Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: Characterization of lentiviral MSCs transduction efficiency. The schemes of plasmids used for lentivirus production for subsequent murine MSCs transduction are shown. The lentiviral backbone plasmid (FUGW) contained the green fluorescent protein (GFP) coding sequence ( A ) that was removed to insert the human BDNF sequence and then FUGW-BDNF plasmid was created ( B ) for relevant lentiviral vectors production. The correct band for BDNF insert (765 bp) was observed under ultraviolet (UV) light in agarose gel ( C ). Quantitative analysis of BDNF levels from MSC-BDNF and unmodified MSC cultures in vitro ( D ). Noninfected control MSCs produced only trace amount of BDNF, whereas production of BDNF in MSC-BDNF culture was approximately 35-fold increased. These data were corroborated by double immunofluorescent staining of BDNF and GFP proteins for their qualitative expression and co-expression analysis ( E ). Scale bar: 20 µm, *** p < 0.001.

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: Transduction, Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis, In Vitro, Control, Produced, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: Long-term follow-up of genetically modified MSC-BDNF and MSC trafficking and homing at different time points post-intravitreal transplantation in rd6 mice. A representative SD-OCT image of chronically degenerated retina of rd6 mouse at the 28th day after intravitreal MSC-BDNF injection ( A ). A hyperreflective streak of the accumulated MSC (white arrow) at the vitreoretinal interface is observed. A representative fluorescence image of degenerated retina of rd6 mouse at 28 days after intravitreal MSC injection ( B ). At this time point, the vast majority of the injected GFP-positive cells (green) were found to be located at the vitreoretinal interface and in the superficial ganglion cell layer. A representative fluorescence images of degenerated retina of rd6 mouse at three months after intravitreal MSC-BDNF injection ( C ). At this time of the experiment, the injected GFP-positive cells (green) were found to be aligned along the RPE-photoreceptor junction and showed double immunostaining against BDNF (red). A representative retinal volume intensity projections of OCT scans of rd6 control mouse ( D ), after intravitreal MSC-BDNF injection ( E ) and MSC alone transplantation ( F ) at the third month of the experiment. At this time of the experiment, the considerable reduction of the retinal white spots that correspond to macrophages and monocytes at the level of retinal pigment epithelium was observed only in eyes after intravitreal MSC-BDNF injection. Green lines indicate the retinal level where the volume intensity projection image (VIP) was captured. Scale bar: 20 µm.

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: Genetically Modified, Transplantation Assay, Injection, Fluorescence, Double Immunostaining, Control

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: Long-term follow-up of BDNF production and its biological function at different time points post-intravitreal MSC-BDNF transplantation. BDNF mRNA ( A ), BDNF, phosphoAkt, totalAkt, phosphoMAPK and totalMAPK protein ( B ) expression was detected in retinas from eyes treated with MSC-BDNF, and their levels were significantly increased post-transplantation compared to other groups: at 28 days in case of mRNA and protein and at three months in the case of BDNF protein only. We also observed increased TrkB gene expression in retinas at 28 days and three months after MSC-BDNF and MSC alone transplantation compared to rd6 and wild type (WT) ( C ). The follow-up of retinal cell proliferation at different time points post-intravitreal transplantation in rd6 mice was also performed. Quantitative analysis of proliferating cell nuclear antigen (PCNA) mRNA expression revealed that their levels were significantly increased in retinas from eyes treated with MSC-BDNF at 28 days post transplantation compared with those in eyes treated with the PBS and MSCs alone ( D ). Double-stained sections for PCNA and GFP (endogenous marker of transplanted MSC) used to visualize and localize proliferating cells revealed the extraordinary PCNA protein concentration in MSC-BDNF transplanted 28 days previously ( E ). Representative images of the performed analyses are shown. Scale bar: 20 µm. Reference gene used for qRT-PCR analysis was glyceraldehyde 3-phospate dehydrogenase (GAPDH). Mean values ± SD are presented in the diagrams, * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 7/group/time point).

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: Transplantation Assay, Expressing, Gene Expression, Staining, Marker, Protein Concentration, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: The expression profile of selected apoptosis-related molecules in retinas treated with MSC-BDNF or MSC alone at different time points (at 28 days and three months post transplantation) and compared to WT and rd6 mice. The mRNA expression of Bcl-xL and BAX genes was determined by the quantitative PCR and the relative ratio Bcl-xL/BAX was calculated ( A ). The concentration of Bcl-xL and Bak protein dimer was determined by specific Luminex ( B ) Similarly, the concentration of Mcl-1/Bak dimer protein was measured ( C ). Caspase-3 protein expression was determined by Western blot, which revealed a lack of expression of this form in WT mice and rd6 after cell transplantation compared to rd6 mice with no treatment ( D ). GAPDH served as loading. A representative image is shown. Mean values ± SDs are presented in the diagrams, * p < 0.05, ** p < 0.01 ( n = 7/group/time point).

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: Expressing, Transplantation Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Luminex, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: Effects of in vivo experimental therapy with genetically transduced MSC-BDNF and unmodified MSC on the retinal function and OCT-based retinal morphology at 28 days post transplantation compared to rd6 mice with no treatment and WT mice. The representative ERG responses recorded after cell injection are shown ( A ). The b-wave amplitude measurements are presented as the mean ± SD immunohistofluorescence co-staining for rhodopsin (green) with opsin blue (red) in ( B ) and rhodopsin (green) with opsin red/green (red) in ( C ) are displayed in the representative retinal specimens from all the groups with semi quantitative evaluation (signal intensity was marked with subsequent methodology: “+” when less than 5 immunopositive cells present in the image, “++” for 6–15 of visible positive cells, “+++” for 16–30 of positive stained cells, “++++” more than 30 immunopositive cells. The control rd6 mouse retinas (left column) are almost completely negative for both cone opsins, whereas MSC-BDNF-treated retinas displayed regeneration of cones as evidenced by positive immunoreactivity for red/green cone opsin (middle column). The representative OCT images of the retinal sections with their typical layered structure are presented in ( D ). Retinal layers were marked both at histological images and OCT as follows: IR- inner retina, INL- inner nuclear layer, OPL- outer plexiform layer, ONL- outer nuclear layer, RPE-PR–retinal pigment epithelium and photoreceptors. Scale bar: 20 µm. * p < 0.05; ** p < 0.01.

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: In Vivo, Transplantation Assay, Injection, Immunohistofluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: Effects of in vivo experimental therapy with genetically transduced MSC-BDNF and unmodified MSC on the retinal function and OCT-based retinal morphology at three months post transplantation compared to rd6 mice with no treatment and WT mice. The representative ERG responses recorded after cell injection are shown ( A ). The b-wave amplitude measurements are presented as the mean ± SD. Immunohistofluorescence co-staining for rhodopsin (green) with opsin blue (red) in ( B ) and rhodopsin (green) with opsin red/green (red) in ( C ) are displayed in the representative retinal specimens from all the groups with semi quantitative evaluation (signal intensity was marked with subsequent methodology: “+” when less than 5 immunopositive cells present in the image, “++” for 6–15 of visible positive cells, “+++” for 16–30 of positive stained cells, “++++” more than 30 immunopositive cells. The control rd6 mouse retinas (left column) are almost completely negative for both cone opsins, whereas MSC-BDNF-treated retinas displayed a regeneration of cones as evidenced by the positive immunoreactivity for red/green cone opsin (middle column). The representative OCT images of the retinal sections with their typical layered structure are presented in ( D ). Retinal layers were marked both at histological images and OCT as follows: IR- inner retina, INL- inner nuclear layer, OPL- outer plexiform layer, ONL- outer nuclear layer, RPE-PR–retinal pigment epithelium and photoreceptors. Scale bar: 20 µm. * p < 0.05.

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: In Vivo, Transplantation Assay, Injection, Immunohistofluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

doi: 10.3390/ijms20030777

Figure Lengend Snippet: List of antibodies employed in double immunofluorescence staining technique.

Article Snippet: We used a primary monoclonal immunoglobulin G (IgG) antibodies directed against the BDNF, Caspase-3, phosphoAkt, totalAkt, phosphoMAPK, totalMAPK as follows: Rabbit anti-BDNF monoclonal antibody (at 1:1000 dilution, Abcam, Cambridge, UK, cat no. ab108319), rabbit anti-Caspase-3 monoclonal antibody (at 1:600 dilution, Santa Cruz Biotechnology, CA, USA, cat no. sc-56046), rabbit anti- phospho-Akt monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4060S), anti-Akt (pan) monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4685S), rabbit anti-phospho-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4370S), rabbit anti-p44/42 MAPK monoclonal IgG antibody (at 1:300 dilution, Cell Signaling Technology, Beverly, MA, USA, cat no. 4695S) and polyclonal anti-GAPDH antibody conjugated with HRP (at a 1:1000 dilution Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat no. sc-20357,).

Techniques: Double Immunofluorescence Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model

doi: 10.1073/pnas.1610383114

Figure Lengend Snippet: A heterozygous loss of β-catenin results in defective adherens junctions, whereas a homozygous β-catenin deletion promotes plakoglobin up-regulation to maintain adherens junctions. (A) Adherens junctions were visualized by IHF staining for E-cadherin and costained with plakoglobin, a common component of both adherens and desmosomal junctions. Arrows point to cells with nuclear plakoglobin signal. Haploid-insufficient β-catenin tumors exhibited a high degree of heterogeneity and therefore, were further categorized into two main groups: poorly differentiated tumors (low plakoglobin) and others (high plakoglobin), consisting of mostly well-differentiated tumors. Ctnnb1+/+ErbB2KI, n = 6; Ctnnb1fl/+ErbB2KI poorly differentiated, n = 5; Ctnnb1fl/+ErbB2KI others, n = 3; Ctnnb1fl/flErbB2KI, n = 5. (Scale bar: 20 μm.) (B) Tumor lysates were subjected to immunoblot analysis for components of adherens junctions. Notably, Ctnnb1fl/+ErbB2KI tumors with high plakoglobin displayed similar levels of all adherens junction proteins assessed. (C) Levels of adherens junction proteins were assessed using a larger sample size by immunoblot analysis and quantified using LI-COR Odyssey Software. Ctnnb1+/+ErbB2KI, n = 7; Ctnnb1fl/+ErbB2KI poorly differentiated, n = 7; Ctnnb1fl/+ErbB2KI others, n = 6; Ctnnb1fl/flErbB2KI, n = 6. Error bar: SEM. **P < 0.005 (Student’s t test); ***P < 0.0005 (Student's t test). (D) Transcript levels of plakoglobin (Jup) and Twist1 were assessed by RT-PCR. Data were normalized to Gapdh. Ctnnb1+/+ErbB2KI, n = 9; Ctnnb1fl/+ErbB2KI poorly differentiated, n = 8; Ctnnb1fl/+ErbB2KI others, n = 5; Ctnnb1fl/flErbB2KI, n = 5. Error bar: SEM. *P < 0.05 (Student’s t test); ***P < 0.0005 (Student’s t test). (E) Ctnnb1fl/flErbB2KI tumor cells were subjected to cellular fractionation to separate nuclear fraction from the cytosol/membrane fraction to show the subcellular localization of plakoglobin. Tubulin and ErbB2 are markers for cytosol and membrane fractions. Laminin A/C is a marker for a nuclear fraction. ns, Not significant.

Article Snippet: Antibody Company Antibody dilution IHC or IHF Western blotting Rabbit anti-ErbB2 Santa Cruz 1/1,000 Rabbit anti-Axin2 EMD Millipore 1/1,000 Rabbit anti-Grb2 Santa Cruz 1/1,000 Rabbit anti-Grb7 Santa Cruz 1/1,000 Rabbit anti-Keratin 14 Covance 1/500 Rabbit anti-Keratin 6 Covance 1/500 Guinea pig anti-Keratin 8/18 Fitzgerald 1/200 Rabbit anti–β-Catenin Cell Signaling 1/200 1/1,000 Rabbit anti-Plakoglobin Cell Signaling 1/1,000 Rabbit anti-Plakoglobin Santa Cruz 1/200 Mouse anti–E-Cadherin BD Biosciences 1/200 1/1,000 Mouse anti–p120-Catenin BD Biosciences 1/200 1/500 Mouse anti–α-Catenin BD Biosciences 1/200 1/500 Mouse anti-Cyclin D1 Sigma-Aldrich 1/500 Mouse anti-Laminin A/C Cell Signaling 1/1,000 Mouse anti-Vinculin EMD Millipore 1/1,000 Mouse anti-Actin Sigma-Aldrich 1/1,000 Rabbit anti–α/β-Tubulin Cell Signaling 1/1,000 Rabbit anti-V5 tag Cell Signaling 1/1,000 All biotin-conjugated antibodies for IHC Jackson Immunoresearch 1/1,000 All Alexa-Fluor–conjugated antibodies for IF Invitrogen 1/1,000 Goat anti-rabbit IRDye 800 LI-COR Biosciences 1/10,000 Goat anti-mouse IRDye 680 LI-COR Biosciences 1/10,000 Open in a separate window List of Antibodies.

Techniques: Staining, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction, Cell Fractionation, Membrane, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model

doi: 10.1073/pnas.1610383114

Figure Lengend Snippet: List of Antibodies.

Article Snippet: Antibody Company Antibody dilution IHC or IHF Western blotting Rabbit anti-ErbB2 Santa Cruz 1/1,000 Rabbit anti-Axin2 EMD Millipore 1/1,000 Rabbit anti-Grb2 Santa Cruz 1/1,000 Rabbit anti-Grb7 Santa Cruz 1/1,000 Rabbit anti-Keratin 14 Covance 1/500 Rabbit anti-Keratin 6 Covance 1/500 Guinea pig anti-Keratin 8/18 Fitzgerald 1/200 Rabbit anti–β-Catenin Cell Signaling 1/200 1/1,000 Rabbit anti-Plakoglobin Cell Signaling 1/1,000 Rabbit anti-Plakoglobin Santa Cruz 1/200 Mouse anti–E-Cadherin BD Biosciences 1/200 1/1,000 Mouse anti–p120-Catenin BD Biosciences 1/200 1/500 Mouse anti–α-Catenin BD Biosciences 1/200 1/500 Mouse anti-Cyclin D1 Sigma-Aldrich 1/500 Mouse anti-Laminin A/C Cell Signaling 1/1,000 Mouse anti-Vinculin EMD Millipore 1/1,000 Mouse anti-Actin Sigma-Aldrich 1/1,000 Rabbit anti–α/β-Tubulin Cell Signaling 1/1,000 Rabbit anti-V5 tag Cell Signaling 1/1,000 All biotin-conjugated antibodies for IHC Jackson Immunoresearch 1/1,000 All Alexa-Fluor–conjugated antibodies for IF Invitrogen 1/1,000 Goat anti-rabbit IRDye 800 LI-COR Biosciences 1/10,000 Goat anti-mouse IRDye 680 LI-COR Biosciences 1/10,000 Open in a separate window List of Antibodies.

Techniques:

Journal: EBioMedicine

Article Title: Mitochondrial dysfunction in human primary alveolar type II cells in emphysema

doi: 10.1016/j.ebiom.2019.07.063

Figure Lengend Snippet: Reduced mitochondrial fusion in ATII cells in emphysema. Freshly isolated ATII cells and lung tissue were obtained from non-smokers (NS), smokers (SM) and emphysema patients (EM). A – MFN1 expression was determined in ATII cells by Western blotting. MFN1 levels were normalized to β-actin and control non-smokers. B – MFN1 mRNA expression in ATII cells. C – MFN1 expression (green) in mitochondria (red) in ATII cells (violet) identified using TOM20 and SP-A antibodies, respectively in lung tissue sections by immunohistofluorescence. MFN1 and TOM20 co-localization in ATII cells is shown using Pearson's correlation coefficient. D – MFN1 levels and quantification in mitochondrial fractions obtained from lung tissue by Western blotting. MFN1 levels were normalized to TOM20 and control non-smokers. E – OPA1 expression in ATII cells identified by proSP-C marker in lung tissue sections. Data is expressed as means ± s.e.m., N = 3–7 lungs per group, * P < .05; ** P < .001.

Article Snippet: We used the following antibodies: DRP1, TOM20, mtTFA, MFN1, MFN2, POLγ, TDP1, DJ-1 (all from Santa Cruz Biotechnology), TOP1-cc (Millipore), p-DRP1 (Ser616) (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Abcam, Cambridge, MA, USA) and β-actin (Sigma, St. Louis, MO, USA).

Techniques: Isolation, Expressing, Western Blot, Control, Immunohistofluorescence, Marker

Journal: EBioMedicine

Article Title: Mitochondrial dysfunction in human primary alveolar type II cells in emphysema

doi: 10.1016/j.ebiom.2019.07.063

Figure Lengend Snippet: Increased mtDNA damage and decreased mtDNA amount and mitochondrial dynamics in emphysema progression. Lung tissue was obtained from areas with mild and severe emphysema (EM) of the same patient. MtDNA amount (mt/nuclear short fragment) (A), mtDNA damage (long/short mt fragment) (B) and common deletion (C) were determined by qPCR. TDP1 (D), p-DRP1 (E) and MFN1 (F) levels were determined in lung tissue by Western blotting. Protein expression was normalized to β-actin. TDP1 (G), DRP1 (H) and MFN1 (I) mRNA levels were determined by RT-PCR. Data is expressed as means ± s.e.m., N = 3–8 lungs per group, * P < .0, ** P < .001.

Article Snippet: We used the following antibodies: DRP1, TOM20, mtTFA, MFN1, MFN2, POLγ, TDP1, DJ-1 (all from Santa Cruz Biotechnology), TOP1-cc (Millipore), p-DRP1 (Ser616) (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Abcam, Cambridge, MA, USA) and β-actin (Sigma, St. Louis, MO, USA).

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

Journal: Nature Communications

Article Title: Zbtb20 promotes astrocytogenesis during neocortical development

doi: 10.1038/ncomms11102

Figure Lengend Snippet: ( a – f ) Plasmids for GFP alone (control) or for both GFP and Zbtb20 were injected into the lateral ventricle of the E15.5 mouse forebrain in utero and were introduced into the dorsolateral region of the neocortex by electroporation. The brain was isolated at P7 or E18.5 and subjected to immunohistofluorescence analysis with antibodies to GFAP, to S100β, to Sox9, to Cux1, to Brn2 and to GFP ( a ), and to GLAST, to BLBP and to GFP ( e ). The percentages of marker + cells among total GFP + cells were determined as means±s.d. ( n =3 to 5) ( b – d , f ). ( g – i ) Plasmids encoding GFP together with either a control shRNA (sh-Luc), a Zbtb20 shRNA (sh-Zbtb20 #1 or #2) or sh-Zbtb20 #1 and an shRNA-resistant mutant of Zbtb20 (mut-Zbtb20) were electroporated into the P0 mouse neocortex. The brain was isolated at P3 or P7 and subjected to immunostaining for GFAP at P7, Tbr2 and Tbr1 at P3, and GFP ( g ). The percentages of marker + cells among total GFP + cells were determined as means±s.d. ( n =4 to 7) ( h , i ). ( j – l ) Plasmids for mCherry alone (control) or for both mCherry and Zbtb20 were electroporated into the E15.5 Aldh1L1-GFP mouse forebrain ( j ) and plasmids encoding mCherry together with either a control shRNA (sh-Luc) or a sh-Zbtb20 #1 were electroporated into the P0 Aldh1L1-GFP mouse neocortex ( k ). The brain was isolated at P7 and subjected to immunostaining for GFP and mCherry. The percentages of GFP + cells among total mCherry + cells were determined as means±s.d. ( n =5 to 7) ( l ). CC, corpus callosum; LV, lateral ventricle; SVZ, subventricular zone. The right-most panels are higher magnification views of the boxed areas ( a , e , g , j , k ). Arrows indicate double-positive cells ( a , e , g , j , k ). * P <0.01 versus corresponding control value; # P <0.01 versus sh-Zbtb20 #1 value. Scale bars, 75 μm ( a ), 50 μm ( e , g , j , k ) and 25 μm (higher magnification views in a , e , g , j , k ).

Article Snippet: Immunostaining was performed with the following antibodies: Zbtb20 (Sigma-Aldrich, HPA016815, rabbit, 1:200 dilution), βIII-tubulin (TuJ1, Covance, MMS-435P, mouse, 1:5,000), GFAP (Millipore, MAB360, mouse, 1:1,000; Dako, Z0334, rabbit, 1:1,000; Abcam, ab4674, chicken, 1:2,000), S100β (Dako, Z0311, rabbit, 1:500; Sigma-Aldrich, SH-B1, mouse, 1:200), O4 (Millipore, MAB345, mouse, 1:1,000), Sox9 (Millipore, AB5535, rabbit, 1:1,000; Abcam, ab76997, mouse, 1:1,000; Santa Cruz Biotechnology, sc17342, goat, 1:100), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), Brn2 (Santa Cruz Biotechnology, sc6029, goat, 1:200), Olig2 (IBL, 18953, rabbit, 1:1,000; Millipore, AB9610, rabbit, 1:1,000; Millipore, MABN50, mouse, 1:500), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:100), Cux1 (Santa Cruz Biotechnology, sc13024, rabbit, 1:200), Tbr1 (Abcam, ab31940, rabbit, 1:2,000), Tbr2 (Abcam, ab23345, rabbit, 1:2,000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1,000), nestin (BD Biosciences, 556309, mouse, 1:200), NeuN (Millipore, MAB377, mouse, 1:200; Millipore, ABN78, rabbit, 1:1,000), HuC/D (Life Technologies, A21271, mouse, 1:200), CC1 (Millipore, OP80, mouse, 1:500), myelin-associated glycoprotein (Millipore, MAB1567, mouse, 1:400), glutathione S -transferase π (BD Biosciences, 610719, mouse, 1:1,000), platelet-derived growth factor receptor-α (BD Biosciences, 558774, rat, 1:200), Ki67 (Abcam, ab15580, rabbit, 1:500), GLAST (Frontier Institute, Af660, rabbit, 1:200), BLBP (Abcam, ab32423, rabbit, 1:500), FoxJ1 (eBioscience, 14-9965, mouse, 1:500), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), RFP (MBL, PM005, rabbit, 1:1,000) and GFP (MBL, 598, rabbit, 1:2,000; Abcam, ab13970, chicken, 1:2,000).

Techniques: Control, Injection, In Utero, Electroporation, Isolation, Immunohistofluorescence, Marker, shRNA, Mutagenesis, Immunostaining

Journal: Nature Communications

Article Title: Zbtb20 promotes astrocytogenesis during neocortical development

doi: 10.1038/ncomms11102

Figure Lengend Snippet: ( a ) Quantitative RT–PCR analysis of relative mRNA abundance for the indicated proteins in Zbtb20-overexpressing and control NPCs. Data are means±s.d. ( n =3). ( b ) E11.5 NPCs were infected with retroviruses for control, Brn2, Zbtb20 or Brn2 plus Zbtb20 and induced to differentiate for 6 days. The percentages of marker + cells among total GFP + cells were determined as means±s.d. ( n =4). ( c ) Luciferase assay of relative Brn2 promoter activity and its concentration-dependent inhibition by Zbtb20 overexpression in NPCs. The effect of Neurog2 was examined as a positive control. Data are means±s.d. ( n =5). ( d ) ChIP analysis of Zbtb20 binding to the Brn2 promoter region in NPCs. Seven different regions (R1–R7) of the Brn2 locus were tested in control cells and cells expressing TY1-tagged Zbtb20. Data are expressed as fold enrichment relative to the corresponding value for control cells and normal mouse immunoglobulin G (IgG). Data are means±s.d. ( n =3 to 5). ORF, open reading frame. ( e – g ) Control, sh-Zbtb20 #1, sh-Zbtb20 #2 or sh-Zbtb20 #1 and mut-Zbtb20 plasmids were electroporated into P0 mouse neocortical NPCs. The P7 brain (CC, corpus callosum) was immunostained for Brn2 ( e ), NeuN, Tbr1 ( g ) and GFP. Arrows indicate marker + /GFP + cells. The boxed region in e is shown at higher magnification in the right-most panel. Scale bars, 50 and 25 μm (higher magnification image in e ). The percentages of Brn2 + cells among total GFP + cells were determined as means±s.d. ( n =5 to 7) ( f ). ( h ) Luciferase assay of relative Brn2 promoter activity in NPCs transfected with plasmids for Zbtb20, Sox9, NFIA, sh-Sox9 #1 or sh-NFIA #1, as indicated. Data are means±s.d. ( n =5). * P <0.01 and ** P <0.05 versus the corresponding control value ( a , b , f , h ), 0 μg of Zbtb20 plasmid ( c ) or TY1-Zbtb20, mouse IgG ( d ); # P <0.01 versus value for Zbtb20 ( b , h ) or sh-Zbtb20 #1 ( f ). ( i ) Model for astrocytogenesis by Zbtb20, Sox9 and NFIA. Zbtb20 and NFIA suppress Brn2 expression.

Article Snippet: Immunostaining was performed with the following antibodies: Zbtb20 (Sigma-Aldrich, HPA016815, rabbit, 1:200 dilution), βIII-tubulin (TuJ1, Covance, MMS-435P, mouse, 1:5,000), GFAP (Millipore, MAB360, mouse, 1:1,000; Dako, Z0334, rabbit, 1:1,000; Abcam, ab4674, chicken, 1:2,000), S100β (Dako, Z0311, rabbit, 1:500; Sigma-Aldrich, SH-B1, mouse, 1:200), O4 (Millipore, MAB345, mouse, 1:1,000), Sox9 (Millipore, AB5535, rabbit, 1:1,000; Abcam, ab76997, mouse, 1:1,000; Santa Cruz Biotechnology, sc17342, goat, 1:100), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), Brn2 (Santa Cruz Biotechnology, sc6029, goat, 1:200), Olig2 (IBL, 18953, rabbit, 1:1,000; Millipore, AB9610, rabbit, 1:1,000; Millipore, MABN50, mouse, 1:500), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:100), Cux1 (Santa Cruz Biotechnology, sc13024, rabbit, 1:200), Tbr1 (Abcam, ab31940, rabbit, 1:2,000), Tbr2 (Abcam, ab23345, rabbit, 1:2,000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1,000), nestin (BD Biosciences, 556309, mouse, 1:200), NeuN (Millipore, MAB377, mouse, 1:200; Millipore, ABN78, rabbit, 1:1,000), HuC/D (Life Technologies, A21271, mouse, 1:200), CC1 (Millipore, OP80, mouse, 1:500), myelin-associated glycoprotein (Millipore, MAB1567, mouse, 1:400), glutathione S -transferase π (BD Biosciences, 610719, mouse, 1:1,000), platelet-derived growth factor receptor-α (BD Biosciences, 558774, rat, 1:200), Ki67 (Abcam, ab15580, rabbit, 1:500), GLAST (Frontier Institute, Af660, rabbit, 1:200), BLBP (Abcam, ab32423, rabbit, 1:500), FoxJ1 (eBioscience, 14-9965, mouse, 1:500), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), RFP (MBL, PM005, rabbit, 1:1,000) and GFP (MBL, 598, rabbit, 1:2,000; Abcam, ab13970, chicken, 1:2,000).

Techniques: Quantitative RT-PCR, Control, Infection, Marker, Luciferase, Activity Assay, Concentration Assay, Inhibition, Over Expression, Positive Control, Binding Assay, Expressing, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

doi: 10.1038/s41419-018-0433-0

Figure Lengend Snippet: a bFGF contents in SF obtained from OA patients ( n = 31) and RA patients ( n = 79) were measured by sandwich ELISA using specific antibodies. Each dot represents the concentration of bFGF from each patient. The median values of bFGF in the RA SF and OA SF denotes the center of interquartile range. * p < 0.01. b , c bFGF induces proliferation of human FLSs ( b ) and human MH7A ( c ) by G1/S cell-cycle transition in a dose-dependent manner compared with untreated controls. Data were obtained from three independent experiments. d , e Synovial tissues from OA patients ( n = 5) and RA patients ( n = 5) were analyzed to measure cell proliferation potential using Ki-67 ( d ) and the activation of RSK2 and FGFR3 using p-RSK2 (T577) and p-FGFR3 (Y724) antibodies ( e ). Scale bars, 50 μm. The photograph is a representative confocal image obtained from an immunohistofluorescence assay; the fluorescence intensities of Ki-67, p-RSK, and p-FGFR3 were normalized by DAPI intensity. The average fold change of the intensity presented in graphs was obtained from five synovial tissues from OA and RA patients. * p < 0.05; ** p < 0.01

Article Snippet: Antibodies for phospho-FGFR3 (Cat#: SC-33041), FGFR3 (Cat#: SC-13121), p-RSK2 T577 (Cat#: SC-16407, SC-377501), RSK2 (Cat#: SC-9986), and β-actin (Cat#: SC-69879) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Sandwich ELISA, Concentration Assay, Activation Assay, Immunohistofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

doi: 10.1038/s41419-018-0433-0

Figure Lengend Snippet: a Representative double immunohistochemical analysis of human RA synovial tissues (×100, n = 3). The tissues were hybridized with a combination of phospho-RSK2 (Thr577) antibody (shown in brown), CD68 (a macrophage marker, shown in red), CD3 (a T-cell marker, shown in red), and CD20 (a B-cell marker, shown in red), as indicated. Boxed area was magnified (×400). The area marked by the blue dotted line indicates the hyperplastic lining layer, and the area marked by the green dotted line indicates the sublining layer in synovium. b , c The knockdown effects of RSK2 on cell proliferation ( b ) and cell migration ( c ) in human RA FLSs. Cell proliferation was measured by MTS assay ( b ), and the migrated area (graph) was quantified by measuring the uncovered area of the wound using Image J (Ver. 1.6). Scale bars, 100 μm ( c ). Data were obtained from three independent experiments, and values are represented as means ± SEM. * p < 0.001 by Student’s t -test. d Representative photographs (×40) of triple immunohistofluorescence analysis of tissues obtained from OA ( n = 5) and RA ( n = 5) patients. p-RSK2 and p-FGFR3 were co-stained with CD4- (a T-cell marker; upper panels) or CD68- (a macrophage marker; bottom panels) specific antibodies as indicated and analyzed by confocal microscopy. Yellow color in the merged photographs indicates the coexistence of CD4 + T-cells or CD68 + macrophages in the synovial tissues of OA and RA patients, respectively. Scale bars, 40 μm

Article Snippet: Antibodies for phospho-FGFR3 (Cat#: SC-33041), FGFR3 (Cat#: SC-13121), p-RSK2 T577 (Cat#: SC-16407, SC-377501), RSK2 (Cat#: SC-9986), and β-actin (Cat#: SC-69879) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Marker, Knockdown, Migration, MTS Assay, Immunohistofluorescence, Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

doi: 10.1038/s41419-018-0433-0

Figure Lengend Snippet: a The efficacy of kaempferol on bFGF-induced human FLS proliferation was measured by MTS assay. b AP-1 transactivation and Cox-2 promoter activities were measured in RSK2 +/+ and RSK2 −/− MEF by transfection of pAP-1-luciferase (top graph) and pCox-2 promoter-luciferase (bottom graph) reporter plasmids as indicated. c Effects of bFGF-induced cell migration in RSK2 +/+ and RSK2 −/− MEFs. The migrated area was quantified by measuring the uncovered area of the wound using Image J (Ver. 1.6). d Efficacy of kaempferol on the cell migration of MH7A and human FLSs. The migrated area (graphs) was quantified by measuring the uncovered area of the wound using Image J (Ver. 1.6). e Efficacy of kaempferol on MMP-9 and MMP-2 activity was analyzed by gelatin zymography using the indicated culture supernatants of human FLSs. f Representative photographs for kaempferol specificity on FGFR3 phosphorylation at Tyr724 induced by bFGF stimulation in MH7A cells. PKC412, an FGFR3 inhibitor; U0126, an MEK inhibitor. Data were obtained from three independent experiments. Each indicated area in the immunocytofluorescence confocal image (×400) was magnified (×630). Scale bars, 20 μm. a – d Data were obtained from three independent experiments, and values are represented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 by Student’s t -test

Article Snippet: Antibodies for phospho-FGFR3 (Cat#: SC-33041), FGFR3 (Cat#: SC-13121), p-RSK2 T577 (Cat#: SC-16407, SC-377501), RSK2 (Cat#: SC-9986), and β-actin (Cat#: SC-69879) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: MTS Assay, Transfection, Luciferase, Migration, Activity Assay, Zymography, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

doi: 10.1038/s41419-018-0433-0

Figure Lengend Snippet: a Inhibitory effects of kaempferol on ex vivo osteoclast formation. The BMMs obtained from CIA + vehicle and CIA + kaempferol mice were analyzed in terms of the osteoclast formation induced by M-CSF or M-CSF + RANKL. TRAP + osteoclasts (≥3 nuclei/TRAP + cell) were counted. Photographs (×100) are representative of TRAP staining obtained from each mouse group (CIA + vehicle, n = 3; CIA + kaempferol, n = 3), and values obtained from the whole well of a 48-well plate are presented as means ± SEM. * p < 0.05. b Inhibitory effects of kaempferol on in vitro osteoclast formation. Naïve murine BMMs were subjected to osteoclast differentiation by combinational stimulation of kaempferol, M-CSF, and RANKL as indicated. TRAP + osteoclasts ( ≥ 3 nuclei/TRAP + cell) were counted. Photographs (×100) are representative of TRAP staining obtained from three independent experiments, and values obtained from the whole well of a 48-well plate are represented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. c Inhibitory effects of kaempferol on osteoclast-specific genes. Naïve murine BMMs stimulated with M-CSF/RANKL and indicated doses of kaempferol for 4 days, and mRNA levels of indicated osteoclast-specific genes were measured by real-time PCR. Data were obtained from three independent experiments, and values are represented as means ± SEM. ** p < 0.01; *** p < 0.001. d Representative photographs of morphological osteoclast analysis. The indicated area shows a multinucleated giant osteoclast cell body after treatment with kaempferol and M-CSF/RANL, as indicated. Scale bars, 40 μm. e Inhibitory effects of kaempferol on osteoclast differentiation. The total nuclear number of multinucleated (≥3 nuclei) giant cells with the phenotypic features of osteoclasts and the number of cells with a single nucleus were counted. Data were obtained from three independent experiments using a four-chamber slide, and values are represented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. f Schematic of the signaling pathway targeted by kaempferol for the inhibition of osteoclast differentiation. bFGF-FGFR3 interaction transduces activation signaling to RSK2, resulting in hyperplasia by the induction of inflammation, FLS proliferation, and cell migration through NF-κB and AP-1. Eventually, the macrophages in synovium differentiate to bone absorbing osteoclasts. Thus, the dual targeting of kaempferol on both FGFR3 and RSK2 may prevent RA in humans

Article Snippet: Antibodies for phospho-FGFR3 (Cat#: SC-33041), FGFR3 (Cat#: SC-13121), p-RSK2 T577 (Cat#: SC-16407, SC-377501), RSK2 (Cat#: SC-9986), and β-actin (Cat#: SC-69879) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Ex Vivo, Staining, In Vitro, Real-time Polymerase Chain Reaction, Inhibition, Activation Assay, Migration

Journal: European Journal of Inflammation

Article Title: Human interaction targets of SARS-COV-2 spike protein: A systematic review

doi: 10.1177/1721727X221095382

Figure Lengend Snippet: Basic characteristics of the included studies.

Article Snippet: Y. M. Hu , The in vitro antiviral activity of lactoferrin against common human coronaviruses and SARS-CoV-2 was mediated by targeting the heparan sulfate co-receptor , Human RD, Huh-7 cell, HEK293T cell, HCT-8 cell, Caco-2 cell, Calu-3 cell, and MRC-5 cell lines , Immunofluorescence, differential scanning fluorimetry, real-time PCR , ACE2 , lactoferrin (LF) had broad-spectrum antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E in cell culture, and bovine lactoferrin (BLF) was more potent than human lactoferrin. BLF bound to heparan sulfate proteoglycans (HSPGs), thereby blocking viral attachment to the host cell. The antiviral activity of BLF could be antagonized by the HSPG mimetic heparin. The antiviral activity of LF was synergistic with remdesivir in cell culture. The N-terminal positively charged region in BLF (residues 17–41) conferred the binding to HSPGs , ( ) .

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Spectroscopy, Immunohistochemistry, Western Blot, Infection, Single Vesicle Fusion Assay, Activation Assay, Pull Down Assay, Affinity Chromatography, Immunofluorescence, Staining, Cell Culture, In Situ, Microarray, In Vitro, RNA Extraction, Functional Assay, Coagulation, Ex Vivo, Transgenic Assay, Transduction, Microscopy, Generated, Protein Binding, Blocking Assay, Isolation, RNA Sequencing Assay, Methylation, Mass Spectrometry, Purification, Neutralization, Imaging, Co-culture Assay, Luciferase, Titration, Depletion Assay, Chromatography, Inhibition, Next-Generation Sequencing, TCID50 Assay, Co-Immunoprecipitation Assay, Transmission Assay, Plaque Assay, Derivative Assay, Immunostaining, SPR Assay, Concentration Assay, Plasmid Preparation, Digital PCR, Real-time Polymerase Chain Reaction, In Vivo, Immunoprecipitation, Cell Attachment Assay, Marker, Variant Assay, Immunohistochemical staining, In Situ Hybridization, Protease Inhibitor, Transfection, Recombinant, Immunohistofluorescence, cDNA Library Assay, Labeling, Microscale Thermophoresis, Cytotoxicity Assay, Conjugation Assay, Raman Spectroscopy, Affinity Precipitation, Fluorescence, Förster Resonance Energy Transfer, Tube Formation Assay, DNA Methylation Assay, Peptide Microarray, TUNEL Assay, Kinase Assay, Confocal Microscopy, Endocytosis Assay, Affinity Purification, Over Expression, Proliferation Assay, Clone Assay, Transcomplementation Assay, Cell-Cell Fusion Assay, Silver Staining, Cell Adhesion Assay, Construct, Electron Microscopy, Produced, shRNA, Angiogenesis Assay, Far Western Blot, Dot Blot, Negative Staining, Enzymatic Assay, Multicolor Immunofluorescence Staining, CRISPR, Modification, XTT Assay

Journal: Molecular Metabolism

Article Title: Sonic Hedgehog receptor Patched deficiency in astrocytes enhances glucose metabolism in mice

doi: 10.1016/j.molmet.2021.101172

Figure Lengend Snippet: Analysis of hedgehog signaling in the mouse hypothalamus. (A) Western blotting analysis of hedgehog (Hh) protein levels in brain tissues from adult mice (n = 3) and HEK293 cells transfected with a mouse Sonic hedgehog (HEK (mShh)) or control (HEK (mock)) vector. N19 and 167Ab Shh antibodies revealed bands at 47 kDa and 22 kDa corresponding to Shh protein precursor and active forms, respectively. Tubulin served as a loading control (SVZ, subventricular zone of the lateral ventricles). (B–F) RNAscope of Patched (Ptc) mRNA combined with immunohistofluorescence for GFAP (C), S100β (D), HuC/D (F), or RNAscope for Glast mRNA (E) on coronal sections of the tuberal region of the hypothalamus from adult mice. Higher magnifications show Ptc mRNA (yellow arrowheads) in GFAP + (C), S100β + (D), Glast + (E), and HuC/D + (F) cells in the hypothalamic parenchyma and presented in merged and single channels with the nuclear marker DAPI. (G–I) RNAscope for Gli1 (G), Gli2 (H), and Gli3 (I) mRNAs combined with RNAscope for Glast mRNA (G and H) and immunohistofluorescence for S100β (G–I) on coronal sections of the tuberal region of the hypothalamus showing the ventromedial hypothalamic nuclei from the adult mice. Magnifications showing expression of Gli1, Gli2, and Gli3 mRNAs in Glast + S100β + cells (G and H) and S100β + cells (I) (white arrowheads), respectively, presented in merged and single channels with the nuclear marker DAPI. Staining was replicated on three mice. Scale bars, 100 μm in (B) and 20 μm in (C–I). 3V, third ventricle.

Article Snippet: HEK293 and NIH3T3 cell lines were from ATCC and 4C20 Smo −/− and P2A6 Ptc −/- mouse embryonic fibroblast lines (MEFs) were kindly provided by Dr. P. Beachy (Stanford University School of Medicine, Stanford, CA, USA).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, RNAscope, Immunohistofluorescence, Marker, Expressing, Staining

Journal: Endocrinology

Article Title: Changes in the Bile Acid Pool and Timing of Female Puberty: Potential Novel Role of Hypothalamic TGR5

doi: 10.1210/endocr/bqae098

Figure Lengend Snippet: Tgr5 mRNA is expressed in the rodent hypothalamus and is regulated during the pubertal transition in female rats. A, qPCR standard curve for FXR using cDNA from rat ARC (blue circles) or liver tissue (red circles) demonstrating the lack of FXR expression in the rat ARC. B, Hypothalamic tissue from female rats were processed for qPCR for tgr5 and leptin receptor ( lepr ) mRNA levels at postnatal day (PN) 14 (Infantile or Inf), PN21(early juvenile or EJ), PN28 (late juvenile or LJ), and during late puberty (LP) (n = 5-11/life stage). Uterine weights (shown in inset) were used to identify individual stages during the pubertal transition. Bars with differing letters (a, b) differ ( P < .05). C, Single cell neurons were isolated from the arcuate nucleus of female mice expressing enhanced green fluorescent protein (GFP) under the control of the 5′-flanking region of the mouse Kiss1 gene and assessed by qPCR for tgr5 mRNA. Abbreviations: AU, arbitrary units; MM, molecular marker; −RT, no reverse transcriptase control; Tissue Control, cDNA from mouse hypothalamus.

Article Snippet: The TGR5-GFP cassette was removed with NdeI and NotI (NEB, Ipswich, MA) and blunted on the 3′ site by incubation with T4 DNA Polymerase (NEB, Ipswich, MA).

Techniques: Expressing, Isolation, Control, Marker, Reverse Transcription

Journal: Endocrinology

Article Title: Changes in the Bile Acid Pool and Timing of Female Puberty: Potential Novel Role of Hypothalamic TGR5

doi: 10.1210/endocr/bqae098

Figure Lengend Snippet: Stimulation of TGR5 within ARC/median eminence fragments results in increased GnRH secretion ex vivo. Hypothalamic fragments containing the ARC nucleus and median eminence were placed in culture and stimulated with kisspeptin (KP), or INT777 (a TGR5 specific agonist) in the absence or presence of the Kisspeptin Receptor/GPR54 antagonist, KP234. GnRH secretion was then assessed by radioimmunoassay. Bars are means and vertical lines represent standard error of the mean. Two-way repeated measures ANOVA, n = 6 rats/group with different letters showing significance, a vs b P < .001 and a vs c P < .05.

Article Snippet: The TGR5-GFP cassette was removed with NdeI and NotI (NEB, Ipswich, MA) and blunted on the 3′ site by incubation with T4 DNA Polymerase (NEB, Ipswich, MA).

Techniques: Ex Vivo, RIA Assay

Journal: Endocrinology

Article Title: Changes in the Bile Acid Pool and Timing of Female Puberty: Potential Novel Role of Hypothalamic TGR5

doi: 10.1210/endocr/bqae098

Figure Lengend Snippet: Gain of function of TGR5 within the ARC nucleus modulates the timing of puberty in rats. A, Western blot validation of lentiviral particles carrying green fluorescent protein (LV-GFP) or LV-TGR5-GFP in infected neuro-2A cells. Antibodies directed against GFP detect 27 kDa band (arrowhead) when using LV-GFP or a 62 kDa band (GFP + TGR5, arrow) when using LV-TGR-GFP. B, Stereotaxic-directed injections of LV-GFP or LV-TGR5-GFP were delivered into the ARC of PND19 female rats. C, Animals were allowed to recover and monitored for age at vaginal opening (VO) and first estrus (FE). Five animals were injected with LV-GFP and 6 with LV-TGR5-GFP. After evaluation of sexual maturation, animals were evaluated for correct delivery of viral particles into the ARC by immunohistofluorescence. Two LV-TGR5-GFP animals showed no GFP signal in the ARC and thus were added as controls into the LV-GFP group.

Article Snippet: The TGR5-GFP cassette was removed with NdeI and NotI (NEB, Ipswich, MA) and blunted on the 3′ site by incubation with T4 DNA Polymerase (NEB, Ipswich, MA).

Techniques: Western Blot, Infection, Injection, Immunohistofluorescence

Journal: Nature Communications

Article Title: Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury

doi: 10.1038/s41467-022-35524-7

Figure Lengend Snippet: A , B Myofiber damage was induced by intramuscular injection of glycerol (Gly) or cardiotoxin (CTX) into the quadriceps. FAP number was quantified in quadriceps-derived SVF by flow cytometry (with the markers CD31, CD45, Sca-1, CD34, CD140α, and podoplanin) from 1 to 9 dpi and compared between control (uninjured, Ctrl), Gly or CTX injected animals ( A , B ) as well as in contralateral non-injured quadriceps ( B ) For A , n = 62 (Ctrl) animals over nine independent experiments at 0 dpi, n = 28 (Gly) and 7 (CTX) animals over four independent experiments at 1 dpi, n = 3 (Gly and CTX) animals over three independent experiments at 3, 7 and 9 dpi. For B, n = 62 (Ctrl), 19 (Gly), 6 (CTX) animals over 9, 3, and 3 independent experiments, respectively. C Detection of in vivo Edu incorporation detected by flow cytometry in FAPs of control and injured animals (Gly, 1 dpi). n = 8 animals at all time points over three independent experiments. D Representative confocal images and immunohistological analysis of injured (Gly and CTX) quadriceps at 1 dpi and quantification of Sca-1 + /Podoplanin + /CD45 − cells in situ. n = 4 (Ctrl and Gly) and 5 (CTX) animals over three independent experiments. Bar scale 50 μm. E , F Clonogenic ( E ) and adipogenic ( F ) assays were performed on total SVF isolated from control or injured (Gly and CTX) muscle at 1 dpi. For E, n = 12 (Ctrl) and 5 (Gly and CTX) animals over three independent experiments. F n = 8 (Ctrl) and 14 (Gly), and 6 (CTX) animals over four independent experiments. G Representative phase contrast images of Ctrl, Gly, or CTX muscle-derived SVF cells under adipogenic culture conditions. Cells were fixed at day 4 and stained with Oil red O. Bar scale 50 μm. H mRNA expression of adipogenic markers measured on total SVF isolated from control or injured (Gly and CTX) muscle at 1 dpi. n = 7 (Gly and CTX) animals over four independent experiments. Results are expressed as a percentage of non-injured control animals with mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs Ctrl.

Article Snippet: ASC sorting: ScAT- or PGAT- derived SVF were depleted in CD45 + and CD31 + cells using anti-CD45-FITC (Miltenyi, 130-116-535) and anti-CD31-FITC antibodies (Miltenyi, 130-123-675) followed by anti-FITC magnetic microbeads (Miltenyi, 130-048-701) using an autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions.

Techniques: Injection, Derivative Assay, Flow Cytometry, Control, In Vivo, In Situ, Isolation, Staining, Expressing

Journal: Nature Communications

Article Title: Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury

doi: 10.1038/s41467-022-35524-7

Figure Lengend Snippet: A Flow cytometry analyses of CD31, CD45, Sca-1, CD34, and podoplanin expression in ScAT-derived SVF of control and injured (Gly and CTX) animals at 1 dpi. n = 106 (Ctrl), 34 (Gly), and 19 (CTX) animals over 15, 15, and 9 independent experiments respectively. B Relative cell numbers in injured muscle and ScAT at 1 dpi. n = 15 (Gly) and 9 (CTX) animals for muscle and 10 (Gly) and 10 (CTX) animals for ScAT over three independent experiments. C ASCs and MSCs content in perigonadic adipose tissue (PGAT) and bone marrow (BM), respectively, using flow cytometry at 1 dpi in Gly-injured animals. n = 25 (PGAT) and 5 (BM) animals over three independent experiments. D ASCs and FAPs phenotypic analysis by flow cytometry. Merged tSNE plot for all control and Gly-injured CD45 − /CD31 − cell among ScAT and muscle SVF-derived cells. The identity of each cluster according to the combinatory expression level of multiple markers is color-coded in the tSNE plot. Cytometry marker expression level plots are presented on the right-hand side. E Comparison of ScAT and muscle tSNE plot in control and injured (Gly and CTX) conditions at 1 dpi; clusters identified in D are black circled. F TUNEL staining of ScAT-derived SVF from Gly- or CTX-injured, or non-injured animals (Ctrl) at 1 dpi. n = 7 (Ctrl), 5 (Gly), and 3 (CTX) animals over three independent experiments. G Phase contrast images of adipogenic challenged ScAT-derived SVF from Ctrl, Gly, and CTX-injured animals. Cells were fixed at day 4 of differentiation and stained with oil red O. Bar scale 50 μm. n = 13 (Ctrl), 11 (Gly), and 9 (CTX) animals over four independent experiments. H mRNA expression of adipogenic genes in ScAT-derived SVF from Ctrl, Gly, and CTX-injured animals at 1 dpi. n = 7 (Ctrl), 7 (Gly), and 7 (CTX) animals over three independent experiments. Results are expressed as percentage of non-injured control animals with mean ± SEM; * p < 0.05, ** p < 0.01,*** p < 0.001 vs Ctrl.

Article Snippet: ASC sorting: ScAT- or PGAT- derived SVF were depleted in CD45 + and CD31 + cells using anti-CD45-FITC (Miltenyi, 130-116-535) and anti-CD31-FITC antibodies (Miltenyi, 130-123-675) followed by anti-FITC magnetic microbeads (Miltenyi, 130-048-701) using an autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Control, Cytometry, Marker, Comparison, TUNEL Assay, Staining

Journal: Nature Communications

Article Title: Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury

doi: 10.1038/s41467-022-35524-7

Figure Lengend Snippet: A Time course evaluation of murine in vitro ASC chemotaxis in response to plasma isolated from Ctrl, CTX- and Gly-injured animals at 1 dpi. n = 5 (Ctrl), 3 (Gly) and 4 (CTX) animals over three independent experiments. B In vitro human ASCs (of three individuals) chemotaxis in response to serum of six individuals collected 1 hour after an acute bout of continuous exercise (60% VO 2 max). C Correlation of human ASC chemotaxis with 1h-post exercise GDF-15 blood levels. n = 6 serums tested on 1 or 2 sets of ASC over three independent experiments. D Model of ScAT grafting from CD34-GFP mouse into WT C57Bl/6 mice (left panel), the figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Immunohistofluorescence image of the ScAT depot 7 days post-graft surgery (right panel, bar scale 200 μm). E Flow cytometry analysis of the SVF from Gly-injured muscle of the grafted mice (1 dpi), GFP + /CD45 − /CD31 − are scated on an histogram for Sca-1 intensity. F – H Immunohistological analysis of Gly-injured (1 dpi) quadriceps in grafted mice with KikGR ScAT in situ (green arrowheads point KikGR + /CD140α + /CD45 − ( F ), KikGR + / Podoplanin + /CD31 − cells ( G ) or KikGR + /CD140α + /Sca-1 + ( H )). Bar scale 10 μm. Results are expressed as mean ± SEM; * p < 0.05.

Article Snippet: ASC sorting: ScAT- or PGAT- derived SVF were depleted in CD45 + and CD31 + cells using anti-CD45-FITC (Miltenyi, 130-116-535) and anti-CD31-FITC antibodies (Miltenyi, 130-123-675) followed by anti-FITC magnetic microbeads (Miltenyi, 130-048-701) using an autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions.

Techniques: In Vitro, Chemotaxis Assay, Clinical Proteomics, Isolation, Generated, Immunohistofluorescence, Flow Cytometry, In Situ

Journal:

Article Title: TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

doi: 10.1016/j.ydbio.2003.12.001

Figure Lengend Snippet: Detection of the TrkB ligands, NT-4 and BDNF, in the developing mouse ovary by immunohistofluorescence-confocal microscopy and conventional light microscopy. Ovaries from 7-day-old trkB +/+ and trkB −/− mice were used. Three animals of each genotype were examined. In panels A– D, immunoreactive cells are seen in green and cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. In panels E– H, NT-4 immunoreactive cells are brown and cell nuclei stained with Gill’s hematoxylin are purple. (A–D) BDNF is present in both oocytes and granulosa cells of newly formed follicles. (A) Oocytes of primordial (short arrows) and small, nongrowing primary follicles (long arrow) contain moderate levels of BDNF immunoreactive material. (B) The oocytes of some growing primary (large single arrowhead) and secondary follicles (large double arrowheads), as well as granulosa cells of some follicles (small arrowheads) contain BDNF. (C) Higher magnification image showing BDNF immunoreactivity in granulosa cells of a growing primary follicle (small arrowheads). (D) Section from a BDNF −/− mouse ovary showing lack of BDNF immunoreactivity. (E–H) Like BDNF, NT-4 is also present in oocytes and granulosa cells of newly formed follicles. (E) NT-4 is detected in oocytes of primordial follicles (short arrows); (F) in growing type 3b primary follicles (large single arrowheads), and (G) secondary follicles (large double arrowheads), NT-4 immunoreactivity is predominantly seen in granulosa cells. (H) Section incubated with NT-4 antibodies preadsorbed with the NT-4 peptide used to raise the antibodies. Scale bars in A–D = 10 μm. Scale bars in E–H = 20 μm.

Article Snippet: Assessment of proliferation The ovaries from 7-day-old trkB +/+ and trkB −/− mice were fixed in Carnoy’s fixative, embedded in paraffin, sectioned at 4 μm, and subjected to immunohistochemistry for PCNA, as reported ( Dissen et al., 2001 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:1000; Santa Cruz Biotechnology) and developing the immunoreaction with a diaminobenzidine/H 2 O 2 /nickel chloride solution, followed by counterstaining with Gill’s hematoxylin (1:10 dilution).

Techniques: Immunohistofluorescence, Confocal Microscopy, Light Microscopy, Staining, Binding Assay, Incubation

Journal:

Article Title: TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

doi: 10.1016/j.ydbio.2003.12.001

Figure Lengend Snippet: Granulosa cell proliferation at the end of the first postnatal week of life is decreased in TrkB-deficient mice, without a concomitant increase in apoptotic cell death. (A–F) Decreased number of cell nuclei positive for the proliferation markers PCNA (A–C) and BrdU (D–F) in granulosa cells of trkB −/− ovaries analyzed at the end of the first postnatal week of life (day 7). (A) Number of PCNA-positive granulosa cells per ovary. (D) Ratio of BrdU positive/total granulosa cells per ovary. Bars are means and numbers on top of bars are number of animals per group. Vertical lines are SEM. (B and C) Microphotographs of ovarian sections from trkB +/+ and trkB −/− mice stained with antibodies to the proliferation marker PCNA and counterstained with Gill’s hematoxylin. (E and F) Microphotographs of ovarian sections from trkB +/+ and trkB −/− mice stained with antibodies to BrdU and counterstained with nuclear fast red. Arrows denote follicles showing a predominance of nonproliferating granulosa cells. Scale bars = 50 μm. (G and H) The stunted development of ovarian follicles associated with the loss of TrkB receptors is not accompanied by increased apoptotic cell death, as determined by a TUNEL fluorescence assay. (I) Follicles from a prepubertal 34-day-old ovary showing numerous apoptotic cell nuclei in the granulosa cell compartment of atretic antral follicles. The extensive apoptosis seen in these cells (green nuclei) is in contrast with the almost complete absence of apoptotic nuclei in the follicles of infantile ovaries (7-day-old) mice having either a normal complement of trkB receptors (+/+, G) or lacking all trkB receptor forms (−/−, H). The images depicted represent observations made in ovaries from three 7-day-old mice per genotype and two wild-type late juvenile mice. Scale bars = 25 μm.

Article Snippet: Assessment of proliferation The ovaries from 7-day-old trkB +/+ and trkB −/− mice were fixed in Carnoy’s fixative, embedded in paraffin, sectioned at 4 μm, and subjected to immunohistochemistry for PCNA, as reported ( Dissen et al., 2001 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:1000; Santa Cruz Biotechnology) and developing the immunoreaction with a diaminobenzidine/H 2 O 2 /nickel chloride solution, followed by counterstaining with Gill’s hematoxylin (1:10 dilution).

Techniques: Staining, Marker, TUNEL Assay, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Expression of Chd7 and Sox2 in the adult mouse spinal cord and in cultured OPCs. A, Double staining for Chd7 and either Olig2, Sox10, PDGFRα-GFP, Sox2, CC1, or GSTπ in the WM and GM of the spinal cord is shown. B, Quantitation of the percentages of Chd7+ cells among marker-positive cells in A. Data are shown as means ± SD (n = 3–9 slices from 3 animals). C, Chd7+/PDGFRα-GFP+/CC1− cells (arrows) and Chd7+/PDGFRα-GFP−/CC1+ cells (arrowheads) in the adult spinal cord. D, Chd7+/GFAP−/CC1+ cells (arrows) and Chd7−/GFAP+/CC1− cells (arrowheads) in the adult spinal cord. E, OPCs derived from the E15.5 mouse forebrain were cultured with FGF2 and PDGF-AA. Double staining for Chd7 and either Olig2, PDGFRα, NG2, Sox10, or Sox2 in cultured OPCs is shown. F, Chd7low/GFAP+ cells (arrows) and Chd7high/GFAP− cells (arrowheads) in cultured OPCs. G, Double staining for Sox2 and either Sox10, PDGFRα-GFP, NG2, CC1, GSTπ, or GFAP in the WM and GM of the spinal cord. H, Triple staining for Chd7, PDGFRα, and Sox2 in the adult spinal cord. I, OPCs were cultured with FGF2 and PDGF-AA. Double staining for Sox2 and either Olig2, Sox10, PDGFRα, or NG2 in cultured OPCs is shown. J, Triple staining for Sox10, PDGFRα, and NFIA in the adult spinal cord. Arrows indicate double-positive (A, E, G, I) and triple-positive (H, J) cells. Scale bars: A, C, D, G, H, J, 50 μm; E, F, I, 25 μm.

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Expressing, Cell Culture, Double Staining, Quantitation Assay, Marker, Derivative Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 is necessary for the induction of expression of Rgcc and PKCθ. A, Double staining for Rgcc and either Chd7 or Sox2 in the adult spinal cord. B, Double staining for PKCθ and either Chd7 or Sox2 in the adult spinal cord. C, Double staining for Rgcc and either Olig2, Sox10, PDGFRα-GFP, or GFAP in the adult spinal cord. D, Double staining for PKCθ and either Olig2, Sox10, PDGFRα-GFP, or GFAP in the adult spinal cord. E–G, Injured spinal cords of control and Chd7 cKO mice were isolated at 3 dpi and immunostained for Rgcc (E), PKCθ (F), and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 9 slices from 3 animals, Rgcc: Chd7 cKO, t(4) = 6.14, p = 0.0040; PKCθ: Chd7 cKO, t(4) = 4.55, p = 0.0100; unpaired Student's t test) (G). cKO, Conditional knock-out. Bottom, Higher-magnification views of the boxed areas in E and F. Arrows indicate double-positive cells (A–F). *p < 0.01, **p < 0.05 versus corresponding control value. Scale bars, 100 μm (E, F), 50 μm (A–D), and 25 μm (higher-magnification views in E, F).

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Expressing, Double Staining, Isolation, Marker, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 is necessary for OPC proliferation after injury and the maintenance of OPC identity. A, Tamoxifen was administered to control (PDGFRα-CreER;Chd7+/+;CAG-CAT-EGFP) and Chd7 cKO (PDGFRα-CreER;Chd7flox/flox;CAG-CAT-EGFP) mice once a day for 5 consecutive days. Three days after the last tamoxifen injection, the spinal cords of control and Chd7 cKO mice were isolated and immunostained for Chd7 and GFP. Arrows and arrowheads indicate Chd7+/GFP+ cells and Chd7−/GFP+ cells, respectively. B–D, Injured spinal cords of control and Chd7 cKO mice were isolated at 3 dpi and subjected to immunohistofluorescence analysis with antibodies to BrdU and GFP (B) and to Ki67 and GFP (C). The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 9 slices from 3 animals, BrdU: Chd7 cKO, t(4) = 5.99, p = 0.0040; Ki67: Chd7 cKO, t(4) = 3.40, p = 0.0270; unpaired Student's t test) (D). E, F, Injured spinal cords of control and Chd7 cKO mice were isolated at 3 dpi and immunostained for cleaved caspase 3 (cl-Casp3) and GFP (E). The percentages of cl-Casp3+ cells among total GFP+ cells were determined as means ± SD (n = 9 slices from 3 animals, cl-Casp3: Chd7 cKO, t(4) = 1.23, p = 0.2840; unpaired Student's t test) (F). G–I, Injured spinal cords of control and Chd7 cKO mice were isolated at 3 dpi and immunostained for NG2 (G), Sox10 (H), and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 9 slices from 3 animals, NG2: Chd7 cKO, t(4) = 5.51, p = 0.0050; Sox10: Chd7 cKO, t(4) = 12.61, p = 0.0001; unpaired Student's t test) (I). cKO, Conditional knock-out; NS, nonsignificant. Bottom, Higher-magnification views of the boxed areas in A–C, E, G, and H. Arrows indicate marker-positive/GFP+ cells (B, C, G, H). *p < 0.01, **p < 0.05 versus corresponding control value. Scale bars, 100 μm (A–C, E, G, H) and 25 μm (higher-magnification views in A–C, E, G, H).

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Injection, Isolation, Immunohistofluorescence, Marker, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 is necessary for OPC differentiation into OLs and remyelination after SCI. A–C, Injured spinal cords of control and Chd7 cKO mice were isolated at 42 dpi and subjected to immunostaining for GSTπ (A), Sox10 (B), and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 15–20 slices from 3–4 animals, GSTπ: Chd7 cKO, t(5) = 6.70, p = 0.0020; Sox10: Chd7 cKO, t(6) = 4.10, p = 0.0060; unpaired Student's t test) (C). D, E, Intact spinal cords of wild-type mice and the injured (42 dpi) spinal cords of control and Chd7 cKO mice were subjected to FluoroMyelin staining (D). The ratios of FluoroMyelin-positive area to the total section area were quantified as means ± SD (n = 9 slices from 3 animals, FluoroMyelin: Chd7 cKO, t(4) = 3.03, p = 0.0390; unpaired Student's t test) (E). F, G, Injured spinal cords of control and Chd7 cKO mice were isolated at 42 dpi and immunostained for GFAP, Sox10, and GFP (F). The percentages of GFAP+ cells among total GFP+ cells were determined as means ± SD (n = 20 slices from 4 animals, GFAP: Chd7 cKO, t(6) = 2.66, p = 0.0370; unpaired Student's t test) (G). H, Open-field locomotor recovery was assessed using the BMS in control and Chd7 cKO mice for 42 d after SCI. Data are median ± SEM (control: n = 5 animals, Chd7 cKO: n = 6 animals, Chd7 cKO: 28 d, p = 0.0300; 35 d, p = 0.0040; 42 d, p = 0.0090; Mann–Whitney U test). cKO, Conditional knock-out. Bottom, Higher-magnification views of the boxed areas in A and B. Arrows indicate marker-positive/GFP+ cells (A, B). Arrowheads and arrow indicate GFAP−/Sox10+/GFP+ cells and GFAP+/Sox10−/GFP+ cells, respectively (F). *p < 0.01, **p < 0.05 versus corresponding control value. Scale bars, 100 μm (A, B, D) and 25 μm (F and higher-magnification views in A, B).

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Isolation, Immunostaining, Marker, Staining, MANN-WHITNEY, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 is necessary for OPC proliferation and identity maintenance in the developing and intact adult spinal cord. A, Schematic diagram of the experimental design. Tamoxifen was administered to pregnant dams at E13.5 (blue arrow) and embryos were analyzed at E15.5. BrdU was injected intraperitoneally to pregnant dams 2 h before sampling (green arrow). B–F, Spinal cord sections of control and Chd7 cKO mouse embryos were immunostained for BrdU (B), Ki67 (C), PDGFRα, NG2, Sox10 (E), and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 9 slices from 3 animals, BrdU: Chd7 cKO, t(4) = 21.70, p = 0.0001; Ki67: Chd7 cKO, t(4) = 10.79, p = 0.0004; PDGFRα: Chd7 cKO, t(4) = 3.73, p = 0.0202; NG2: Chd7 cKO, t(4) = 4.42, p = 0.0114; Sox10: Chd7 cKO, t(4) = 4.64, p = 0.0097; unpaired Student's t test) (D, F). G, Schematic diagram of the experimental design. Tamoxifen was administered to adult control and Chd7 cKO mice once a day for 5 consecutive days (blue arrows). After tamoxifen treatment, BrdU was administered to the mice via the drinking water (green line) and by intraperitoneal injections for 8 d (green arrows). Two hours after the last BrdU injection, the intact spinal cords of control and Chd7 cKO mice were isolated and immunostained for BrdU (H), Ki67 (I), cleaved caspase 3 (cl-Casp3) (K), NG2, Sox10 (L), GSTπ, GFAP, Sox10 (N), and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 9–12 slices from 3 to 4 animals, BrdU: Chd7 cKO, t(4) = 3.71, p = 0.0206; Ki67: Chd7 cKO, t(4) = 5.18, p = 0.0065; cl-Casp3: Chd7 cKO, t(4) = 0.26, p = 0.8061; NG2: Chd7 cKO, t(6) = 3.01, p = 0.0236; Sox10: Chd7 cKO, t(4) = 9.35, p = 0.0007; GSTπ: Chd7 cKO, t(4) = 6.62, p = 0.0026; GFAP: Chd7 cKO, t(6) = 3.68, p = 0.0102; unpaired Student's t test) (J, K, M, O). cKO, Conditional knock-out; NS, nonsignificant. Bottom, Higher-magnification views of the boxed areas in B and H. Arrows indicate marker-positive/GFP+ cells (B, C, E, H, I, L, and top in N). Arrowheads and arrow indicate GFAP−/Sox10+/GFP+ cells and GFAP+/Sox10−/GFP+ cells, respectively (bottom in N). *p < 0.01, **p < 0.05 versus corresponding control value. Scale bars, 100 μm (B, H) and 25 μm (C, E, I, L, N and higher-magnification views in B, H).

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Injection, Sampling, Marker, Isolation, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 is necessary for OPC proliferation, the maintenance of OPC identity, and OL differentiation in vitro. A, B, OPCs derived from Chd7flox/flox mice were infected with retroviruses encoding GFP alone (control) or GFP plus Cre and then cultured with FGF2 and PDGF-AA. Three days after infection, the cells were immunostained for Chd7 and GFP (A). Arrows and arrowheads indicate Chd7+/GFP+ cells and Chd7−/GFP+ cells, respectively. The cells were also harvested 3 d after infection and the expression level of Chd7 mRNA was measured by quantitative RT-PCR analysis (B). Data are expressed relative to the control value and are means ± SD (n = 3 experiments, Chd7: Cre, t(4) = 57.32, p = 0.0001; unpaired Student's t test). C–H, Chd7flox/flox OPCs infected with retroviruses for control or Cre were cultured with FGF2 and PDGF-AA. Three days after infection, the cells were labeled with EdU for 2 h and were stained for cleaved caspase 3 (cl-Casp3) (C), EdU, Ki67 (E), PDGFRα, Sox10 (G), and GFP. The percentages of marker-positive cells among total GFP+ cells were quantified as means ± SD (n = 3 experiments, cl-Casp3: Cre, t(4) = 0.81, p = 0.462; n = 5 experiments, EdU: Cre, t(8) = 5.77, p = 0.0004; Ki67: Cre, t(8) = 4.99, p = 0.0011; n = 3 experiments, PDGFRα: Cre, t(4) = 23.26, p = 0.0001; Sox10: Cre, t(4) = 12.60, p = 0.0002; unpaired Student's t test) (D, F, H). I, J, Chd7flox/flox OPCs infected with retroviruses for control or Cre were cultured with FGF2 and PDGF-AA. Two days after infection, the cells were induced to differentiate without FGF2 and PDGF-AA, and with T3 for 5 d, after which the cells were immunostained for Sox10, MBP, GFAP, and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 3 experiments, Sox10: Cre, t(4) = 27.05, p = 0.0001; MBP: Cre, t(4) = 12.99, p = 0.0002; GFAP: Cre, t(4) = 7.25, p = 0.0019; unpaired Student's t test). K, Chd7flox/flox OPCs infected with retroviruses for control or CreERT2 were cultured with FGF2 and PDGF-AA. Two days after infection, the cells were induced to differentiate without FGF2 and PDGF-AA and with T3 and treated with 4-OHT 8 h after induction of differentiation. After 5 d, the cells were immunostained for MBP and GFP. The percentages of marker-positive cells among total GFP+ cells were determined as means ± SD (n = 3 experiments, MBP: control + 4-OHT, t(4) = 0.19, p = 0.8563; CreERT2, t(4) = 0.71, p = 0.5144; CreERT2 + 4-OHT, t(4) = 18.83, p = 0.0001; unpaired Student's t test). NS, Nonsignificant. Arrows indicate marker-positive/GFP+ cells (C, E, G, I). *p < 0.01 versus corresponding control value. Scale bars: I, 50 μm; A, C, E, G, 25 μm.

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: In Vitro, Derivative Assay, Infection, Cell Culture, Expressing, Quantitative RT-PCR, Labeling, Staining, Marker

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Sox2 is necessary for OPC proliferation and identity maintenance in vitro. A, OPCs were infected with retroviruses encoding GFP together with either a control shRNA (sh-Luc) or a Sox2 shRNA (sh-Sox2 #1) and were then cultured with FGF2 and PDGF-AA. Three days after infection, the cells were immunostained for Sox2 and GFP. Arrows and arrowheads indicate Sox2high/GFP+ cells and Sox2low/GFP+ cells, respectively. B, OPCs were infected with retroviruses for control, sh-Sox2 #1, or sh-Sox2 #2. The cells were harvested 3 d after infection and knock-down efficiency of the shRNAs was determined by quantitative RT-PCR analysis. Data are expressed relative to the control value and are means ± SD (n = 3 experiments, Sox2: sh-Sox2 #1, t(4) = 26.19, p = 0.0001; sh-Sox2 #2, t(4) = 25.04, p = 0.0001; unpaired Student's t test). C–H, OPCs infected with retroviruses for control, sh-Sox2 #1, or sh-Sox2 #2 were cultured with FGF2 and PDGF-AA. Three days after infection, the cells were labeled with EdU for 2 h and were stained for cleaved caspase 3 (cl-Casp3) (C), EdU, Ki67 (E), PDGFRα, Sox10 (G), and GFP. The percentages of marker-positive cells among total GFP+ cells were quantified as means ± SD (n = 3 experiments, cl-Casp3: sh-Sox2 #1, t(4) = 0.50, p = 0.6399; sh-Sox2 #2, t(4) = 0.06, p = 0.95; EdU: sh-Sox2 #1, t(4) = 8.78, p = 0.0009; sh-Sox2 #2, t(4) = 5.43, p = 0.0056; Ki67: sh-Sox2 #1, t(4) = 8.53, p = 0.0010; sh-Sox2 #2, t(4) = 10.99, p = 0.0004; PDGFRα: sh-Sox2 #1, t(4) = 6.93, p = 0.0023; sh-Sox2 #2, t(4) = 4.73, p = 0.0091; Sox10: sh-Sox2 #1, t(4) = 6.75, p = 0.0025; sh-Sox2 #2, t(4) = 6.15, p = 0.0035; unpaired Student's t test) (D, F, H). I–K, OPCs infected with retroviruses for control, Cre, sh-Sox2 #1, or Cre plus sh-Sox2 #1 were cultured with FGF2 and PDGF-AA. Three days after infection, the cells were labeled with EdU for 2 h and were stained for cl-Casp3 (I), EdU, Ki67 (J), PDGFRα, Sox10 (K), and GFP. The percentages of marker-positive cells among total GFP+ cells were quantified as means ± SD (n = 3 experiments, cl-Casp3: Cre, t(4) = 0.40, p = 0.7050; sh-Sox2 #1, t(4) = 0.14, p = 0.8924; Cre + sh-Sox2 #1, t(4) = 0.37, p = 0.7280; EdU: Cre, t(4) = 9.71, p = 0.0006; sh-Sox2 #1, t(4) = 8.30, p = 0.0011; Cre + sh-Sox2 #1, t(4) = 11.36, p = 0.0003; Ki67: Cre, t(4) = 18.18, p = 0.0001; sh-Sox2 #1, t(4) = 24.37, p = 0.0001; Cre + sh-Sox2 #1, t(4) = 11.15, p = 0.0004; PDGFRα: Cre, t(4) = 12.62, p = 0.0002; sh-Sox2 #1, t(4) = 13.12, p = 0.0002; Cre + sh-Sox2 #1, t(4) = 12.19, p = 0.0003; Sox10: Cre, t(4) = 8.83, p = 0.0009; sh-Sox2 #1, t(4) = 10.34, p = 0.0005; Cre + sh-Sox2 #1, t(4) = 12.91, p = 0.0002; unpaired Student's t test). L, 293T cells were transfected with plasmids for Chd7 and Sox2. The cell lysates were subjected to co-immunoprecipitation and Western blot analysis. M, Lysates of OPCs cultured with FGF2 and PDGF-AA were subjected to coimmunoprecipitation and Western blot analysis. N, OPCs cultured with FGF2 and PDGF-AA were subjected to PLA with antibodies to Chd7 and to Sox2, normal rabbit IgG, and normal goat IgG. PLA signals (red) indicate the interaction between Chd7 and Sox2. Cell nuclei were stained with DAPI. NS, Nonsignificant; IP, immunoprecipitation; WCL, whole-cell lysate; Gt, goat; Rb, rabbit. Arrows indicate marker-positive/GFP+ cells (C, E, G). *p < 0.01 versus corresponding control value. Scale bars: A, C, E, G, 25 μm; N, 10 μm.

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: In Vitro, Infection, shRNA, Cell Culture, Quantitative RT-PCR, Labeling, Staining, Marker, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Chd7 regulates the expression of OL-related genes. A, Volcano plot of microarray data showing gene expression changes between control and Chd7 knock-down cells. Red and green dots represent genes significantly upregulated (fold change >1.5, p < 0.05) and downregulated (fold change <0.66, p < 0.05) in Chd7 knock-down cells, respectively. B, Heat map representing the expression of OL-related genes in control and Chd7 knock-down cells from three independent cultures. The color scale represents normalized gene expression levels (red, upregulated expression levels; green, downregulated expression levels). C, OPCs infected with retroviruses for control, sh-Chd7 #1, or sh-Sox2 #1 were cultured with FGF2 and PDGF-AA. The cells were harvested 3 d after infection and the relative mRNA abundance for the indicated proteins was measured by quantitative RT-PCR analysis. Data are shown as means ± SD (n = 3 experiments, PDGFRα: sh-Chd7 #1, t(4) = 14.82, p = 0.0001; sh-Sox2 #1, t(4) = 7.03, p = 0.0021; Myt1: sh-Chd7 #1, t(4) = 12.39, p = 0.0002; sh-Sox2 #1, t(4) = 5.32, p = 0.0060; CSPG4: sh-Chd7 #1, t(4) = 15.18, p = 0.0001; sh-Sox2 #1, t(4) = 12.47, p = 0.0002; Hes5: sh-Chd7 #1, t(4) = 11.40, p = 0.0003; sh-Sox2 #1, t(4) = 11.37, p = 0.0003; Sox9: sh-Chd7 #1, t(4) = 6.01, p = 0.0038; sh-Sox2 #1, t(4) = 2.63, p = 0.0577; Id2: sh-Chd7 #1, t(4) = 15.43, p = 0.0001; sh-Sox2 #1, t(4) = 1.39, p = 0.2346; Sox10: sh-Chd7 #1, t(4) = 26.29, p = 0.0001; sh-Sox2 #1, t(4) = 7.77, p = 0.0015; Olig2: sh-Chd7 #1, t(4) = 5.19, p = 0.0065; sh-Sox2 #1, t(4) = 10.32, p = 0.0005; unpaired Student's t test). NS, Nonsignificant. *p < 0.01 versus control value. D, GO analysis of genes downregulated in Chd7 knock-down cells. The number of genes belonging to each category is shown in parentheses.

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

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

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Rgcc and PKCθ are necessary for OPC proliferation and the maintenance of OPC identity. A, OPCs infected with retroviruses encoding GFP together with either a control shRNA (sh-Luc), an Rgcc shRNA (sh-Rgcc #1 or #2), or a PKCθ shRNA (sh-PKCθ #1 or #2) were cultured with FGF2 and PDGF-AA. Three days after infection, knock-down efficiency of the shRNAs was determined by quantitative RT-PCR analysis. Data are expressed relative to the control value and are means ± SD (n = 3 experiments, Rgcc: sh-Rgcc #1, t(4) = 40.92, p = 0.0001; sh-Rgcc #2, t(4) = 18.26, p = 0.0001; PKCθ: sh-PKCθ #1, t(4) = 12.09, p = 0.0003; sh-PKCθ #2, t(4) = 12.92, p = 0.0002; unpaired Student's t test). B–G, OPCs infected with retroviruses for control, sh-Rgcc #1, sh-Rgcc #2, sh-PKCθ #1, or sh-PKCθ #2 were cultured with FGF2 and PDGF-AA. Three days after infection, the cells were labeled with EdU for 2 h and were stained for EdU, Ki67 (B), PDGFRα, Sox10 (D), cleaved caspase 3 (cl-Casp3) (F), and GFP. The percentages of marker-positive cells among total GFP+ cells were quantified as means ± SD (n = 3 experiments, EdU: sh-Rgcc #1, t(4) = 6.33, p = 0.0032; sh-Rgcc #2, t(4) = 4.94, p = 0.0078; sh-PKCθ #1, t(4) = 6.56, p = 0.0028; sh-PKCθ #2, t(4) = 5.98, p = 0.0039; Ki67: sh-Rgcc #1, t(4) = 15.62, p = 0.0001; sh-Rgcc #2, t(4) = 5.29, p = 0.0061; sh-PKCθ #1, t(4) = 12.37, p = 0.0002; sh-PKCθ #2, t(4) = 11.91, p = 0.0003; PDGFRα: sh-Rgcc #1, t(4) = 11.62, p = 0.0003; sh-Rgcc #2, t(4) = 11.59, p = 0.0003; sh-PKCθ #1, t(4) = 24.78, p = 0.0001; sh-PKCθ #2, t(4) = 14.35, p = 0.0001; Sox10: sh-Rgcc #1, t(4) = 9.65, p = 0.0006; sh-Rgcc #2, t(4) = 6.86, p = 0.0024; sh-PKCθ #1, t(4) = 6.97, p = 0.0022; sh-PKCθ #2, t(4) = 5.54, p = 0.0052; cl-Casp3: sh-Rgcc #1, t(4) = 0.10, p = 0.9182; sh-Rgcc #2, t(4) = 0.15, p = 0.8836; sh-PKCθ #1, t(4) = 0.27, p = 0.7983; sh-PKCθ #2, t(4) = 0.03, p = 0.9715; unpaired Student's t test) (C, E, G). NS, Nonsignificant. Arrows indicate marker-positive/GFP+ cells (B, D, F). *p < 0.01 versus corresponding control value. Scale bars, 25 μm (B, D, F).

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Infection, shRNA, Cell Culture, Quantitative RT-PCR, Labeling, Staining, Marker

Journal: The Journal of Neuroscience

Article Title: Chd7 Collaborates with Sox2 to Regulate Activation of Oligodendrocyte Precursor Cells after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1109-17.2017

Figure Lengend Snippet: Overexpression of Rgcc or PKCθ rescues the Chd7 deletion phenotypes. A, B, OPCs were infected with retroviruses encoding GFP alone (control), GFP plus Rgcc, or GFP plus PKCθ, and then cultured with FGF2 and PDGF-AA. Three days after infection, the expression level of Rgcc (A) and PKCθ (B) mRNAs was measured by quantitative RT-PCR analysis. Data are expressed relative to the control value and are means ± SD (n = 3 experiments, Rgcc: Rgcc, t(4) = 24.97, p = 0.0001; PKCθ: PKCθ, t(4) = 6.91, p = 0.0023; unpaired Student's t test). C, D, OPCs infected with retroviruses for control, Cre, Rgcc, Cre plus Rgcc, PKCθ, Cre plus PKCθ, or Cre plus Rgcc plus PKCθ were cultured with FGF2 and PDGF-AA. Three days after infection, the cells were labeled with EdU for 2 h and stained for EdU, Ki67 (C), PDGFRα, Sox10 (D), and GFP. The percentages of marker-positive cells among total GFP+ cells were quantified as means ± SD (n = 3 experiments, EdU: Cre, t(4) = 5.38, p = 0.0057; Rgcc, t(4) = 4.62, p = 0.0099; Cre + Rgcc, t(4) = 2.85, p = 0.0460 vs control; t(4) = 4.91, p = 0.0080 vs Cre; PKCθ, t(4) = 4.86, p = 0.0082; Cre + PKCθ, t(4) = 2.94, p = 0.0420 vs control; t(4) = 4.72, p = 0.0091 vs Cre; Cre + Rgcc + PKCθ, t(4) = 1.24, p = 0.2820 vs control; t(4) = 7.86, p = 0.0014 vs Cre; Ki67: Cre, t(4) = 16.63, p = 0.0001; Rgcc, t(4) = 5.66, p = 0.0048; Cre + Rgcc, t(4) = 6.42, p = 0.0030 vs control; t(4) = 6.49, p = 0.0029 vs Cre; PKCθ, t(4) = 5.10, p = 0.0069; Cre + PKCθ, t(4) = 15.84, p = 0.0001 vs control; t(4) = 6.01, p = 0.0038 vs Cre; Cre + Rgcc + PKCθ, t(4) = 1.92, p = 0.1264 vs control; t(4) = 13.52, p = 0.0002 vs Cre; PDGFRα: Cre, t(4) = 10.42, p = 0.0005; Rgcc, t(4) = 1.31, p = 0.2592; Cre + Rgcc, t(4) = 4.58, p = 0.0102 vs control; t(4) = 5.31, p = 0.0060 vs Cre; PKCθ, t(4) = 4.36, p = 0.0120; Cre + PKCθ, t(4) = 6.01, p = 0.0038 vs control; t(4) = 7.65, p = 0.0016 vs Cre; Cre + Rgcc + PKCθ, t(4) = 0.22, p = 0.8335 vs control; t(4) = 9.78, p = 0.0006 vs Cre; Sox10: Cre, t(4) = 16.85, p = 0.0001; Rgcc, t(4) = 0.46, p = 0.6642; Cre + Rgcc, t(4) = 6.10, p = 0.0036 vs control; t(4) = 8.38, p = 0.0011 vs Cre; PKCθ, t(4) = 7.39, p = 0.0018; Cre + PKCθ, t(4) = 4.22, p = 0.0134 vs control; t(4) = 9.41, p = 0.0007 vs Cre; Cre + Rgcc + PKCθ, t(4) = 1.21, p = 0.2926 vs control; t(4) = 8.00, p = 0.0013 vs Cre; unpaired Student's t test) *p < 0.01, **p < 0.05 versus corresponding control value. #p < 0.01 versus Cre value. E, Model for OPC activation by Chd7. In response to injury, the expression level of Chd7 and Sox2 is upregulated in activated OPCs, in which they form a complex and induce directly the expression of PKCθ and Rgcc, which are essential for OPC proliferation and the maintenance of OPC identity.

Article Snippet: Immunostaining was performed with the following antibodies: Chd7 (Abcam, ab31824, rabbit, 1:500 dilution; OriGene, TA309607, rabbit, 1:500; Abcam, ab134832, goat, 1:200; Santa Cruz Biotechnology, sc390742, mouse, 1:200), Olig2 (Millipore, AB9610, rabbit, 1:1000; Millipore, MABN50, mouse, 1:500), Sox10 (Santa Cruz Biotechnology, sc17342, goat, 1:500; Abcam, ab155279, mouse, 1:5000), Sox2 (Santa Cruz Biotechnology, sc17320, goat, 1:200), CC1 (Millipore, OP80, mouse, 1:500), glutathione S-transferase π (GSTπ) (BD Biosciences, 610719, mouse, 1:1000), GFAP (Millipore, MAB360, mouse, 1:1000; Millipore, AB5804, rabbit, 1:1000; Abcam, ab4674, chicken, 1:2000), NG2 (Millipore, AB5320, rabbit, 1:200), PDGFRα (BD Biosciences, 558774, rat, 1:200), myelin-associated glycoprotein (MAG) (Millipore, MAB1567, mouse, 1:400), NFIA (Sigma-Aldrich, HPA006111, rabbit, 1:200), Myelin basic protein (MBP) (Bio-Rad, MCA409S, rat, 1:200), Galactocerebroside (GalC) (Millipore, MAB342, mouse, 1:500), Ki67 (Abcam, ab15580, rabbit, 1:1000), cleaved caspase 3 (Cell Signaling Technology, 9661, rabbit, 1:1000), Rgcc (Sigma-Aldrich, SAB1101621, rabbit, 1:200), PKCθ (Abcam, ab109481, rabbit, 1:200), and GFP (MBL, 598, rabbit, 1:2000; Abcam, ab13970, chicken, 1:2000).

Techniques: Over Expression, Infection, Cell Culture, Expressing, Quantitative RT-PCR, Labeling, Staining, Marker, Activation Assay

Journal: Regenerative Therapy

Article Title: A silk fibroin/chitosan/nanohydroxyapatite biomimetic bone scaffold combined with autologous concentrated growth factor promotes the proliferation and osteogenic differentiation of BMSCs and repair of critical bone defects

doi: 10.1016/j.reth.2022.08.006

Figure Lengend Snippet: Histological analysis of bone regeneration in vivo (A) Hematoxylin–eosin (HE) staining, Masson staining, immunohistochemistry of Col-1 and CD31 and immunohistofluorescence of Col-1 (B) New bone formation area quantified by Masson staining (n = 6) (C–D) Expression of COL-1 and CD31 quantified by immunohistochemistry (n = 6) (E) Expression of COL-1 quantified by immunohistofluorescence (n = 6). F, fibrous tissue; C, type I collagen; TB, trabecular bone; NB, new bone; Black arrow, remaining scaffold; Black star, transition zone between the remaining scaffold and the new bone. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the BM group, #p < 0.05, ##p < 0.01 compared with the BM-SCN group.

Article Snippet: The expression of Col-1 (Proteintech, 67288-1-Ig, 1:200 dilution) and CD31 (Proteintech, 11265-1-AP, 1:100 dilution) in each group was detected by immunohistochemistry.

Techniques: In Vivo, Staining, Immunohistochemistry, Immunohistofluorescence, Expressing