Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: The differential activation of CD14 and CD16 monocytes responding to endothelial and epithelial dysfunction. The profile scores of molecules mediating the interplays of classic monocytes with vascular endothelial cells (A-B) and non-classic monocytes with alveolar epithelial cells (C—D). E. Immunostaining for CDH5 and γH2AX in coronary artery cryo-section between KD patient and healthy donor, indicating endothelial injuries in KD. F. Expression level of CCR1 , DYSF , SELL , LMNB1, and XAF1 in CD14 classical monocytes among five groups. G. UMAP projection of CCR1 , DYSF , SELL , LMNB1, and XAF1 positive cells, respectively . H. UMAP projection of CCR1 , SELL and XAF1 triple positive monocytes among five groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Wilcoxon rank-sum test, adjusted for Bonferroni post hoc test, had been applied.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Expressing

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: SELL+/CCR1+/XAF1+ CD14 monocytes enhanced the adhesion and damages to endothelial cells. A. Flow cytometry identified the higher percentages and median fluorescence intensity of SELL, CCR1, and LMNB1 in KD and COV. B. Immunostaining for CD14 and DYSF, XAF1 in isolated PBMCs. C. The ratio of DYSF positive CD14 monocytes in total classic monocytes among KD, COV, FLU, and healthy donors. And the median fluorescence intensity of XAF1 in CD14 monocytes among KD, FLU, and healthy donors. D-E. THP-1 had been stained with Calcein AM and transfected siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 before co-culture with HUVECs. Then the numbers of adhesion THP-1 with HUVECs were counted by every 20× field view. F. The expressions of TNFa and IL6 in HUVECs, and the ratio of γH2AX + cells in HUVECs after co-cultured with THP-1, which had been transfected with siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 . G. The inhibition of SELL , CCR1 and XAF1 together in THP-1 significantly reduced the adhesion between monocytes and endothelial cells. H. Collaborated inhibiton of SELL , CCR1 and XAF1 in THP-1 decreased the expression of IL6 and TNF-α in HUVECs with lower ratio of rH2AX+ cells after co-culture with THP-1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data. Bar, 100 μm.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Flow Cytometry, Fluorescence, Immunostaining, Isolation, Staining, Transfection, Co-Culture Assay, Cell Culture, Inhibition, Expressing

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Polarization of HepG2/C3A subclone F2 cells on semipermeable collagen inserts. (A to D) Immunostaining of F2 cells on semipermeable collagen inserts. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Bar =10 μm. (A and B) Maximum-intensity projections of x-y stacks (A) and x-z sections (B) for the tight-junction protein ZO-1. (C and D) x-y (C) and x-z (D) sections for DPP4. (E) Percentages of albumin exported from F2 cells. The albumin in the apical (black bars) and basolateral (gray bars) supernatants was quantified by ELISA. The data shown are means and standard deviations (SD) (n = 12) of the results of four experiments performed in triplicate. *, P < 0.05; ***, P < 0.001. (F) Total bile acids from 21-day cultures of F2 cells quantified by LC-MS. The results are expressed as percentages of exported bile acids in the apical (black bars) and basolateral (gray bars) supernatants. The data shown are means and SD (n = 4). *, P < 0.05. (G) Bile acids were quantified by LC-MS on day 21 postseeding. The amounts of each detected species in the apical (hatched bars) and basolateral (gray bar) supernatants are shown (n = 4). The bile acids quantified were allolithocholic acid (alloLCA), alpha-muricholic acid (aMCA), beta-muricholic acid (bMCA), cholic acid (CA), 3-sulfo-cholic acid (CA-3S), chenodeoxycholic acid (CDCA), 3-sulfo-cheno deoxycholic acid (CDCA-3S), deoxycholic acid (DCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), gamma-muricholic acid (gMCA), glycoursodeoxycholic acid (GUDCA), hyodeoxycholic acid (HDCA), isodeoxycholic acid (isoDCA), isolithocholic acid (isoLCA), lithocholic acid (LCA), tauroalfamuricholic acid (TaMCA), taurobetamuricholic acid (TbMCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), 3-sulfotaurolithocholic acid (TLCA-3S), ursodeoxycholic acid (UDCA), and omega muricholic acid (wMCA). For clarity, only detected bile acids are shown. (H) HepG2/C3A (circles) and F2 (squares) cells were infected with nHEV genotype 3 (1.35 × 106 HEV RNA copies/106 cells; white symbols) or eHEV genotype 3 (3.3 × 106 HEV RNA copies/106 cells; black symbols). Supernatants were collected every 2 days, and HEV RNA was quantified by RT-PCR. (I) HepG2/C3A (circles) and F2 (squares) cells were infected with eHEV genotype 3 (2.5 × 108 HEV RNA copies/106 cells). Supernatants were collected on day 15 postinfection, and HEV RNA was quantified by RT-PCR. The data shown are from three independent experiments performed in triplicate. The horizontal bars represent medians. *, P < 0.05. (J) Immunofluorescence of ORF2 protein in HepG2/C3A (white bars) and F2 (black bars) cells 21 to 30 days postinfection. Nuclei were stained with DAPI. The results are expressed as percentages of cells containing ORF2. The data shown are means and SD of the results of three independent experiments. ***, P < 0.001.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Staining, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Infection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Colocalization of ORF2 with Rab27A and DPP4 at the apical sides of hepatocytes. (A to E) Immunostaining of ORF2 protein (red) and Rab27a (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells on the merged image indicate the paths of the 2-pixel-wide scans shown in panel E. (C and D) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (C) or Manders (D) coefficients were calculated on each section. The mean proportion of ORF2 intensities in Rab27a (red) and of Rab27a intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. (F to J) Immunostaining of ORF2 protein (red) and DPP4 (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells in the merged image indicate the paths of the 2-pixel-wide line scans shown in panel J. (H and I) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (H) or Manders (I) coefficients were calculated on each section. The mean proportions of ORF2 intensities in DPP4 (red) and of DPP4 intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. Scale bars = 10 μm.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Infection

Journal: The Journal of Biological Chemistry

Article Title: A Novel Suppressive Effect of Alcohol Dehydrogenase 5 in Neuronal Differentiation *

doi: 10.1074/jbc.C114.561860

Figure Lengend Snippet: ADH5 counteracted differentiation of human neural stem cells toward neurons. A, immunostaining of the neural progenitor markers Nestin, Sox2, Pax6, and Ki67 in hNSCs. Scale bar, 50 μm. B, scheme of differentiating hNSCs toward neurons. Briefly, hNSCs were transduced with lentiviral vectors (ADH5, ADH5(R115D), or empty vector) at day 0. At day 4, the transduced hNSCs subcultured in a 24-well microplate were induced to differentiation toward neurons. Immunofluorescence and qPCR were performed at day 40. C, qPCR analysis of ADH5 expression in hNSCs and their neuron derivatives. D, qPCR analysis of SOX2 and Nestin in hNSCs transduced with lentivirus expressing either EGFP (Vector) or ADH5. E, Transwell assay to analyze the relative number of migrated hNSCs transfected with lenti-EGFP or lenti-ADH5 (wt). F, immunofluorescence staining of differentiated neurons indicated in B in the absence or presence of ADH5 inhibitor C3. Scale bar, 50 μm. G, qPCR analysis of ADH5, MAP2, and GAD67, in the differentiated neurons indicated in B and F. H, qPCR analysis of synapsin as a marker of neuron maturation in the differentiated neurons indicated in B and F. I, qPCR analysis of glial fibrillary acidic protein (GFAP), α-smooth muscle actin (SMA), and α-fetoprotein (AFP) lineage-specific markers in the differentiated neurons indicated in B and F. J, scheme of differentiating hNSCs toward neurons in the presence of ADH5 inhibitor C3. Briefly, hNSCs subcultured in a 24-well microplate at day 0 were induced to differentiation toward neurons at day 3 by differentiation medium together with C3. Immunofluorescence and qPCR were performed at day 9. K, qPCR analysis of neuron-specific markers, MAP2 and GAD67, in the differentiated hNSCs treated with C3 for 6 days. All the values are shown as means ± S.E. n = 3. Single asterisk, p < 0.05; triple asterisk, p < 0.001. NS, not significant.

Article Snippet: The following antibodies were used: β-actin (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), ADH5 (Proteintech), Sox2 (Abcam), Pax6 (Vector Laboratories), Nestin (Millipore), Ki67 (Covance), MAP2 (Millipore), and Tuj1 (Sigma-Aldrich).

Techniques: Immunostaining, Transduction, Plasmid Preparation, Immunofluorescence, Expressing, Transwell Assay, Transfection, Staining, Marker

Journal: Frontiers in Oncology

Article Title: PET/CT Imaging of Activated Cancer-Associated Fibroblasts Predict Response to PD-1 Blockade in Gastric Cancer Patients

doi: 10.3389/fonc.2021.802257

Figure Lengend Snippet: Association between the expression of FAP and immunosuppressive TME. (A) Representative images of IHC staining of FAP, macrophage M2 (CD163+), MDSC (CD11b+/CD33+) infiltration, and PD-1 expression in GC tissue microarrays. Scale bar = 100 µm. (B) Quantitative results of IHC for CD163, CD11b, CD33, and PD-1 in groups with low intensity and high intensity of FAP in GC tissue microarrays. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides and GC tissue microarrays ( n = 31) were stained with rabbit anti-FAP (Abcam, ab53066; Cambridge, MA, USA), anti-CD11b (Abcam, ab133357), anti-CD33 (Abcam, ab269456), anti-CD163(Abcam, ab182422), and anti-PD-1 (Abcam, ab243644) primary antibodies overnight at 4°C and incubated with anti-rabbit secondary antibody for 1 h at room temperature.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: PET/CT Imaging of Activated Cancer-Associated Fibroblasts Predict Response to PD-1 Blockade in Gastric Cancer Patients

doi: 10.3389/fonc.2021.802257

Figure Lengend Snippet: FAPI imaging characterizes the immunosuppressive TME in GC. (A) Representative 68 Ga-FAPI-04 PET/CT images of patients with gastric cancer and IHC staining images for tumor specimens from gastroscopical biopsy of FAP, MDSC (CD11b+/CD33+), and macrophage M2 (CD163+) infiltration. Scale bar = 100 µm. The primary lesions are marked by red arrows, and the metastatic lesions are marked by white arrows. (B) Correlation analysis of TMEscore and the corresponding mean 68 Ga-FAPI-04 uptake ratios in primary gastric cancer or liver metastases.

Article Snippet: The slides and GC tissue microarrays ( n = 31) were stained with rabbit anti-FAP (Abcam, ab53066; Cambridge, MA, USA), anti-CD11b (Abcam, ab133357), anti-CD33 (Abcam, ab269456), anti-CD163(Abcam, ab182422), and anti-PD-1 (Abcam, ab243644) primary antibodies overnight at 4°C and incubated with anti-rabbit secondary antibody for 1 h at room temperature.

Techniques: Imaging, Positron Emission Tomography-Computed Tomography, Immunohistochemistry

Journal: Cell Reports

Article Title: Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening

doi: 10.1016/j.celrep.2021.109218

Figure Lengend Snippet: Establishment of human alveolospheres (A) Schematics of sorting-based isolation and establishment of alveolospheres from human lung tissues. (B) Representative bright-field images of an alveolosphere (left) and airway organoid (right). (C) Immunostaining of HT2-280 (green, left), SFTPC (green, middle), and AQP5 (green, right) in alveolospheres. (D) Representative gating strategy for sorting HT2-280 + EPCAM + human AT2 cells. (E) Transmission electron microscopy images of alveolospheres. Microvilli (MVs) were observed in spheres at the apical lumen. The inset (right) shows a lamellar body (LB). (F) An MA plot comparing the transcriptomes between alveolospheres (n = 4) and airway organoids (n = 2) (passages 4–6). Genes with false discovery rate (FDR) < 0.01 are shown in dark gray. Representative genes that are upregulated in alveolospheres (green) and airway organoids (blue) are highlighted. Dotted lines show log 2 (fold change) values of 1 and −1. (G) Co-staining of SFTPB (green) and AQP5 (red) in alveolospheres. Nuclear counterstaining, Hoechst 33342 (C and G). Scale bar: 2 μm (E, right), 10 μm (E, left), 25 μm (B, right, C, and G), and 100 μm (B, left). See also Figure S1 .

Article Snippet: Rabbit monoclonal anti-AQP5 , Abcam , Cat#ab92320, RRID: AB_2049171.

Techniques: Isolation, Immunostaining, Transmission Assay, Electron Microscopy, Staining

Journal: Cell Reports

Article Title: Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening

doi: 10.1016/j.celrep.2021.109218

Figure Lengend Snippet: A human alveolosphere-based platform for SARS-CoV-2 infection (A) Schematics of SARS-CoV-2 infection to alveolospheres and measurement of viral RNA copy number in the supernatant. (B) Analysis of SARS-CoV-2 RNA copy number in the culture medium of alveolospheres (passage 3) (multiplicity of infection [MOI] = 10). Copy numbers are shown as RNA copies per 1 μL of culture medium. (C) TCID 50 assay using the culture medium of alveolospheres (passage 3, MOI = 10). Dotted line indicates detection limit. The data of (B) and (C) are representative of at least three independent experiments. (D) An MA plot comparing the transcriptomes between SARS-CoV-2-infected (n = 2) and mock-infected alveolospheres (n = 2) (MOI = 2). Genes with FDR < 0.01 are shown in red. Dotted lines show log 2 (fold change) values of 1 and −1. (E) Gene Ontology analysis of genes with FDR < 0.01 in SARS-CoV-2-infected alveolospheres. (F) Co-staining of SARS-CoV-2 nucleocapsid (N) protein (red) and HT2-280 (top, green) or AQP5 (bottom, green) in mock-infected (left) or SARS-CoV-2-infected (right) alveolospheres (passage 5) (MOI = 5). (G) Transmission electron microscopy images of human alveolospheres (passage 3) infected with SARS-CoV-2 (MOI = 5). Black arrowhead (top left) indicates secreted virions. White arrow indicates budding viral particles in ERGIC. Inset shows higher magnification. Data are demonstrated as mean ± SEM (B and C). Scale bars, 100 nm (G, right), 1 μm (G, left), and 25 μm (F). Nuclear counterstaining, Hoechst 33342 (F). See also Figure S3 .

Article Snippet: Rabbit monoclonal anti-AQP5 , Abcam , Cat#ab92320, RRID: AB_2049171.

Techniques: Infection, Staining, Transmission Assay, Electron Microscopy

Journal: Cell Reports

Article Title: Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening

doi: 10.1016/j.celrep.2021.109218

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-AQP5 , Abcam , Cat#ab92320, RRID: AB_2049171.

Techniques: Recombinant, Lysis, Blocking Assay, RNA Sequencing Assay, Infection, Plasmid Preparation, Expressing, Software