Journal: The Journal of Clinical Investigation

Article Title: Peripherally derived FGF21 promotes remyelination in the central nervous system

doi: 10.1172/JCI94337

Figure Lengend Snippet: (A) Representative images of β-klotho expression in the mouse spinal cord 3 days after LPC injection. (B) Representative images of spinal cord sections double-labeled for PDGFRα and Ki67. Graph shows quantitations as indicated in images (n = 3 for control, 3 for CKO); *P < 0.05. Arrows indicate Ki67+ cells colabeled with PDGFRα. (C) Representative images of spinal cord sections labeled for MBP. Graph shows quantitations as indicated in the images (n = 4 each); **P < 0.01. (D) Representative images of brain sections double-labeled for PDGFRα and Ki67 in the mouse cortex, 7 days after traumatic brain injury. FGF21 was administered i.c.v. 24 hours after LPC injection (n = 4); *P < 0.05. Arrows indicate Ki67+ cells colabeled with PDGFRα; arrowheads indicate Ki67+ cells not labeled with PDGFRα. (E) Representative images of brain sections labeled for MBP in the mouse cortex, 14 days after traumatic brain injury. Graph shows quantitations as indicated in the images (n = 6 for vehicle, n = 4 for FGF21); *P < 0.05. (F) Representative immunoelectron microscopy images of myelin in the mouse cortex, 14 days after traumatic brain injury. Graphs show quantitations of g-ratio indicated in the images (n = 4); **P < 0.01 as determined by Student’s t test. Error bars represent SEM. Scale bars: 20 μm (A); 50 μm (B and D); 200 μm (C and E); 2 μm (F).

Article Snippet: Sections were incubated with primary antibodies against mouse anti–human PDGFRα (1:50; 0100-0220, Serotec) and rabbit anti–human β-klotho (1:50; AV53325, Sigma-Aldrich) and Alexa Fluor 488–conjugated goat anti-mouse IgG and/or Alexa Fluor 647–conjugated goat anti-rabbit IgG (Invitrogen) secondary antibodies.

Techniques: Expressing, Injection, Labeling, Immuno-Electron Microscopy

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: Human glial chimeras were maintained on either a cuprizone (CZN)-containing or control diet from 12–24 weeks of age. Twelve weeks later, at 36 weeks, the mice were killed and their resident hGPCs isolated by CD140a-based FACS, which were then subjected to RNA-seq (n = 6).

Article Snippet: AB_396285 CD140a-PE PE-conj mouse anti-human CD140a, AR1 10 μL/ 10 6 cells 556002 BD Biosci AB_396286 Secondaries Rat anti-Mouse IgM MicroBeads 20 μL/10 7 cells 130-047-301 Miltenyi AB_244358 Rat anti-Mouse IgG2a+b MicroBeads 10 μL/10 7 cells 130-047-201 Miltenyi AB_244356 AlexaFluor 568 Goat anti-Mouse IgG (H+L) 1:400 A-11031 Invitrogen AB_144696 AlexaFluor 568 Goat anti-Mouse IgG1 1:400 A-21124 Invitrogen AB_2535766 AlexaFluor 488 Goat anti-Mouse IgG (H+L) 1:400 A-11029 Invitrogen AB_2534088 AlexaFluor 488 Goat anti-Mouse IgG1 1:400 A-21121 Invitrogen AB_2535764 AlexaFluor 647 Goat anti-Mouse IgG (H+L) 1:400 A-21235 Jackson AB_2535813 AlexaFluor 568 Goat anti-Rabbit IgG (H+L) 1:400 A-11036 Invitrogen AB_2534094 AlexaFluor 488 Goat anti-Rabbit IgG (H+L) 1:400 A-11034 Invitrogen AB_2576217 AlexaFluor 568 Goat anti-Rat IgG (H+L) 1:400 A-11077 Invitrogen AB_2534121 AlexaFluor 488 Goat anti-Rat IgG (H+L) 1:400 A-11006 Invitrogen AB_2534074 Open in a separate window

Techniques: Control, Isolation, RNA Sequencing

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet:

Article Snippet: AB_396285 CD140a-PE PE-conj mouse anti-human CD140a, AR1 10 μL/ 10 6 cells 556002 BD Biosci AB_396286 Secondaries Rat anti-Mouse IgM MicroBeads 20 μL/10 7 cells 130-047-301 Miltenyi AB_244358 Rat anti-Mouse IgG2a+b MicroBeads 10 μL/10 7 cells 130-047-201 Miltenyi AB_244356 AlexaFluor 568 Goat anti-Mouse IgG (H+L) 1:400 A-11031 Invitrogen AB_144696 AlexaFluor 568 Goat anti-Mouse IgG1 1:400 A-21124 Invitrogen AB_2535766 AlexaFluor 488 Goat anti-Mouse IgG (H+L) 1:400 A-11029 Invitrogen AB_2534088 AlexaFluor 488 Goat anti-Mouse IgG1 1:400 A-21121 Invitrogen AB_2535764 AlexaFluor 647 Goat anti-Mouse IgG (H+L) 1:400 A-21235 Jackson AB_2535813 AlexaFluor 568 Goat anti-Rabbit IgG (H+L) 1:400 A-11036 Invitrogen AB_2534094 AlexaFluor 488 Goat anti-Rabbit IgG (H+L) 1:400 A-11034 Invitrogen AB_2576217 AlexaFluor 568 Goat anti-Rat IgG (H+L) 1:400 A-11077 Invitrogen AB_2534121 AlexaFluor 488 Goat anti-Rat IgG (H+L) 1:400 A-11006 Invitrogen AB_2534074 Open in a separate window

Techniques: In Vivo, In Vitro

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AB_396285 CD140a-PE PE-conj mouse anti-human CD140a, AR1 10 μL/ 10 6 cells 556002 BD Biosci AB_396286 Secondaries Rat anti-Mouse IgM MicroBeads 20 μL/10 7 cells 130-047-301 Miltenyi AB_244358 Rat anti-Mouse IgG2a+b MicroBeads 10 μL/10 7 cells 130-047-201 Miltenyi AB_244356 AlexaFluor 568 Goat anti-Mouse IgG (H+L) 1:400 A-11031 Invitrogen AB_144696 AlexaFluor 568 Goat anti-Mouse IgG1 1:400 A-21124 Invitrogen AB_2535766 AlexaFluor 488 Goat anti-Mouse IgG (H+L) 1:400 A-11029 Invitrogen AB_2534088 AlexaFluor 488 Goat anti-Mouse IgG1 1:400 A-21121 Invitrogen AB_2535764 AlexaFluor 647 Goat anti-Mouse IgG (H+L) 1:400 A-21235 Jackson AB_2535813 AlexaFluor 568 Goat anti-Rabbit IgG (H+L) 1:400 A-11036 Invitrogen AB_2534094 AlexaFluor 488 Goat anti-Rabbit IgG (H+L) 1:400 A-11034 Invitrogen AB_2576217 AlexaFluor 568 Goat anti-Rat IgG (H+L) 1:400 A-11077 Invitrogen AB_2534121 AlexaFluor 488 Goat anti-Rat IgG (H+L) 1:400 A-11006 Invitrogen AB_2534074 Open in a separate window

Techniques: Marker, In Vitro, Recombinant, Clinical Proteomics, Software, Microscopy, Lysis

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Representative immunofluorescence images of AIDS-KS lesion tissues (lower) and their adjacent normal skin tissues (upper) stained with antibodies against mesenchymal (Nestin, PDGFRA, or α-SAM in green), endothelial (PDPN, CD31 or VEGFR2 in red), and KSHV (LANA in yellow) markers. The nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. Images of each individual channel for mesenchymal, endothelial and KSHV marker, respectively, are shown in . ( B) Triple labeling for mesenchymal, endothelial, and KSHV markers, demonstrating the colocalization of these three fluorescent signals in the same KS tumor cell, as revealed by the plot of fluorescence intensity profiles across a white arrow in panel A. (C) Co-expression of mesenchymal (Nestin, PDGFRA, and α-SAM), endothelial (PDPN, CD31, and VEGFR2), and KSHV (LANA) markers in KS early (patch and plaque) and late lesions (nodular). Boxed areas are enlarged. Scale bar, 50 μm. ( D) Number of LANA, Nestin, CD31, PDGFRA, PDPN, α-SAM, and VEGFR2 positive cells was counted from 4–6 individual fields (180μm x 150μm) composed mostly of spindle tumor cells and vessels in KS early (n = 7 samples) or late lesions (n = 5 samples). Numbers were compared by Chi-2 test. ( E) Spearman’s test shows a correlation between LANA expression and cells positive for PDPN in early and late KS tumors. ( F) Percentage of Nestin-/CD31-, Nestin+/CD31-, Nestin+/CD31+, Nestin-/CD31+ and PDGFRA-/PDPN-, PDGFRA+/PDPN-, PDGFRA+/PDPN+, PDGFRA-/PDPN+, and α-SAM-/VEGFR2-, α-SAM+/VEGFR2-, α-SAM+/VEGFR2+, α-SAM-/VEGFR2+ cells in LANA+ spindle tumor cells in early or late KS lesions.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Immunofluorescence, Staining, Marker, Labeling, Fluorescence, Expressing

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) The difference in morphology and structure between normal vessels (left) and KS specialized vessels (right). Higher magnifications of the black-boxed areas are shown underneath. Scale bar, 200 μm (upper), 50 μm (lower). Asterisk, KS abnormal vessels. (B) Expression patterns of mesenchymal and endothelial markers in normal and KS abnormal vessels. Samples were stained with antibodies against Nestin/PDGFRA/α-SAM (green), CD31/PDPN /VEGFR2 (red), and LANA (yellow), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 50 μm. Asterisk, KS abnormal vessels. (C) LANA+ abnormal hybrid vascular density in early and late KS tumors. The number of vessels was quantified from 4–6 individual fields (354μm x 246μm) for each KS tumor sample. Error bars represent mean ± SEM. All statistical analyses were performed using the Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Cell lysates from mock- and KSHV-infected PDLSCs at indicated time points were immunoblotted for PDGFRA, COL1A1, α-SAM, TAGLN, PROX1, PDPN, VEGFA, and β-actin. ( B) Relative mRNA levels of mesenchymal and endothelial related genes in mock- and KSHV-infected PDLSCs (K-PDLSCs) after 4 days infection. ( C) Mock- and KSHV-infected PDLSCs (2-D) were immunostained for LANA, TAGLN, PDPN, vWF, and CD31 at 4 days post-infection. Scale bar, 50 μm. ( D) A time course of K-PDLSC aggregating to form spheroid in non-adherent plates. Scale bar, 500 μm. ( E) Expression of LANA, TAGLN, PDPN, vWF, and CD31 in mock- and KSHV-infected PDLSC spheroid (3-D) at 4 days post-infection. Scale bar, 50 μm. ( F) The expression of endothelial markers in mock- and KSHV-infected PDLSCs under 2-D or 3D cell culture for 4d. ( G) The mRNA expression level of TAGLN , α-SAM , Nestin , PDGFRA , PDPN , ICAM , PROX1 , CD31 , and VEGFA was analyzed by RT- qPCR in K-PDLSC spheroids in comparison with their parallel 2D culture.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Infection, Expressing, Cell Culture, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Mock- and KSHV-infected PDLSCs and LECs were examined for PDGFRA and PDPN expression profile by flow cytometry analysis. Three subpopulations (xM, hybrid M/E, and xE) were quantified based on the PDGFRA and PDPN profiles ( n = 3 independent experiments). Statistical analyses were performed using two-tailed Student’s test and P-values were calculated by GraphPad Prism. *p < 0.05, **p < 0.01, ***p < 0.001. ( B) PDGFRA and PDPN expression in PDLSC and K-PDLSC spheroids at 4 days post-infection were analyzed by IFA. Scale bar, 50 μm. ( C) K-PDLSCs were stained for PDGFRA and PDPN and sorted by flow cytometry. The purified xM and M/E populations were cultured for 10 days and their PDGFRA/PDPN profiles were examined for their phenotypic plasticity. ( D) Western blot analysis of xM, M/E, and LECs for their mesenchymal and endothelial markers. ( E) The expression profiles of mesenchymal and endothelial markers in hybrid M/E and xM state cells were analyzed at the mRNA level by RT-qPCR. ( F) IFA analysis of LANA, TAGLN, and VCAM in xM and M/E state cells. Scale bar, 50 μm.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Infection, Expressing, Flow Cytometry, Two Tailed Test, Staining, Purification, Cell Culture, Western Blot, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Soft agar colony formation assay to determine anchorage-independent cell growth in PDLSCs, K-PDLSCs, xM, and M/E state cells. Representative cell colonies in soft agar are shown. Scale bar, 200 μm. (B) Images of PDLSC and K-PDLSC spheroids 48h after transferring onto nonadherent and adherent plates. Scale bar, 200 μm. (C) FASC analysis of the migrated cells detached from K-PDLSC spheroids for PDGFRA/PDPN profiles. The cells that were stained with mouse and rat IgG were used as a control for creating flow cytometry gates. (D) Illustration of spheroids Transwell invasion assay. ( E) Quantitation of the number of invaded cells from PDLSC and K-PDLSC spheroids. (F) PDPN/PDGFRA expression profiles of the invaded cells from K-PDLSC spheroids. (G) Transwell migration and invasion assays of PDLSC, K-PDLSC, xM, and M/E state cells. Quantitation of cell migration and invasion was shown on the right. (H) Tubule formation assays were performed with xM and M/E cells. HUVECs were included as a positive control. Error bars represent mean ± SEM (n = 3). Statistical analyses were performed using the two-tailed Student’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Soft Agar Assay, Transferring, Staining, Flow Cytometry, Transwell Invasion Assay, Quantitation Assay, Expressing, Migration, Positive Control, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Schematic diagram illustrating the process of transplanting PDLSC and K-PDLSC spheroids under the renal capsule of nude mice. ( B) The kidneys transplanted with PDLSC and K-PDLSC spheroids were harvested after 28 days. ( n = 3–4 mice for each group). ( C) Representative images of H&E staining of PDLSC and K-PDLSC spheroids transplants. Scale bar, 200 μm. (D) IHC staining of PDLSC and K-PDLSC spheroids transplants for LANA and Ki-67. (E) Immunofluorescent overview of Nestin, CD31, PDGFRA, PDPN, and LANA expression in PDLSC and K-PDLSC spheroids under the renal capsule. Scale bar, 50 μm.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) PDLSCs were transduced with different viral gene expression vectors as indicated. The expression of some of these viral genes in cells was verified by Western analysis . The effects of these viral gene products on the expression of PDGFRA, PDPN, ICAM, and VEGFA in PDLSCs were analyzed by Western blotting. (B) vFLIP-, vCyclin- and LANA-expressing PDLSCs were examined for hybrid M/E state cells using FASC. ( C) Immunofluorescence staining of vFLIP-expressing PDLSCs for PDPN expression. Scale bar, 20 μm. ( D) vFLIP-expressing PDLSCs were subjected to a tubule formation assay. Scale bar, 200 μm. ( E) vFLIP-expressing PDLSC spheroids were examined by IFA for PDGFRA/PDPN expression profiles. Scale bar, 50 μm. (F) Representative images of H&E staining of PDLSC and vFLIP-PDLSC spheroids transplanted in mice under the kidney capsule. Scale bar, 100 μm (upper) and 20 μm (lower). ( n = 3–4 mice for each group). (G) Immunofluorescent overview of PDGFRA/PDPN expression in PDLSC and vFLIP-PDLSC spheroids under the renal capsule. Scale bar, 50 μm.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: Transduction, Expressing, Western Blot, Immunofluorescence, Staining, Tube Formation Assay

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: ( A) Schematic diagram depicting the CRISPR/Cas9 editing sites in the KSHV genome. ( B) The CRISPR/Cas9-mediated knockout efficiency of ORF71 (vFLIP) gene from the KSHV genome in KSHV-infected PDLSCs was verified by PCR with primers Pr1 and Pr2. (C) The vFLIP expression in ORF71 knockout cells was determined by RT-qPCR. ( D) The effect of vFLIP knockout (vFLIP-KO) on the generation of hybrid M/E state cells, as well as the restoration of vFLIP function by ectopic expression of sgRNA-resistant vFLIP gene , was quantitated by flow cytometry. ( E) The effect of vFLIP-KO on the expression of PDGFRA and PDPN was examined with K-PDLSC spheroids by IFA. Scale bar, 50 μm. (F) The effect of vFLIP-KO on malignant transformation ability was examined using colony formation assay. Scale bar, 200 μm. ( G ) Images of WT and vFLIP-KO KSKV-infected PDLSC spheroids showing the effect of vFLIP-KO on cell migration capability. Scale bar, 200 μm. (H) The effect of vFLIP-KO on the angiogenic ability of K-PDLSCs was assayed by tubule-formation assay. Scale bar, 200 μm. (I) Representative images of H&E staining of WT and vFLIP-KO KSHV-infected PDLSC spheroid transplants in mice under the kidney capsule. Scale bar, 200 μm. Higher magnifications of the black boxes are shown on the right. Scale bar, 50 μm. ( n = 3–4 mice for each group). (J) Immunofluorescence assay of WT and vFLIP-KO KSHV-infected PDLSC transplants for LANA, PDGFRA, and PDPN. Scale bar, 50 μm. (K) Schematic model for the role of KSHV vFLIP in promoting MEndT to generate hybrid M/E state cells for KS tumorigenesis and aberrant angiogenesis. Error bars represent mean ± SEM. n = 3, unless otherwise indicated. Statistical analyses were performed using the two-tailed Student’s test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Then the cells were fixed with 4% paraformaldehyde for 20 min and incubated with APC-anti-human PDPN (eBioscience, 17-9381-42), and PE-anti-human PDGFRA (Sino Biological, 10556-MM02-P) for 30 min at 4°C.

Techniques: CRISPR, Knock-Out, Infection, Expressing, Quantitative RT-PCR, Flow Cytometry, Transformation Assay, Colony Assay, Migration, Tube Formation Assay, Staining, Immunofluorescence, Two Tailed Test