Journal: PLoS ONE

Article Title: Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling

doi: 10.1371/journal.pone.0059252

Figure Lengend Snippet: (A) Plated EBs differentiating in the presence of both PMA and CHIR for 6 days. (B) Phase-contrast images of neural epithelial cells on the indicated days after splitting. (C) Immunostaining of hESC-derived neural epithelial cells with antibodies raised against the indicated neural progenitor markers. Nuclei are counterstained with Hoechst. (D) Immunostaining of spontaneously differentiated neural epithelial cells for TUBBIII and NEUN, for GFAP and S100-beta after astrocyte differentiation, as well as O4 and OLIG2 after spontaneous differentiation, indicating oligodendrocyte formation. Scale bars are 100 µm. See also .

Article Snippet: The primary antibodies used in this study are mouse anti-NESTIN (1∶150, R&D), goat anti-SOX1 (1∶150, R&D), rabbit anti-PAX6 (1∶300, Covance), goat anti-SOX2 (1∶200, Santa Cruz), mouse anti-FOXA2 (1∶100, Santa Cruz), rabbit anti-TH (1∶500, Pel Freez), sheep anti-TH (1∶400, Pel Freez), mouse anti-TUBBIII (1∶1000, Covance), rabbit anti-TUBBIII (1∶2000, Covance), rabbit anti-MAP2 (1∶1000, Santa Cruz), mouse anti-O4 (1∶100, Millipore), rabbit anti-ISLET1 (1∶1500, Abcam), mouse anti-HB9 (5 µg/mL own preparation from DSHB hybridoma), goat anti-CHAT (1∶100, Millipore), mouse anti-BRN3A (1∶500, Santa Cruz), rabbit anti-PERIPHERIN (1∶200, Millipore), rabbit anti-GFAP (1∶1000, Dako), mouse anti GFAP (1∶500, Millipore), mouse anti S100-beta (1∶100, Thermo), mouse anti-SMA (1∶150, Dako), goat anti-T (1.200, Santa Cruz), mouse anti-Vimentin (1∶150, Dako), rabbit anti-CD9 (1.100, Santa Cruz), mouse anti-PAX7 (1∶100, Neuromics), mouse anti-SLUG (1∶100, Millipore), mouse anti-OSTEOCALCIN (R&D Systems, 5 µg/mL), goat anti-FABP4 (R&D Systems, 5 µg/mL) mouse anti-FORSE 1 (DSHB, 10 µg/mL), mouse anti-PAX3 (DSHB, 5 µg/mL), mouse anti-NKX2.2 (DSHB, 5 µg/mL), mouse anti-NKX6.1 (DSHB, 5 µg/mL), mouse anti TFAP2A (DSHB, 2 µg/mL), rabbit anti cleaved CASPASE3 (1∶1000, CellSignaling).

Techniques: Immunostaining, Derivative Assay

Journal: PLoS ONE

Article Title: Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling

doi: 10.1371/journal.pone.0059252

Figure Lengend Snippet: Summary of the markers used in this study as well as the characteristics of NSCs, lt-hESNSCS, R-NCs, pNSCs, and smNPCs.

Article Snippet: The primary antibodies used in this study are mouse anti-NESTIN (1∶150, R&D), goat anti-SOX1 (1∶150, R&D), rabbit anti-PAX6 (1∶300, Covance), goat anti-SOX2 (1∶200, Santa Cruz), mouse anti-FOXA2 (1∶100, Santa Cruz), rabbit anti-TH (1∶500, Pel Freez), sheep anti-TH (1∶400, Pel Freez), mouse anti-TUBBIII (1∶1000, Covance), rabbit anti-TUBBIII (1∶2000, Covance), rabbit anti-MAP2 (1∶1000, Santa Cruz), mouse anti-O4 (1∶100, Millipore), rabbit anti-ISLET1 (1∶1500, Abcam), mouse anti-HB9 (5 µg/mL own preparation from DSHB hybridoma), goat anti-CHAT (1∶100, Millipore), mouse anti-BRN3A (1∶500, Santa Cruz), rabbit anti-PERIPHERIN (1∶200, Millipore), rabbit anti-GFAP (1∶1000, Dako), mouse anti GFAP (1∶500, Millipore), mouse anti S100-beta (1∶100, Thermo), mouse anti-SMA (1∶150, Dako), goat anti-T (1.200, Santa Cruz), mouse anti-Vimentin (1∶150, Dako), rabbit anti-CD9 (1.100, Santa Cruz), mouse anti-PAX7 (1∶100, Neuromics), mouse anti-SLUG (1∶100, Millipore), mouse anti-OSTEOCALCIN (R&D Systems, 5 µg/mL), goat anti-FABP4 (R&D Systems, 5 µg/mL) mouse anti-FORSE 1 (DSHB, 10 µg/mL), mouse anti-PAX3 (DSHB, 5 µg/mL), mouse anti-NKX2.2 (DSHB, 5 µg/mL), mouse anti-NKX6.1 (DSHB, 5 µg/mL), mouse anti TFAP2A (DSHB, 2 µg/mL), rabbit anti cleaved CASPASE3 (1∶1000, CellSignaling).

Techniques:

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Fibroblast-secreted galectin-1 (Gal-1) significantly promotes multiple CIC features in CRC cells. (A) Expression of endogenous Gal-1 in MRC-5 and WS1 fibroblasts as detected through Western blot (top panel; recombinant human Gal-1 protein (rhGal-1) used as positive control) and ELISA (bottom panel). (B) Invasion capacity of KM12C with the addition of increasing doses of rhGal-1. (C) qPCR analysis for the gene expression of Twist1 and E-cadherin and (D) Western blot for protein expression of Slug and E-cadherin in KM12C with the addition of increasing doses of rhGal-1. (E) IF staining of E-cadherin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence); scale bar, 10 μm. (F) Sphere formation capacity of KM12C with the addition of increasing doses of rhGal-1; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (G) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with increasing dosages of rhGal-1 for 24 hours. Cell viability was assessed 48 hours after drug treatment. (H) Left panel: Validation of Gal-1 knockdown using small interfering RNA (siRNA) specific for Gal-1 (siRNA-I & siRNA-II) in WS1 fibroblasts. Non-target siRNA was used as a negative control. After 48 hours, CM from siRNA-I, siRNA-II, and siC were removed and analyzed by ELISA. Right panel: Invasion capacity of KM12C after culturing in siGal-WS1-CM compared to siC-WS1-CM for 48 hours. (I) Sphere formation capacity of KM12C cultured in KM12C-CM, siC-WS1-CM, or siGal-WS1-CM for 72 hours; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (J) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with KM12C-CM, siGal-WS1-CM, or siC-WS1-CM for 24 hours. Cell viability was assessed 48 hours after drug treatment; control, KM12C without cisplatin treatment. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Small Interfering RNA, Negative Control, Cell Culture

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Fibroblast-secreted Gal-1 significantly increases metastasis and tumor dissemination of CRC cells in vivo . (A) Body weight of NOD-SCID mice 40 days after tail vein injection of KM12C only (3 x 10 5 cells); KM12C (3 x 10 5 cells) admixed with WS1 silenced for with short-hairpin RNA (shRNA) of non-target sequences (shC-WS1; 3 x 10 5 cells); KM12C (3 x 10 5 cells) admixed with WS1 silenced with shRNA specific for Gal-1 (shGal-WS1; 3 x 10 5 cells); or shC-WS1 only (3 x 10 5 cells). Each condition consisted of three mice, with their body weight measured every 7 days. Immunohistochemistry (IHC) staining for human histone H1 (brown nuclei) in (B) mouse lung and (C) spleen tissue sections; representative sections (top panel) and quantitative results (bottom panel) are shown, with arrows indicating human Histone H1(+) cells; scale bar, 20 μm. (D) Visualization of fluorescently labeled co-cultured cells KM12C (3 x 10 5 cells; green fluorescence, labeled with DiO), and siC- or siGal-WS1 (3 x 10 5 cells; red fluorescence, labeled with with DiI) in lung sections 24 hours after injection into the tail vein of C57BL/6 mice. Top panel, representative images; bottom panel; quantitative results. Arrows indicate KM12C; scale bar, 100 μm. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05, and *** p < 0.005 compared to the control. (E) Analyses of Gal-1 ( LGALS1 ) and β-catenin ( CTNNB1 ) expression with regard to disease recurrence in the public dataset GSE33113 and GSE17536 of gene expression omnibus (GEO); * p < 0.05; N.S., not significant.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: In Vivo, Injection, shRNA, Immunohistochemistry, Labeling, Cell Culture, Fluorescence, Expressing

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Gal-1 promotes β-catenin expression, nuclear translocation, and activity in CRC cells. (A) IF staining for β-catenin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence). Arrows show nuclear β-catenin; scale bar, 10 μm. (B) Western blot for β-catenin levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with the addition of increasing doses of rhGal-1 for 48 hours; for nuclear fraction, histone H1 is used as the positive control and α-Tubulin as the negative control. (C) Luciferase reporter assay for β-catenin activity in KM12C with the addition of increasing doses of rhGal-1. TOPFlash plasmids (β-catenin promoter reporter construct containing TCF/LEF binding sites; please see Materials and Methods) and TOPFlash mutant plasmids (β-catenin promoter reporter construct containing mutated TCF/LEF binding sites; please see Materials and Methods) were transduced into KM12C, with the luciferase activity measured 48 hours later; addition of the Wnt/β-catenin agonist CHIR-99021 (CHIR; 0.3 µM) was used as a positive control. (D) qPCR analysis for the gene expression of Twist1 in KM12C after treatment with rhGal-1 (100 ng/ml) and without or with the Wnt/β-catenin antagonist XAV-939 (XAV; 10 µM) for 24 hours. All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Translocation Assay, Activity Assay, Staining, Fluorescence, Western Blot, Positive Control, Negative Control, Luciferase, Reporter Assay, Construct, Binding Assay, Mutagenesis

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: SOX9 is a critical mediator involved in Gal-1-induced upregulation of β-catenin activity and CIC features. (A) Ingenuity Pathway Analysis (IPA) for the prediction of candidate mediators within the LGALS1/CTNNB1/Twist1 axis. IPA database revealed the several major pathways which might be involved in tumor development and metastasis. According to the IPA results and literature review, SOX9 was selected and confirmed whether it is the downstream gene of Gal-1 by Western blot. (B) Western blot for the analysis of SOX9 protein levels in KM12C after culturing in MRC-5- or WS1-CM; KM12C-CM was used as the control. Internal control: GAPDH. (C) Western blot for SOX9 levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with addition of increasing doses of rhGal-1 for 48 hours; for nuclear protein blot, histone H1 is used as the positive control and α-Tubulin as the negative control. (D) Sphere formation capacity of shC-KM and shSOX9-II-KMC12 (shSOX9-KM) after treating with rhGal-1 (100 ng/ml) for 72 hours. (E) Drug resistance capacity of shC- and shSOX9-KM to cisplatin (25 µM) after pretreatment with rhGal-1 (100 ng/ml) for 24 hours. Cell viability was assessed 48 hours after drug treatment. (F) qPCR analysis for the gene expression of Twist1 in shC- and shSOX9-KM after treatment with Gal-1 (100 ng/ml) and XAV (10 µM). (G) Invasion capacity of shC- and shSOX9-KM with addition of rhGal-1 (100 ng/ml) and XAV (10 µM). All results are shown as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01, and *** p < 0.005 compared to the control. N.S., not significant.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Activity Assay, Western Blot, Positive Control, Negative Control, Expressing

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: High expression of Gal-1 and SOX9 correlate with clinical CRC outcome. (A) ONCOMINE assessment of the expression levels of LGALS1 and SOX9 in the Kaiser Colon database. (B) Analysis of LGALS1 or SOX9 expression levels in tumor tissue compared to adjacent normal tissue using GSE9348 (top panel) and The Cancer Genome Atlas (TCGA) databases (bottom panel). * p < 0.05 and *** p < 0.005 for early-stage lesions compared to adjacent normal tissue. (C) Analysis of LGALS1 and SOX9 expression levels to the CRC stage using the GSE17536 and TCGA datasets. (D) Immunohistological staining of Gal-1 and SOX9 on CRC tumor samples, which included 40 primary lesions (primary), 10 metastatic lesions (metastatic), and 9 normal colon samples; scale bar, 100 μm. (E) Kaplan-Meier survival curves of four groups of CRC patients as stratified by median expression levels of SOX9 and Gal-1 in tumor tissue: SOX9 low /Gal-1 low , n = 38; SOX9 low /Gal-1 high & SOX9 high /Gal-1 low , n = 100; and SOX9 high /Gal-1 high , n = 39. Survival analyses was performed for two groups: SOX9 high /Gal-1 high versus SOX9 low /Gal-1 low (left-side graph); or for three groups: SOX9 high /Gal-1 high versus SOX9 high /Gal-1 low + SOX9 low /Gal-1 high versus SOX9 low /Gal-1 low (right-side graph).

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: Stromal Galectin-1 Promotes Colorectal Cancer Cancer-Initiating Cell Features and Disease Dissemination Through SOX9 and β-Catenin: Development of Niche-Based Biomarkers

doi: 10.3389/fonc.2021.716055

Figure Lengend Snippet: Direct targeting of stromal-secreted Gal-1 on CRC cells promote CIC features and disease progression through SOX9 and β-catenin.

Article Snippet: Briefly, mouse monoclonal anti-Gal-1 antibody (1:500; Cat. No.437400; Invitrogen) was coated in 96-well plates at 4°C overnight.

Techniques:

Journal: Bioscience Reports

Article Title: Targeting intra-viral conserved nucleocapsid (N) proteins as novel vaccines against SARS-CoVs

doi: 10.1042/BSR20211491

Figure Lengend Snippet: ( A ) N-protein alignment from 4 different Coronaviruses (CoVs): 1. Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); 2. Severe acute respiratory syndrome coronavirus (SARS-CoV); 3. BAT severe acute respiratory (BAT SARS); 4. BAT coronavirus (BAT-CoV). Yellow color indicates the similarity of sequences among four viruses. The location of Peptide #1, #2, #3 (30 amino acid sequences) is also mentioned. ( B–E ) The whole N-protein vaccination was repeated four times (2-week interval). Red arrow indicates the time points for vaccination. Blood samples were taken before vaccination followed by every 2 weeks until 22nd week. Serum antibodies were detected by using anti-IgM, -IgG1, -IgG2 and anti IgG (H+L) horseradish peroxidase (HRP) conjugated antibodies. Antibody responses can be detected after 2nd vaccination and sustained till last sample collection in mouse#1 (B), #2 (C), #3 (D) and #4 (E). ( F ) Mean data of antibody productions in the BALB/c mice ( n =4). ( G ) Mean antibody production of N-protein vaccination in FVB mice ( n =3). Data represent mean ± S.D.

Article Snippet: Anti N-protein IgG (whole IgG) were detected by goat anti-mouse IgG-HRP (H+L) secondary antibody (Invitrogen 31430).

Techniques:

Journal: Bioscience Reports

Article Title: Targeting intra-viral conserved nucleocapsid (N) proteins as novel vaccines against SARS-CoVs

doi: 10.1042/BSR20211491

Figure Lengend Snippet: ( A ) Peptide sequences were selected based on N-protein conserved regions highlighted in yellow ( A). ( B ) Binding of anti N-polyclonal Abs serum and peptides were tested by ELISA. Whole N-protein was used as control. Peptides, #1, #2, #3 and N-protein were coated respectively with 5 ng and 20 ng/well. Anti-N polyclonal Abs serum (from A mouse at 1:1000 and 1:2000 dilutions) was incubated in each well followed by washing, and incubation with secondary anti-mouse IgG (H+L) (HRP). The optical density (OD) was measured. Anti-N polyclonal Abs bind not only whole N-protein but also enriched binding to Peptide #3, the highest OD compared with Peptide #1 and #2. ( C ) BALB/c mice were immunized with Peptide#3 in 2-week interval, 3 repeats. Red arrow indicates each vaccine time point. Antibody subtype in Anti-Peptide #3 Ab serum were classified by using anti-IgM, -IgG1, -IgG2a and -IgG (H+L) horseradish peroxidase (HRP) conjugated antibodies. Data represents mean ± S. D, n =3. ( D ) Comparison osf antibody subtype induction in N-protein vaccine and Peptide #3 vaccine after vaccination for three times. ( E ) Anti-Peptide #3 Ab serum (from C) were tested by Elisa for the binding capacity. Anti-Peptide #3 Ab serum binds to Peptide #3 and whole N-protein, detected by anti-mouse IgG (H+L) (HRP).

Article Snippet: Anti N-protein IgG (whole IgG) were detected by goat anti-mouse IgG-HRP (H+L) secondary antibody (Invitrogen 31430).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Bioscience Reports

Article Title: Targeting intra-viral conserved nucleocapsid (N) proteins as novel vaccines against SARS-CoVs

doi: 10.1042/BSR20211491

Figure Lengend Snippet: ELISA was done to analyze the binding affinity of peptides and N-protein (SARS-CoV2) to in house produced mouse anti SARS-CoV Ab (clone 6H3) . ELISA plate was coated with 5 ng and 20 ng/ well of different peptides and N-protein (SARS-CoV2). Mouse 6H3 antibodies were diluted at 1:1000 and 1:5000. The binding of antibody was detected by anti-mouse IgG (H+L) (HRP). The optical density (OD) was measured.

Article Snippet: Anti N-protein IgG (whole IgG) were detected by goat anti-mouse IgG-HRP (H+L) secondary antibody (Invitrogen 31430).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Produced