represents proteins common to only two or three groups. Blue represents proteins unique to each group. F. Proximity ligation assay (PLA) to examine direct interaction between adducin (pT445) and sarcomeric proteins (α-actinin 2) in P4 heart tissue. The red dots (arrows) are fluorescent signals which indicate a close interaction between two antigens. Samples include Interaction between α-actinin and troponin T (as positive control) or phospho-adducin (pT445) and α-actinin. Scale bar=10 μm. G. Immuno-EM image to show the subcellular location of phospho-adducin in cardiomyocytes with disassembled sarcomeres in neonatal MI hearts at day 3 after surgery. Two red boxes in left image are magnified on the right. (i) adducin is localized to z-disks. (ii) phospho-adducin is heavily localized to z-disks associated with plasma membrane in cardiomyocytes with disassembled sarcomeres. Red arrows indicate location of adducin.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Purification, Transformation Assay, Two Tailed Test, Pull Down Assay, Labeling, Proximity Ligation Assay, Positive Control, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

doi: 10.1101/2022.11.29.518315

Figure Lengend Snippet: (a) Immunoblotting of cleaved caspase 7 following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNα and 1000 U/mL TNFα) (Mix) for 96 h. Tubulin was used as loading control (n=3). (b) Immunoblotting of IκBα, phosphorylated STAT1, and phosphorylated JNK following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNγ and 1000 U/mL TNFα) alone or in combination (Mix) for 30 min. GAPDH was used as loading control. Representative image (n=3). Lysates from INS-1E cells with/without cytokine exposure were used as positive control. Gene expression of (c) DDIT3/CHOP, (d) HRK/DP5 and (e) CXCL10 in cells exposed to individual cytokines for 48 h. GAPDH was used as housekeeping gene. Data are means ± SEM (n=4), *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-MHC-I (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-GAPDH (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076) or anti-rabbit (#7074) (Cell Signaling) IgG antibodies.

Techniques: Western Blot, Control, Positive Control, Gene Expression

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: bioRxiv

Article Title: A Diversity Outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

doi: 10.1101/2021.10.07.463570

Figure Lengend Snippet: A) Tumor volumes (mean mm 3 ± SE) of CCB6F1 with (dashed lines) and without (solid lines) ICI treatment on days 3, 6, and 10. Significance determined by Student’s T-test of tumor volume at day 15 or 16, prior to mice from either group being euthanized. B) qRT-PCR of CD8, IFNγ, and downstream IFNγ response genes PD-L1 and STAT1 relative to GAPDH from flash frozen tumors in ICI treated and untreated mice. Nondetects in samples were set to a Ct of 55 to reduce bias. Significance is determined by Student’s T-test. C) Averaged qRT-PCR results from grouped predicted non-responder mice (CC44, CC17) versus predicted responder mice (CC36, CC42, CC51) for transcripts in (B) . Significance determined by Student’s T-test. D) Mean Fluorescence Intensity (MFI) of anti-B16F0 IgG in serum of CCB6F1 mice measured by flow cytometry. Statistical significance determined via Student’s T-test.

Article Snippet: Tumor tissue from mice was flash-frozen in liquid nitrogen and homogenized in Trizol as described by manufacturer (Thermo Fisher, Waltham, MA). cDNA was synthesized using a ProtoScript First Strand cDNA synthesis kit (New England Biolabs, MA, E6300S) using PolyDT primers. qRT-PCR was conducted with Taqman probes (Thermo Fisher) for GAPDH (Mm99999915_g1), CD8α (Mm01188922_m1), IFN-γ (Mm01168134_m1), PD-L1 (Mm03048248_m1), STAT1 (Mm01257286_m1).

Techniques: Quantitative RT-PCR, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: A Diversity Outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

doi: 10.1101/2021.10.07.463570

Figure Lengend Snippet: A) B16F0 tumor volume in CC007B6F1 mice with (n=7) and without (n=7) ICI on days 3, 6, and 10 (mean mm 3 ± SE). B) Manhattan plot from binary analysis of presence or absence of tumor development in treated DOB6F1 mice . QTL effects for the Chr6 peak indicate that the A/J genotype is a positive driver for complete response. C) qRT-PCR performed on flash frozen tumors from (A) for CD8, IFNγ, and downstream IFNγ response genes PD-L1 and STAT1. D) Chr6 genotype in CC007 mice (bracket indicates locus of interest). Modified from (13).

Article Snippet: Tumor tissue from mice was flash-frozen in liquid nitrogen and homogenized in Trizol as described by manufacturer (Thermo Fisher, Waltham, MA). cDNA was synthesized using a ProtoScript First Strand cDNA synthesis kit (New England Biolabs, MA, E6300S) using PolyDT primers. qRT-PCR was conducted with Taqman probes (Thermo Fisher) for GAPDH (Mm99999915_g1), CD8α (Mm01188922_m1), IFN-γ (Mm01168134_m1), PD-L1 (Mm03048248_m1), STAT1 (Mm01257286_m1).

Techniques: Quantitative RT-PCR, Modification

Journal: bioRxiv

Article Title: A Diversity Outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

doi: 10.1101/2021.10.07.463570

Figure Lengend Snippet: A) Schematic showing dates of pellet implantation, treatment dates, and approximate pellet depletion. All dates relative to tumor inoculation at day 0. B) B16F0 tumor volume in B6 mice receiving a subcutaneous placebo pellet (left) or 30-day slow release prolactin pellet (right) 7 days prior to inoculation. Mice were treated with combined ICI on days 3, 6, and 10 after tumor inoculation. C) qRT-PCR from flash frozen tumors from (B) for CD8, IFNγ, and downstream IFNγ response genes PD-L1 and STAT1.

Article Snippet: Tumor tissue from mice was flash-frozen in liquid nitrogen and homogenized in Trizol as described by manufacturer (Thermo Fisher, Waltham, MA). cDNA was synthesized using a ProtoScript First Strand cDNA synthesis kit (New England Biolabs, MA, E6300S) using PolyDT primers. qRT-PCR was conducted with Taqman probes (Thermo Fisher) for GAPDH (Mm99999915_g1), CD8α (Mm01188922_m1), IFN-γ (Mm01168134_m1), PD-L1 (Mm03048248_m1), STAT1 (Mm01257286_m1).

Techniques: Quantitative RT-PCR