Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and group quantitation for circulating Ly6C low CD169 + Tim4 + macrophages in sham-operated and MI wild type (WT) C57BL/6 mice 1 d post-MI; n=4/group, statistics: unpaired t test. B. Left , Flow histograms demonstrating surface expression of CCR3 and CCR4 in Ly6C low CD169 + Tim4 + macrophages from the same groups; statistics: unpaired t test, NTM, normalized to mode, y-axis represents cell counts. Right , chemokine gene expression by RT-PCR (normalized to 18s) in the myocardial border zone (BZ) 1 d after MI or sham operation; n=4-5/group, statistics: unpaired t test. *p<0.05, **p<0.01, ***p<0.001 versus sham. C . Left , Circulating Ly6C low CD169 + Tim4 + cell frequency prior to and 1 d after MI in Spx mice; n=9, statistics: paired t test. NS, not significant. D. FACS density plots, histograms, and quantitation of cardiac Ly6C low CD169 + Tim4 + macrophages in WT and Spx mice 1 d post-MI or sham operation; n=4-7/group, statistics: unpaired t test. E . Immunostains and FACS dot plots of splenic CD169 + MMMs (red) 24 h after MI or sham operation, and quantitation of MMM frequency by FACS and spleen weight (Wt); n=5-7/group, statistics: unpaired t test. TL, tibia length. F . Left , FACS dot plots and histograms, and corresponding quantitation, of blood and heart Ly6C low CD169 + Tim4 + Bioparticle + cells from sham and MI mice given 10 mg/kg Texas Red-conjugated bioparticles i.v. 3 h before sacrifice; n=3-4/group, statistics: unpaired t test for blood and non-parametric Mann-Whitney U test for heart (non-normal distribution). Right, quantitation of splenic BioParticle + MMMs in the same experimental mouse groups; statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left, FACS pseudocolor plots of cardiac CD169 + Tim4 + macrophages and separation based on LYVE1 surface expression in naïve and 1 d post-MI mice. Right , quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in hearts from naïve and 1 d post-MI mice. n=4-6/group; statistics: unpaired t test. B . Heat map of 462 significant (p adjusted<0.05) DEGs by RNAseq analysis in sorted LYVE1 hi and LYVE1 low CD169 + Tim4 + cardiac macrophages 1 d post-MI. C. PCA plots using the top 500 DEGs after rlog transformation of RNAseq data from these macrophages sorted from the indicated sites in naïve and 1 d post-MI mice. D. Left , Flow histograms depicting LYVE1 surface expression on cardiac CD169 + Tim4 + macrophages (green) and total Autofluorescence + CD64 + MHCII + macrophages (brown) in WT and Spx mice, 1 d post-MI. Right , FACS quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in the hearts of WT and Spx mice, 1 d post-MI; n=5-6/group. Statistics: unpaired t test. E. Representative confocal micrograph of border zone (BZ) myocardium immunostained for CD169 (red) and Tim4 (green) 1 d post-MI in WT and Spx mice; nuclear staining with DAPI (blue). Scale bar, 200 μm. Inset shows magnified images of CD169 and Tim4 staining. Yellow arrows indicate double positive cells; scale bar 10 μm.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Expressing, Quantitation Assay, Transformation Assay, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Protocol for LXRα agonist T0901317 treatment (40 mg/kg i.p.) from 1 d prior to 5 d post-MI, with 10 d post-MI follow-up, in WT and Spx mice. B . FACS contour plots and quantitation of cardiac CD169 + Tim4 + macrophages 1 d post-MI and blood CD169 + Tim4 + macrophages 10 d post-MI in untreated and T0901317-treated WT and Spx mice. N=5-6/group, statistics: one-way ANOVA, Bonferroni post-test. C . Kaplan-Meier survival curves post-MI in untreated and T0901317-treated WT and Spx mice. Statistical comparisons by log-rank test, group sizes as indicated. D . Representative end-diastolic long-axis 2-dimensional echocardiograms and group data for LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) in the same mouse groups at 10 d post-MI; n=5-10/group, statistics: one-way ANOVA, Bonferroni post-test. E . Top , Kaplan-Meier survival curves over 8 w post-MI in WT mice treated with either vehicle or T0901317 from 1 d before MI to 5 d post-MI (statistical comparison by log-rank test, n=12-17/group as indicated) and group data for LVEF, LVEDV, LVESV, and normalized heart and lung weight at 8 w post-MI; n=8-9/group for echocardiography, n=4-7/group for gravimetry. Statistical comparisons: unpaired t test. HF, heart failure; TL, tibia length; NS, not significant. Bottom Left , Representative Masson’s trichrome staining of LV short-axis sections (2x magnification) and infarct border zone (BZ, scale bar 500 μm), along with quantitation of cardiac fibrosis (BZ and remote zone [RZ], blue staining) in vehicle- and T0901317-treated WT HF mice. Also shown are confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the RZ of hearts from vehicle-and T0901317-treated HF mice (8 w post-MI) and quantitation of iNOS + CD206 + and iNOS − CD206 + macrophages (Mφ) per mm 2 . Double positive (CD206 + iNOS + ) cells appear yellow (arrows). DAPI (blue) was used for nuclear staining. N=4-8/group, statistics: unpaired t test. Bottom Right , Representative FACS contour plots to identify Ly6C hi monocytes in vehicle- and T0901317-treated WT HF mice (8 w post-MI), and corresponding quantitation. N=5-10/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Comparison, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Control, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown may lead to increase in BMP5 expression. ( A ) Regulatory factors suppressing tumor aggressiveness were identified by using microarray analysis. ( B ) Expression levels for each gene were compared (ratios between gene expression levels of non-silencing control). In two G9a knockdown cells (shG9a #1 and shG9a #2), expression levels for each gene were compared to those of non-silencing control. Then the most upregulated and downregulated genes were screened. ( C ) G9a knockdown cells showed increase in BMP5 expression. ( D ) Reduced G9a occupancy was found at the BMP5 promoter region. G9a knockdown cells also showed decreased H3K9me2 occupancy at the promoter region of BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Expressing, Microarray, Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: BMP5 contributes to reduce migration and invasion abilities of tumor cells. ( A ) Low BMP5 expression is associated with poor survival outcomes of breast cancer patients. ( B ) Patients with stage 3 disease showed higher BMP5 levels than those of stage 2 patients. ( C , D ) Migration/invasion abilities were decreased by treatment of recombinant BMP5; however, the capabilities were enhanced following BMP5 downregulation. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Migration, Expressing, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown facilitates Smad protein phosphorylation via BMP5 activation. ( A , B ) G9a-knockdown-induced increase in BMP5 expression had no effect on the total level of either Smad1 or Smad5. G9a knockdown increased phosphorylation of Smad1/5/9. ( C ) ICC demonstrated that nuclear translocation of pSmad1/5/9 was increased in G9a-depleted MCF7 cells. ( D ) Similar tendency was found after treatment of recombinant BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Phospho-proteomics, Activation Assay, Expressing, Translocation Assay, Recombinant

Journal: Scientific Reports

Article Title: Identified endoplasmic reticulum stress-related molecular cluster and immune characterization in endometriosis

doi: 10.1038/s41598-025-22400-9

Figure Lengend Snippet: qRT-PCR and IHC validation the hub gene expression level. ( a ) Hub ERS genes mRNA expression of normal, eutopic and ectopic endometrium. ( b ) Immunostaining of EPAS1, F8, VCAM1, VWF protein expression. ( c ) Semi-quantitative analysis of IHC results. qRT-PCR, real-time quantitative reverse transcription; IHC, immunohistochemistry. * p < 0.05, ** p < 0.01, ∗ ∗ ∗ p < 0.001, **** p < 0.0001.

Article Snippet: Briefly, the sections were stained with the primary antibody against F8 (1:500, affinity, USA), VCAM1(1:200, Proteintech, Wuhan, China), VWF (1:400, Proteintech, Wuhan, China), and EPAS1(1:50, Proteintech, Wuhan, China) and then scanned with a digital scanner.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Expressing, Immunostaining, Reverse Transcription, Immunohistochemistry

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: The spot annotated within the dashed box (absent in the control IP) was identified as human ATXN3 by subsequent MS/MS analysis .

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Control, Tandem Mass Spectroscopy

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: HEK-293 cells were transfected with either PNKP siRNA or ATXN3 siRNA or control siRNA and the nuclear extracts (1 mg) prepared from those cells were IP’d with anti-PNKP Ab (BioBharati Life Science Pvt. Ltd, Kolkata, India; 2A ) or anti-ATXN3 Ab (Proteintech, 2B ) or with IgG as a control, and tested for the presence of PNKP- and ATXN3-associated proteins with Abs to the proteins shown on the right. (C) Detection of PNKP’s interaction with ATXN3 in SH-SY5Y cells by proximity ligation assays using a Duolink kit (Olink Bioscience, Uppsala, Sweden). Nuclei were counterstained with DAPI (blue). ATXN3-depleted (by siRNA) cells were used as a control to show the specificity of the interaction (middle panel). Right panel, Non-specific Ab (IgG) control (D) GST-PNKP pull-down of ATXN3 (WT and mutant) using purified GST-tagged full-length or three domains (FHA-, Kinase- and Phosphatase-domain) of PNKP, probed with anti-ATXN3. Bottom panel, Coomassie-stained gel of the corresponding PNKP domains, a second gel run in parallel.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Transfection, Control, Ligation, Mutagenesis, Purification, Staining

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) A 32 P-labeled 3’-phosphate-containing oligo substrate (5 pmol) was incubated at 37°C for 10 min in buffer A (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol and 0.1 μg/μl acetylated BSA) with PNKP alone (50 fmol, ln 2) or ATXN3-Q29 alone (400 fmol, ln 8) or PNKP plus increasing amounts of Q29 (25, 50, 100, 200 and 400 fmol; lns 3–7). Ln 1, substrate only. Generation of the 32 P-labelled 3’P-containing oligo substrate was described previously . (B) Effect of mutant ATXN3 (Q72) on PNKP’s 3’-phosphatase activity. 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with PNKP at 37°C for 20 min alone (50 fmol, ln 2), ATXN3-Q72 alone (200 fmol, ln 3), PNKP (50 fmol) plus increasing amounts of Q72 (50, 100 and 200 fmol, lns 4–6). Ln 1, no protein, substrate only. Quantitation of the products (released phosphate) is represented in the histogram (lower panel), with the activity of PNKP alone arbitrarily set as 1 (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Labeling, Incubation, Mutagenesis, Activity Assay, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Western blot analysis showing the level of expression of ATXN3 in SH-SY5Y cells stably expressing control vs. ATXN3-shRNA. Lamin B was used as a loading control. (B) A representative gel (n = 3) showing the 3’-phosphatase activity in the nuclear extract (200 ng) of control (ln 3) and ATXN3-depleted cells (ln 4). Lns 5–8, increasing amounts (10, 25, 50 and 100 fmol) of purified ATXN3 (WT) were added back to ATXN3-depleted nuclear extract. Quantitation of the products is represented in the histogram (n = 3, ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Western Blot, Expressing, Stable Transfection, Control, shRNA, Activity Assay, Purification, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: Long amplicon qPCR (LA-QPCR) was used to evaluate genomic DNA SB levels in control vs. ATXN3-depleted HEK-293 cells. Representative gel showing PCR-amplified fragments of the HPRT (left panel) and POLB (right panel) genes. Amplification of each large fragment (upper panels) was normalized to that of a small fragment of the corresponding gene (bottom panels) and the data were expressed as lesion frequency/10 Kb DNA as described previously . Histograms represent the DNA damage quantitation for control vs ATXN3-depleted cells (n = 3, ** = P< 0.01). Error bars indicate standard error of means.

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Amplification, Control, Quantitation Assay

Journal: PLoS Genetics

Article Title: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

doi: 10.1371/journal.pgen.1004749

Figure Lengend Snippet: (A) Upper panel, 32 P-labelled 3’-phosphate-containing oligo substrate (2.5 pmol) was incubated with the NE (250 ng) isolated from rat mesenchymal (CSM14.1) cells conditionally expressing vector alone (lane 1), WT (Q-23, lane 2) or mutant ATXN3 (Q-70, lane 3). Ln 4, no protein. S: substrate, P: product (released 32 P i ). Middle panel: Western analysis of the corresponding NE (10μg) showing PNKP expression. GAPDH (Ab from Genetex Inc.) was used as loading control. Bottom panel: quantitation of the products (P) is represented in a histogram. (n = 3, ** = P< 0.01) (B) Comet assays showing the accumulation of DNA SBs in mutant (Q-70) ATXN3 cells. (C) Comparative (WT vs. SCA3 mice) 3’-phosphatase activity in the NEs from four different brain regions. 7.5 pmol of 3’-P-containing substrate was incubated at 37°C for 10 min with WT or transgenic mouse brain NE (200 ng; PN-Pontine Nuclei, SN-Substantia Nigra, DCN-Deep Cerebellar Nuclei, and HP-Hippocampus). Quantitation of the products is shown in the histogram with the age-matched WT arbitrarily set as 1 (n = 5; ** = P< 0.01).

Article Snippet: In situ Proximity Ligation Assay (PLA) between PNKP (anti-mouse Ab, a gift from Michael Weinfeld) and ATXN3 (anti-rabbit Ab, Proteintech) was carried out according to the protocol as described [ ] using a Duolink PLA kit (QLink Bioscience Cat# LNK 92101 K101, Uppsala, Sweden).

Techniques: Incubation, Isolation, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Control, Quantitation Assay, Activity Assay, Transgenic Assay

Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: Primers for RT‒qPCR.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques:

Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: Itraconazole inhibited the HH signaling pathway in HemECs and HUVECs. HemECs and HUVECs were treated with itraconazole (HemECs: 0.5, 2 μM; HUVECs: 5, 20 μM) for 24 and 48 h. The protein levels of SHH, PTCH1, SMO, and GLI1 in HemECs and HUVECs were detected through western blotting ( A , B ). The mRNA levels of SHH, PTCH1, SMO, and GLI1 in HemECs and HUVECs were measured through RT‒qPCR ( C , D ). * P < 0.05, ** P < 0.01, compared with untreated cells.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques: Western Blot

Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: rhSHH attenuated the effects of itraconazole on angiogenesis and downregulation of the HH signaling pathway. HemECs and HUVECs were treated with 2 μg/ml rhSHH, 2 μM itraconazole, or 2 μg/ml rhSHH +2 μM itraconazole for 24 h. HUVECs were treated with 2 μg/ml rhSHH, 20 μM itraconazole, or 2 μg/ml rhSHH +20 μM itraconazole for 24 h. Vascular angiogenesis of HemECs and HUVECs was determined using the tube formation assay ( A , B ). Scale bar = 0.1 mm. The protein levels of VEGFA were detected using ELISAs ( C ). The protein and mRNA levels of SHH, PTCH1, SMO, and GLI1 in HemECs ( D ) and HUVECs ( E ) were determined through western blotting and RT‒qPCR, respectively. * P < 0.05, ** P < 0.01, compared with untreated cells. # P < 0.05, ## P < 0.01, compared with the itraconazole group.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques: Tube Formation Assay, Western Blot

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a Cells were treated with 0.2 mM of 15 N-TMZ for 2 h. Intracellular 15 N-AICA were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Control vs. HPRT1 shRNA, P = 5.27e-10; WT vs. D138N, P = 1.63e-09; WT vs. K166A, P = 1.95e-09; MES28, Control vs. HPRT1 shRNA, P = 2.98e-11; WT vs. D138N, P = 2.61e-12; WT vs. K166A, P = 1.96e-12. b Cells were treated with 0.2 mM of 15 N-TMZ for 2 h. Intracellular 15 N-AICAR levels were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Control vs. HPRT1 shRNA, P = 4.75e-08; WT vs. D138N, P = 1.18e-10; WT vs. K166A, P = 1.32e-10; MES28, Control vs. HPRT1 shRNA, P = 1.61e-09; WT vs. D138N, P = 1.67e-08; WT vs. K166A, P = 1.31e-08. c Representative chromatograms of products of the HPRT1 kinase assay. d Michaelis-Menten curve of HPRT1 for AICA. Reactions were performed by mixing purified active HPRT1 and AICA. Data represent the mean ± SD from sextuplicate experiments. e and f Cells with or without shRNA-mediated HPRT1 depletion were treated with or without 0.2 mM of TMZ ( e ) or 0.25 of mM AICA for 2 h ( f ), respectively. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. g HPRT1-depleted cells with reconstituted expression of WT Flag-HPRT1, Flag-HPRT1 D138N, or Flag-HPRT1 K166A were treated with or without 0.2 mM of TMZ for 2 h. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. Statistics: a , b unpaired Student’s t -test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Control, shRNA, Kinase Assay, Purification, Western Blot, Expressing, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a – e, g – i Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. a – i Three biological repeats were repeated independently with similar results. a Cells pretreated with or without 0.25 mM of AICA were treated with or without 0.2 mM of TMZ for the indicated time points. b Cells pretreated with or without 0.25 mM of A769662 were treated with or without 0.2 mM of TMZ for the indicated time points. c Cells were treated with or without 0.2 mM of TMZ for 2 h. d In vitro phosphorylation and SDS-PAGE analysis and autoradiography were performed by mixing purified WT His-RRM1 or His-RRM1 T52A protein with active AMPK in the presence of [γ- 32 P]ATP. e Bacterially purified WT His-RRM1 or His-RRM1 T52A was incubated with or without active AMPK in the presence or absence of ATP. f Stoichiometry of RRM1 phosphorylation by AMPK. Bacterially purified His-RRM1 was incubated with active AMPK in the presence of [γ- 32 P]ATP. The radioactive intensity of incorporated 32 P was measured and the incorporation of 32 P into RRM1 was calculated. Data represent the mean ± SD of triplicate samples. g The indicated cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with the indicated dose of TMZ for 2 h. h AMPKα1/2 double knockout (DKO) U87 cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ for 2 h. C1 and C2, two clones of AMPKα1/2 DKO U87 cells. i The indicated cells with or without HPRT1 depletion were transfected with vectors expressing WT Flag-RRM1 or Flag-RRM1 T52A. The cells were further treated with 0.2 mM of TMZ for 2 h.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Immunoprecipitation, Western Blot, In Vitro, Phospho-proteomics, SDS Page, Autoradiography, Purification, Incubation, Expressing, Double Knockout, Clone Assay, Transfection

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a and b WT His-RRM1 or His-RRM1 T52A mutant proteins were mixed with His-RRM2 and incubated with active AMPK in the presence of ATP for 20 min followed by HPLC-MS analysis. ( a ) Ribonucleotide reductase (RNR) activity was measured according to dCDP production. ( b ) K d was calculated. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. a WT + AMPK vs. T52A + AMPK, P = 2.91e-08; b His-RRM1 WT vs. His-RRM1 WT + AMPK, P = 3.73e-05; His-RRM1 WT + AMPK vs. His-RRM1 T52A + AMPK, P = 0.00032. c Cells with or without HPRT1 depletion were transfected with Flag-RRM1 and treated with or without 0.2 mM of TMZ for 2 h. RNR activity was measured according to dCDP production. Immunoblotting analysis was performed to confirm the AMPK-mediated phosphorylation status of RRM1. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 3.18e-08; MES28, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 1.56e-11. d Cells with or without HPRT1 depletion were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, TMZ vs. HPRT1 shRNA + TMZ, P = 1.12e-08; MES28, TMZ vs. HPRT1 shRNA + TMZ, P = 1.29e-06. e RRM1-depleted cells with reconstituted expression of Flag-RRM1 WT or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. ** P < 0.001. U87, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 3.68e-05; MES28, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 4.7e-09. Statistics: a – e unpaired Student’s t -test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Mutagenesis, Incubation, Activity Assay, Transfection, Western Blot, Phospho-proteomics, shRNA, Expressing, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a Luciferase-expressing MES28 and U87 cells with or without HPRT1 depletion were intracranially injected into nude mice ( n = 10 for each group). Shown are luminescence intensity of tumors in representative mice at the indicated time points. b The survival time of the indicated groups of mice was recorded. ** P < 0.001. MES28, Control shRNA+TMZ vs. HPRT1 shRNA+TMZ, P = 1.97e-05; U87, Control shRNA+TMZ vs. HPRT1 shRNA+TMZ, P = 2.81e-05. c and d Representative IHC images of ACC1 pS79 ( c ) and γ-H2AX ( d ) were shown. Scale bars, 60 μm. n = 10 for each group. e Representative TUNEL images were shown. Scale bars, 60 μm. n = 10 for each group. f Luciferase-expressing MES28 and U87 cells with RRM1 depletion and reconstituted expression of WT Flag-RRM1 or the Flag-RRM1 T52A mutant were intracranially injected into nude mice ( n = 10 for each group). Shown are luminescence intensity of tumors in representative mice at the indicated time points. g The survival time of the indicated groups of mice was recorded. ** P < 0.001. MES28, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 4.42e-05; U87, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 0.00025. h Representative IHC images of RRM1 pT52 were shown. Scale bars, 60 μm. n = 10 for each group. i Representative TUNEL images were shown. Scale bars, 60 μm. n = 10 for each group. Statistics: b , g Log-rank test for two-group comparison. b Control shRNA + TMZ vs. HPRT1 shRNA + TMZ, g Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Luciferase, Expressing, Injection, Control, shRNA, TUNEL Assay, Mutagenesis, Comparison

Journal: Nature Communications

Article Title: Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas

doi: 10.1038/s41467-023-41663-2

Figure Lengend Snippet: a and b Kaplan–Meier survival analysis based on HPRT1 expression from collected primary GBM samples ( a ) and the indicated GBM datasets ( b ). c Kaplan–Meier survival analysis based on HPRT1 expression from the indicated glioma datasets. d – f Kaplan–Meier survival analysis based on HPRT1 ( d ), AMPK pT172 ( e ), and RRM1 pT52 ( f ) expression from collected recurrent GBM samples. g – i The Pearson correlation test was used to analyze the correlation among HPRT1, AMPK pT172, and RRM1 pT52. Statistics: a – f Log-rank test for two-group comparison. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ACC1 pS79 (11818), ACC1 (3676), AMPKα pT172 (50081), AMPKα (5831), AMPKα1 (4148), AMPK β1 (12063), γH2AX (9718), and α-Tubulin (3873) were purchased from Cell Signaling Technology (Beverly, MA, USA); Antibody against Flag (F3165) was purchased from Sigma-Aldrich (Shanghai, China); Antibodies against MGMT (ab108630) and UPRT (ab251653) were purchased from Abcam (Shanghai, China); Antibodies against APRT (21405-1-AP), OPRT (14830-1-AP), QPRT (25174-1-AP), HPRT1 (15059-1-AP), and RRM1 (10526-1-AP) were from Proteintech (Wuhan, China).

Techniques: Expressing, Comparison