Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: A loss of XPB NTD integrity induces large-scale chromatine decondensation. a Schematic representation of the lacO/LacR tethering system used in U2OS17 cells. See the Materials and methods section for a full description of the cell line. b Schematic representation of wild-type and mutant XPB-LacR-GFP constructs. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) are omitted. c Proteins from whole-cell extracts (15 μg) of U2OS17 cells transiently transfected with wild-type or mutant XPB-LacR-GFP constructs were resolved by SDS-PAGE and immunoblotted using either polyclonal rabbit anti-GFP (upper panel), polyclonal rabbit anti-XPB (middle panel) or monoclonal mouse anti-Actin antibodies (lower panel). Source data are provided as a Source Data file. d U2OS17 cells were transiently transfected with 1 μg of expression vectors for the following proteins: LacR-GFP, XPB WT -LacR-GFP, XPB 320–782 -LacR-GFP, XPB F99S -LacR-GFP, XPB T119P -LacR-GFP, XPB 1–550 -LacR-GFP. In parallel, U2OS17 cell line was transiently transfected with 5 μg of expression vector for LacR-GFP. GFP was observed by fluorescence microscopy 24 h post transfection. Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition). Significant p -values are indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Mutagenesis, Construct, Transfection, SDS Page, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Software, Whisker Assay

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: XP-B/CS F99S patient-derived cells have a global increase in the H3K9ac histone mark. a Extracts from either XP-B/CS F99S or XP-B/CS F99S + XPB WT cells were resolved by SDS-PAGE and immunoblotted with a monoclonal mouse anti-XPB antibody. Source data are provided as a Source Data file. (6-4)PP removal measurements were carried out in XP-B/CS F99S , stably transfected XP-B/CS F99S + XPB WT and wild-type MRC5 cells harvested at different time points after UV irradiation at 30 J/m 2 as indicated. Cells were labeled with a monoclonal mouse anti-(6-4)PP antibody and signals were measured using a INCell 1000 analyzer (GE Healthcare). The graph represents the percentage of lesions remaining in the genome at a given time (error bars represent SD from three independent experiments). For each time point, about 20000–40000 cells were analyzed. b Histones were extracted from either XP-B/CS F99S , XP-B/CS F99S + XPB WT or wild-type MRC5 fibroblasts, resolved by SDS-PAGE and immunoblotted with either polyclonal rabbit anti-histone H3, polyclonal rabbit anti-histone H3ac, monoclonal mouse anti-histone H3K9ac or polyclonal rabbit anti-histone H3K9me2. Source data are provided as a Source Data file. c XP-B/CS F99S or XP-B/CS F99S + XPB WT cells were treated 3 h with DMSO or SP (50 μM) and histones were extracted, resolved by SDS-PAGE and immunoblotted either with a polyclonal rabbit anti-histone H3 or with monoclonal mouse anti-histone H3K9ac antibodies. In parallel, cell extracts were resolved by SDS-PAGE and western blotted with either a monoclonal mouse anti-XPB or polyclonal rabbit anti-tubulin antibodies. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Derivative Assay, SDS Page, Stable Transfection, Transfection, Irradiation, Labeling, Western Blot

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: XPB recruits KAT2A-containing HAT complexes to chromatin. a TFIIH-XPB WT was immunoprecipitated from nuclear extracts of stably transfected cells using anti-GFP antibodies (GFP-Trap) and washed with 200 mM salt (Lanes 2 and 4). Control IPs were performed with anti-GFP on an XP-B/CS F99S cell line stably expressing GFP alone (Lanes 1 and 3). Proteins on the resin were resolved by SDS-PAGE and immunoblotted using polyclonal rabbit anti-GFP, monoclonal mouse anti-p62, polyclonal rabbit anti-CDK7, polyclonal rabbit anti-KAT2A (SCBT) (Supplementary Figure ), polyclonal rabbit anti-KAT2B, polyclonal rabbit anti-WDR5, or polyclonal rabbit anti-SUPT7L antibodies. Source data are provided as a Source Data file. b The U2OS17 cell line was transiently transfected with 1 μg of expression vectors for the following proteins: LacR-GFP, XPB WT -LacR-GFP, XPB F99S -LacR-GFP, or XPB 320–782 -LacR-GFP together with 1 μg of expression vector for Flag-KAT2A. Colocalization of KAT2A with GFP was detected by immunofluorescence staining using a polyclonal rabbit anti-flag antibody. The values on the graph represent the percentage of colocalization of KAT2A with GFP on the array (error bars represent SD from three independent quantifications). c Purified wild-type recombinant core TFIIH (cIIH-XPB WT ) was incubated with purified Flag-KAT2A (400 ng) and pull-down assays were performed using either unspecific anti-IgG or polyclonal rabbit anti-KAT2A (SCBT) antibody (Supplementary Figure ). After washing, proteins on the resin were resolved by SDS-PAGE and immunoblotted using mouse monoclonal anti-XPB or anti-p52 antibodies (two subunits of TFIIH) or monoclonal mouse anti-KAT2A (IGBMC) antibody. Source data are provided as a Source Data file. d Bacterially expressed recombinant KAT2A (rKAT2A) (400 ng) was tested for its ability to interact with either rXPB WT (lanes 1–3) or rXPB F99S (lanes 4–6) (500 ng) in a pulled-down assay (IP-XPB). After washing, proteins on the resin were resolved by SDS-PAGE and immunoblotted using mouse monoclonal anti-XPB and mouse monoclonal anti-KAT2A antibodies (IGBMC). Lanes 1 and 4, immunoprecipitations with an irrelevant antibody (IP-IgG). Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Immunoprecipitation, Stable Transfection, Transfection, Control, Expressing, SDS Page, Plasmid Preparation, Immunofluorescence, Staining, Purification, Recombinant, Incubation

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Loss of XPB NTD integrity induces an increase in KAT2A HAT activity. a rKAT2A and the recombinant HAT-ATAC module (rHAT-ATAC) containing KAT2A, ADA3, ADA2a, and SGF29 were resolved by SDS-PAGE followed by Coomassie staining. Core TFIIH containing p62, p52, p44, p34 and either XPB WT (cIIH-XPB WT ) or XPB F99S (cIIH-XPB F99S ) were resolved by SDS-PAGE followed by Coomassie staining. Source data are provided as a Source Data file. b One hundred nanograms of core TFIIH containing either XPB WT (cIIH-XPB WT ) or XPB F99S (cIIH-XPB F99S ) were incubated with 50 ng rKAT2A together with histone H3.3 and cold acetyl-CoA. Following resolution by SDS-PAGE, proteins were immunoblotted using polyclonal rabbit anti-H3, monoclonal mouse anti-H3K9Ac, mouse monoclonal anti-XPB, and polyclonal rabbit anti-KAT2A (SCBT) antibodies. Quantification of H3K9Ac was performed using ImageJ software and normalized with H3. Source data are provided as a Source Data file. c Twenty nanograms of rXPB WT or rXPB F99S were incubated with 50 ng of rKAT2A together with histone H3.3 and cold acetyl-CoA. Following incubation, reactions were treated as described in panel ( b ). Source data are provided as a Source Data file. d Twenty nanograms of rXPB WT or rXPB F99S were incubated with 200 ng of the rHAT-ATAC module together with histone H3.3 and cold Acetyl-CoA. Following incubation, reactions were treated as described in panel ( b ). Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Recombinant, SDS Page, Staining, Incubation, Software

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Higher-order chromatin decondensation induced by XPB mutants is due to KAT2A HAT activity. a U2OS17 cells were transfected either with siCTL or with siKAT2A for 72 h and cell extracts were resolved by SDS-PAGE and immunoblotted using polyclonal rabbit anti-KAT2A or polyclonal rabbit anti-tubulin antibodies. siCTL or siKAT2A transfected cells were re-transfected with either LacR-GFP, XPB WT -LacR-GFP, XPB F99S -LacR-GFP or XPB 320–782 -LacR-GFP for 24 h and the relative array volumes (Vol) were quantified as described above. Significant p -value are indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file. b XP-B/CS F99S and XP-B/CS F99S + XPB WT cells were treated or not with MB-3 (200 μM) for 15 h and were subsequently labeled with polyclonal rabbit anti-tubulin and stained with DAPI. Cells were then reconstructed in 3D using Imaris Software (Bitplane) and the volumes of the cells and their nuclei were measured. The whisker box plot shows the volume of the nucleus/volume (Vol) of the cell (at least 30 cells for each condition, error bars represent SD from three independent experiments). The significant p -value is indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Transfection, SDS Page, Labeling, Staining, Software, Whisker Assay

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: KAT2A HAT activity induces inappropriate gene activation in XP-B/CS F99S cells. a RNA-seq analysis scatter plots comparing XP-B/CS F99S vs XP-B/CS F99S + XPB WT transcription profiles. Points show significantly over- (bottom) or under-(top) represented mRNA in XP-B/CS F99S cells compared to XP-B/CS F99S + XPB WT . All data were evaluated with the DESeq2 R package. For a given gene, its value is the normalized gene expression value relative to the mean of all samples belonging to the same condition. b A Comparative analysis of RNA-seq data from XP-B/CS F99S and XP-B/CS F99S + XPB WT . The pie chart indicates the number of genes that are up- and downregulated in the XP-B/CS F99S vs XP-B/CS F99S + XPB WT . c Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated with either DMSO or DRB, as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. d Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated either with DMSO or with MB-3 (200 μM) as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. e Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated with either siCTL or siKAT2A, as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. f Pre-mRNA levels (error bars represent SD from three independent experiments) of CYP26 and LRRC42 were analyzed in XP-B/CS F99S and XP-B/CS F99S + XPB WT cells as well as on stable clone 14 and clone 5 that were selected after transfection of cDNA coding for XPB T119P . Clone 14 does not express the transgene while clone 5 expresses only the transgene. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Activation Assay, RNA Sequencing, Gene Expression, Expressing, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Chromatin modification and RNA Pol II TIC formation at the promoters of overexpressed genes in XP-B/CS cells. a , b Schematic representations of the RARβ2 ( a ) and RNF130 ( b ) gene regions, including the upstream (Us) and promoter (Pr 1 and 2, respectively) regions that were amplified (see in facing half-arrowheads). c – j ChIPs monitoring occupancy by KAT2A (using a polyclonal rabbit anti-KAT2A antibody (EpiGentek) Supplementary Figure ) ( c , d ), H3K9ac/H3 (using monoclonal mouse anti-H3K9Ac and polyclonal rabbit anti-H3 antibodies) ( e , f ), TFIIB (using a polyclonal rabbit anti-TFIIB antibody) ( g , h ) and Pol II pS5 (using a monoclonal rat anti-Pol II pS5 antibody) ( i , j ) of either the Us or the Pr regions of RARβ2 ( c , e , g , i ) or the Pr region of RNF130 ( d , f , h , j ) in XP-B/CS F99S and XP-B/CS F99S + XPB WT cells treated either with DMSO or with MB-3 (200 μM). k – n ChIPs monitoring occupancy by the TFIIH core subunits p62 (using polyclonal rabbit anti-p62 antibody) ( k , l ) and XPB F99S (using polyclonal rabbit anti-XPB antibody) ( m , n ) of the Us or Pr regions of RARβ2 ( k – m ) or of the Pr region of RNF130 ( l – n ) in XP-B/CS F99S treated with either DMSO or MB-3

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Modification, Amplification

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: A microscopic and molecular assay to measure parasite load. (A) Confocal microscopy images of THP-1 cell-derived macrophage-like cells infected or not with L. donovani promastigotes at the MOI shown at 100× magnification. The infection was carried out for 24 h before fixing and staining. DAPI stain was used to stain both the nuclear and parasite DNA (the pseudocolor red is shown instead of the blue color of DAPI for better visualization; laser wavelength: 405.0, power: 4.3). The arrow indicates an amastigote inside a cell. Images were obtained under the DAPI channel using the NIS-Elements Imaging software (version 5.20.00). (B) The images obtained in A were manually scanned to count the number of cells infected and the number of amastigotes/cell and plotted. Then, 100–200 cells were counted per condition. (C) Parasite count in THP-1-derived cells infected with L. donovani (L. d) or not (C, control). The Ct values obtained from qPCR carried out on DNA isolated from infected cells were compared with the same represented as a standard curve obtained from qPCR carried out on promastigote DNA that was isolated from serially diluted parasite cultures in M199 medium; from this standard curve, the parasite numbers were deduced and shown as mean and SD from two experiments. (D) A new method to estimate parasite load after normalizing for host DNA concentration. The kDNA was measured from DNA isolated from infected cells by qPCR and normalized to copies of GAPDH DNA. The mean is shown from three separate experiments along with SD. NC, no infection control. (E) RAW264.7 cells infected with L. donovani were stained with Geimsa stain for LD body counting. Images were obtained in a bright field using Cell D software at 100× magnification. The arrow indicates an amastigote inside a cell. The number of amastigotes in 100 macrophage cells was counted using oil immersion lenses. (F) Data obtained from counting cells in E are plotted, showing the mean and SD. (G) Parasite load was estimated as kDNA normalized to gapdh from two experiments (shown as mean and SD) that were performed simultaneously with those shown in E. L. d, L. donovani; NC, no infection control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Confocal Microscopy, Derivative Assay, Infection, Staining, Imaging, Software, Control, Isolation, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani growth in cells. (A) Human (left) or mouse(right) IFN-λ3 at the shown concentration was added along with parasites during infection, and kDNA was measured after 24 h. The data are from two experiments in THP-1-derived cells and four experiments from RAW264.7 cells, showing the mean and SD. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Human (left) or mouse (right) IFN-λ3 at 100 ng/mL was added at different time points after L. donovani infection, and kDNA was measured after 24 h of infection. The data are from two experiments in both cell lines, shown as the mean and SD. *P < 0.05. (C) IFN-λ4 at 6 µg/mL was added, and the experiment was as in A showing data from two experiments with mean and SD (**P < 0.01). A high concentration of IFN-λ4 was required as the specific activity of the recombinant protein preparation is low, and 6 µg/mL would give a comparable activity to 100 ng/mL of IFN-λ3, as detailed in our previous work (16). (D) Effect of IFN-λ3 and IFN-λ4 on cytokine secretion in L. donovani infected M2-macrophage-like cells. M2 macrophages were differentiated for 2 days from THP-1 cells as described in our previous work (16), and IFN-λ3 at 1 µg/mL or IFN-λ4 at 6 µg/mL were added during parasite infection. After 24 h, the supernatants and cells were collected, and qPCR and ELISA were carried out. The data are from two experiments, showing the mean and SD. *P < 0.05; **P < 0.01. L. d, L. donovani; NT, no treatment control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Concentration Assay, Infection, Derivative Assay, Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani by increasing ROS production in cells. (A) (Top) RAW264.7 cells infected with L. donovani for different time points as shown were subject to qPCR for kDNA (normalized to Gapdh) and Ifnl3 (normalized to Rps29). (Bottom) Immunoneutralization of secreted mIFN-λ3 increases parasite load. The experiment was similar to the one described in the top, except that a rat anti-mouse IFN-λ3 antibody (shown as α-mIFN-λ3) was added or not at 24 h pi, at increasing concentrations as shown (1.5, 3.0, and 4.5 µg/mL) in the medium, and kDNA copies in the cells were measured after an additional 12 h (i.e., 36 h pi) of incubation. The data from both the top and bottom are from two experiments, showing the mean and SD. *P < 0.05i, post-infection. (B) ROS levels are induced by IFN-λ3, as measured by the NBT assay. (Top) The indicated treatment (IFN-λ3, LPS, or L. d ± IFN-λ3) was for 2 h; IFN-λ3 was at 100 ng/mL and LPS was at 1 µg/mL. (Bottom) L. d infection was for indicated time points. The means from three experiments (top) and two experiments (bottom) are shown; error bars denote SD. NT, no treatment. *P < 0.05; **P < 0.001; L.D, L. donovani. (C) mIFN-λ3 increases ROS production in RAW264.7 cells in a dose-dependent manner, as measured by a fluorescence-based assay. RAW264.7 cells were incubated with the shown concentrations of mIFN-λ3 or LPS (1 µg/mL) or NAC (100 mM) + mIFN-λ3 (0.5 µg/mL) for 2 h before staining with DCFDA. The fluorescence intensity quantified is shown at the top (data from two experiments with mean and SD are depicted; *P < 0.05), and the bottom shows representative images taken at 20× magnification of the cells under the conditions shown. (D) IFN-λ3 can inhibit parasite load even when ROS production is inhibited. (top) L. donovani (L. d) infection was given in THP-1-derived cells as before for 24 h in the absence (NT, no treatment) or presence of IFN-λ3 and in the presence or absence of an increasing concentration of NAC added to the medium. The parasite load was estimated after 24 h pi. (Bottom) As in top, but the kDNA copies are shown after normalizing to NT and NAC at 50 mM; data are from two experiments showing the mean and SD. **P < 0.01. (E) (Top) THP-1 cells were pretreated with 50 ng/mL of IFN-λ3 for 48 h (incubated along with PMA), and then the media was removed and L. donovani (L. d) or mock infection (1:10 MOI) was given for 2 h. ROS levels were measured by the NBT assay. Data from two experiments with mean and SD are shown. *P < 0.05. NT, no treatment. (Bottom) kDNA levels shown in THP-1 cells that were pretreated with 100 ng/mL IFN-λ3 or not (incubated along with PMA for 48 h) were washed off the media and infected or not with L. donovani for 24 h, and kDNA was measured by qPCR as before. The data are from two experiments, showing the mean and SD. Similar experiments with IFN-λ4 are shown in Fig. S2B.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Incubation, Fluorescence, Staining, Derivative Assay, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 targets an early event in parasite uptake. (A) IFN-λ3 at the indicated doses (the color key for the doses shown is the same for A, B, and C) were incubated along with the promastigotes, and slides were stained with DAPI, and the amastigotes presence and numbers were manually counted. The data are from three (for no treatment) and four (for IFN-λ3 treatment) independent experiments, each with 100–200 individual cells shown as the mean and SD. *P < 0.05. (B) (Left) Fluorescence microscope image of THP-1-derived cells incubated with CFSE-stained heat-killed L. donovani showing different stains. The arrow indicates a parasite taken up by the cell by phagocytosis. (Right) Manual counting of the parasite inside the cells and numbers was done as above and plotted. The data are from four independent experiments, each involving 100 to 200 individual cells, showing the mean and SD. For B and C, live parasites inside the cells were observed with the DAPI channel, while heat-killed parasites were observed under the GFP channel. Images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Texas Red (imparts gray pseudocolor). (C) (Top) Fluorescence microscopic image of THP-1-derived macrophage-like cells that have taken up zymosan in a phagocytosis assay as described in the Materials and Methods; images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Brightfield. The arrow indicates zymosan inside a cell. (Bottom) Manual counting of the cells with zymosan was performed as in B and plotted. The data are from two separate experiments with ~250 cells in each, showing the mean and SD. For B and C, image analysis was done using Leica Application Suite X software (version 3.7.2.22383) at 100× magnification. (D) Schematic representation of an experimental design with five (I-V) strategies. L donovani (L. d) infection (downward arrow) was given at 1:20 MOI for only 6 h, after which the uninfected promastigotes were removed after rigorous washing with PBS. IFN-λ3 treatment (inverted dark triangle) was given at the depicted time points, and after the indicated incubation periods, cells were collected and quantified for kDNA copies by qPCR (downward arrow with round head). (E and F). IFN-λ3 targets an early event during parasite uptake. Experiments as designed in the schematic representation shown in D were carried out, and results are presented in E (strategies I-IV) for 24 (E, top), 48 (E, middle), or 72 h pi (E, bottom) and F (strategies I for 48 h pi and V). 1:10 MOI and continuous infection conditions as described for previous experiments were also carried out, but the incubation periods included 48 and 72 h pi along with 24 h pi (shown in E middle, bottom, and top, respectively). qPCR was performed to estimate kDNA copies and ISG expression. The kDNA copies were normalized to NT (no IFN-λ3 treatment) and shown in E, while the actual fold changes are shown in F. The data in both E and F are from two experiments showing the mean and SD. *P < 0.05; **P < 0.01. The data shown in E are also shown with actual fold changes and without normalization to NT control samples in Fig. S2E and F.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Incubation, Staining, Fluorescence, Microscopy, Derivative Assay, Phagocytosis Assay, Software, Infection, Expressing, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 before infection fails to inhibit parasite load in the mouse model of VL. (A) and (B) Gene expression changes measured by qPCR from mice spleen (A) or liver (B) pretreated with 4 µg/mouse of mIFN-λ3 or PBS 18 h before infection with L. donovani. The infection was allowed for 6 weeks, and gene expression was quantified using primers listed in Table S1 and SYBR green (Applied Biosystems) protocol. kDNA was also measured and normalized using the 2−∆∆Ct method. Six female mice were used in each group, and 18 data points are shown for the six samples carried out in technical triplicate. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal. **P < 0.01. No significant differences were observed in the body weight and organ weights for the two groups.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Gene Expression, SYBR Green Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 treatment during infection fails to inhibit parasite load in the mouse model of VL. (A) Liver and spleens of the mice treated with PBS or 4 µg/mouse of mIFN-λ3 (in two doses of 2 µg/mouse in each dose given 24 h apart) at 12 weeks of infection were collected and weighed before subjecting them to qPCR for kDNA (liver and spleen) and other genes (spleen only). Each group had six animals (n = 6); 18 data points are shown for the six samples carried out in technical triplicates for qPCR data. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal; for the organ weights, single measurements were taken for each organ belonging to each animal. *P < 0.01. (B) The sera from mice were subjected to ELISA for the three mouse cytokines shown.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: A distinct innate immune signature marks progression from mild to severe COVID-19

doi: 10.1016/j.xcrm.2020.100166

Figure Lengend Snippet:

Article Snippet: EQ Four Element Calibration Beads , Fluidigm , Cat# 201078.

Techniques: Purification, Control, Recombinant, Blocking Assay, Electron Microscopy, Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Mass Cytometry, Software, Diffusion-based Assay

Journal: iScience

Article Title: SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1β pathway

doi: 10.1016/j.isci.2024.109733

Figure Lengend Snippet: The effect of NR1D1 on the activation of NLRP3 inflammatory bodies and the secretion of IL-1β (A) The mRNA expression of NR1D1, NLRP3, ASC, CASP1 and IL-1β in the different groups. (B and C) The protein expressions and quantitative analysis of NR1D1, NLRP3, ASC, CASP1 and IL-1β in the different groups. (D) TUNEL assay and Quantitative analysis results of NPMSCs. (White scale bar = 50 μm). (E) The ChIP-seq and ChIP-qPCR of NR1D1 and NLRP3; NR1D1 and IL-1β. All data are expressed as the mean ± SD. ∗∗ : p < 0.01 compared with the control group; # : p < 0.01 compared with the LPS+ATP-NC-siRNA group.

Article Snippet: The P 3 NPMSC were fixed with paraformaldehyde (4%) for 20 min. Then they were washed twice with PBS containing 0.5% Triton X-100 for 20 min. After that the cells were incubated with 15% bovine serum albumin for 2 h at room temperature, rinsed with PBS and incubated with primary antibodies: collagen II (Bioss, China, catalog no. bs-10579R) (1:200), matrix metalloproteinase 13 (MMP-13) (Bioss, China, catalog no. bs-10240R) (1:200), NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M) overnight (4°C).

Techniques: Activation Assay, Expressing, TUNEL Assay, ChIP-sequencing, ChIP-qPCR, Control

Journal: iScience

Article Title: SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1β pathway

doi: 10.1016/j.isci.2024.109733

Figure Lengend Snippet: Primers used for reverse transcription quantitative polymerase chain reaction analysis of gene expression

Article Snippet: The P 3 NPMSC were fixed with paraformaldehyde (4%) for 20 min. Then they were washed twice with PBS containing 0.5% Triton X-100 for 20 min. After that the cells were incubated with 15% bovine serum albumin for 2 h at room temperature, rinsed with PBS and incubated with primary antibodies: collagen II (Bioss, China, catalog no. bs-10579R) (1:200), matrix metalloproteinase 13 (MMP-13) (Bioss, China, catalog no. bs-10240R) (1:200), NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M) overnight (4°C).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Sequencing

Journal: iScience

Article Title: SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1β pathway

doi: 10.1016/j.isci.2024.109733

Figure Lengend Snippet: SR9009 regulates the pyroptosis level of NPMSC stimulated by LPS (A) Pyroptosis level of NPMSCs detected by flow cytometry. (B) Quantitative analysis of the pyroptosis rate of NPMSCs. (C) TUNEL assay results of NPMSCs (White scale bar = 50 μm; red scale bar = 25 μm). (D) Quantitative analysis of TUNEL staining positive cells. (E) and (F) Immunofluorescence staining of NLRP3, ASC, CASP1 and NR1D1, IL-1β, GSDMD (Scale bar = 50 μm). (G) Quantitative analysis of the fluorescence expressions of NLRP3, ASC, CASP1 and NR1D1, IL-1β, GSDMD. (H), (I), and (J) The protein expression and quantitative analysis of NLRP3, ASC, CASP1 and NR1D1, IL-1β, GSDMD in the different groups. All data are the mean ± SD. ∗∗ : p < 0.01 compared with the control group; # : p < 0.01 and ## : p < 0.01 compared with the LPS+ATP group; SLA: SR9009+LPS+ATP group.

Article Snippet: The P 3 NPMSC were fixed with paraformaldehyde (4%) for 20 min. Then they were washed twice with PBS containing 0.5% Triton X-100 for 20 min. After that the cells were incubated with 15% bovine serum albumin for 2 h at room temperature, rinsed with PBS and incubated with primary antibodies: collagen II (Bioss, China, catalog no. bs-10579R) (1:200), matrix metalloproteinase 13 (MMP-13) (Bioss, China, catalog no. bs-10240R) (1:200), NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M) overnight (4°C).

Techniques: Flow Cytometry, TUNEL Assay, Staining, Immunofluorescence, Fluorescence, Expressing, Control

Journal: iScience

Article Title: SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1β pathway

doi: 10.1016/j.isci.2024.109733

Figure Lengend Snippet:

Article Snippet: The P 3 NPMSC were fixed with paraformaldehyde (4%) for 20 min. Then they were washed twice with PBS containing 0.5% Triton X-100 for 20 min. After that the cells were incubated with 15% bovine serum albumin for 2 h at room temperature, rinsed with PBS and incubated with primary antibodies: collagen II (Bioss, China, catalog no. bs-10579R) (1:200), matrix metalloproteinase 13 (MMP-13) (Bioss, China, catalog no. bs-10240R) (1:200), NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M) overnight (4°C).

Techniques: Recombinant, RNA Sequencing, Sequencing, Software, Flow Cytometry, Transmission Assay, Microscopy, CCK-8 Assay, TUNEL Assay

Journal: Nature

Article Title: A mesocortical dopamine circuit enables the cultural transmission of vocal behavior

doi: 10.1038/s41586-018-0636-7

Figure Lengend Snippet: a , Tutor-naive juvenile male finch PAG activity aligned to the onset of 35 presentations of song playback in the presence of an adult female bird (top: averaged sound spectrogram, middle: spike raster plot, bottom: mean firing rate). b , Mean firing rate (FR) during presentation of song playback in the presence of a female bird, normalized to baseline FR (two-sided paired t -test: t (7) = 0.620, P = 0.555; n = 8 neurons from 2 birds). c , PAG activity during a tutor song bout (top: sound spectrogram, middle: voltage recording, bottom: firing rate, blue bar: song motif). d , PAG unit activity aligned to the offset of a live tutor’s song bouts (red bar: live song), shown as in a . e , A max-projected image of serial sagittal sections visualized with a confocal microscope, showing the site of tetrode recordings in PAG (~0.8 mm lateral of the midline). f , PAG unit activity aligned to the onset of live tutor’s song motifs, shown as in a . Note that the tutor often sings multiple motifs within a single bout, thus some motifs precede (and follow) the alignment time. Error bars indicate mean ± SEM.

Article Snippet: Sections were coverslipped with Fluoromount-G (SouthernBiotech), and then imaged with a confocal microscope (SP8; Leica) through a 20x objective lens controlled by LAS X software (Leica).

Techniques: Activity Assay, Microscopy

Journal: Nature

Article Title: A mesocortical dopamine circuit enables the cultural transmission of vocal behavior

doi: 10.1038/s41586-018-0636-7

Figure Lengend Snippet: a , From left to right, a max-projected image of serial sagittal sections visualized with a confocal microscope, showing HVC with TH immunolabeling (~2.4 mm lateral), HVC shelf and caudolateral nidopallium (NCL) just ventral to HVC with TH immunolabeling (~2.4 mm lateral), and HVC with dopamine beta-hydroxylase (DBH) immunolabeling (~2.4 mm lateral) in control birds, which received injection of vehicle into HVC. Similar results were obtained in 5 independently repeated experiments (orientation is similar to b ). b , From left to right, a max-projected image of serial sagittal sections visualized with a confocal microscope, showing HVC with TH immunolabeling (~2.4 mm lateral), HVC shelf and NCL just ventral to HVC with TH immunolabeling (~2.4 mm lateral), and HVC with DBH immunolabeling (~2.4 mm lateral) in birds that received injection of 6-OHDA into HVC 2 days before tissue fixation. Similar results were obtained in 4 independently repeated experiments (D: dorsal, R: rostral). c , Density of TH-positive (TH+) fibers in HVC of control birds ( n = 5 hemispheres from 3 birds) was higher than that of birds that received injections of 6-OHDA 2 days before fixation (Tukey-Kramer test: P = 0.002) ( n = 4 hemispheres from 2 birds), and that of birds that received injections of 6-OHDA ~60 days before fixation, as in (Tukey-Kramer test: P = 0.002) ( n = 6 hemispheres from 4 birds). d , Density of TH+ fibers in HVC shelf and NCL in control birds ( n = 5 hemispheres from 3 birds), birds that received injection of 6-OHDA 2 days before fixation ( n = 4 hemispheres from 2 birds), and birds that received injection of 6-OHDA ~60 days before fixation, as in ( n = 6 hemispheres from 4 birds). e , Density of DBH-positive (DBH+) fibers in HVC in control birds ( n = 4 hemispheres from 2 birds) and birds that received injection of 6-OHDA 2 days before injection ( n = 4 hemispheres from 2 birds) was not significantly different (two-sided unpaired t -test: t (7) = 0.379, P = 0.716). Error bars indicate mean ± SEM.

Article Snippet: Sections were coverslipped with Fluoromount-G (SouthernBiotech), and then imaged with a confocal microscope (SP8; Leica) through a 20x objective lens controlled by LAS X software (Leica).

Techniques: Microscopy, Immunolabeling, Control, Injection

Journal: Nature

Article Title: A mesocortical dopamine circuit enables the cultural transmission of vocal behavior

doi: 10.1038/s41586-018-0636-7

Figure Lengend Snippet: a , From left to right, a max-projected image of serial sagittal sections visualized with a confocal microscope, showing a lateral part of PAG (lPAG) (~1.0 mm lateral), a medial part of PAG (mPAG, ~0.2 mm lateral), SNc (~1.2 mm lateral), and VTA (~0.2 mm lateral), each of which was labeled with dextran injected into HVC (green) and an antibody for TH (pseudo-colored magenta). Similar results were obtained in 4 independently repeated experiments (R: rostral, V: ventral). b , Proportion of HVC-projecting neurons in PAG and VTA/SNc ( χ 2 -test: χ 2 (1) = 406.54, P < 0.001, n = 4 hemispheres from 3 birds). c , Proportion of TH-positive (TH+) neurons in HVC-projecting neuron subsets in PAG and VTA/SNc ( χ 2 -test: χ 2 (1) = 204.62, P < 0.001, n = 4 hemispheres from 3 birds). d , From left to right, a max-projected image of serial sagittal sections visualized with a confocal microscope, showing PAG (~0.6 mm lateral), SNc (~0.6 mm lateral), and VTA (~0.2 mm lateral), each of which was labeled with dextran injected into Area X (green) and an antibody for TH (pseudo-colored magenta). Similar results were obtained in 3 independently repeated experiments. e , Proportion of double-labeled neurons (dextran and TH) in PAG and SNc/VTA ( χ 2 -test: χ 2 (1) = 493.92, P < 0.001, n = 3 hemispheres from 3 birds) in birds that received injection of dextran into Area X. f , Proportion of Area X-projecting neurons in PAG and VTA/SNc ( χ 2 -test: χ 2 (1) = 472.07, P < 0.001, n = 3 hemispheres from 3 birds). g , Proportion of TH+ neurons in Area X-projecting neuron subsets in PAG and VTA/SNc ( χ 2 -test: χ 2 (1) = 55.14, P < 0.001, n = 3 hemispheres from 3 birds). Error bars indicate mean ± SEM.

Article Snippet: Sections were coverslipped with Fluoromount-G (SouthernBiotech), and then imaged with a confocal microscope (SP8; Leica) through a 20x objective lens controlled by LAS X software (Leica).

Techniques: Microscopy, Labeling, Injection

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Knockdown, Over Expression, Western Blot, Control, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Knockdown, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Control, Western Blot