Journal: Science translational medicine

Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

doi: 10.1126/scitranslmed.3003713

Figure Lengend Snippet: Survivin oncoprotein mRNA expression is increased in MCV-positive MCC. (A) Digital transcriptome subtraction comparison of 64 genes involved in programmed cell death and cell cycle regulation showed that survivin (BIRC5) mRNA transcripts (highlighted) were sevenfold higher in an MCV-positive compared with an MCV-negative MCC cDNA library. The relative expression of genes was normalized to total sequence reads for each MCC library (also see table S1). (B) MCV T antigen is required for survivin expression. Lentiviral MCV T antigen exon 1 knockdown (panT1) decreased survivin protein expression (left) but did not alter XIAP, BCL-2, BAX, or p53 protein levels (right) in four MCV-positive MCC cell lines: MKL-1, MKL-2, MS-1, and WaGa. UISO, MCV-negative cell line; shCntrl, scrambled shRNA control lentivirus; LT, large T antigen. (C) MCV T antigen is required for survivin transcription. Survivin mRNA levels were reduced in the MKL-1 but not the UISO cell line after T antigen knockdown, indicating that T antigen activates survivin transcription. Survivin mRNA was measured by qRT-PCR and normalized to β-actin mRNA. The experiments were performed in triplicate and repeated twice (mean ± SEM, two-tailed t test). (D) Survivin expression is required for MCV-positive MCC tumor cell survival. Survivin was targeted for knockdown using two shRNA lentiviral vectors, shsur1 and shsur2, in MKL-1 and UISO cells. MKL-1 cells initiate apoptosis after survivin knockdown, with increased expression of cleaved PARP (cPARP) and caspase 3 (cCasp3), whereas UISO cells are resistant to survivin knockdown-induced apoptosis. α-Tubulin is used as a loading control.

Article Snippet: Cell culture Seven MCC cell lines [MKL-1, MKL-2, MS-1, UISO, MCC13, MCC26, and WaGa (gift of J. Becker) ( 29 , 54 , 55 )], NCI-H69 small-cell lung cancer cell line [American Type Culture Collection (ATCC)], 293 human embryonic kidney cells (ATCC), U2OS osteosarcoma cell line (gift of O. Gjoerup), BJhTERT immortalized foreskin fibroblast cell line (gift of O. Gjoerup), and BJ primary foreskin fibroblasts (ATCC) were used to screen and evaluate the small molecules examined in this study ( 29 , 54 , 55 ).

Techniques: Expressing, Comparison, cDNA Library Assay, Sequencing, Knockdown, shRNA, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Science translational medicine

Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

doi: 10.1126/scitranslmed.3003713

Figure Lengend Snippet: The survivin promoter inhibitor YM155 inhibits MCV-positive MCC cell line growth. (A) Dose-dependent growth curves at 48 hours for YM155-treated cell lines. MCV-negative MCC13, BJ, and BJhTERT cells showed relative resistance to YM155 treatment, whereas all MCV-positive cell lines (MKL-1, MKL-2, MS-1, and WaGa) were sensitive to YM155. MCV-negative UISO and MCC26 cell lines had intermediate sensitivity to YM155. (B) Trypan blue vital dye exclusion assay showed dose-dependent cell killing at 48 hours for MKL-1 cells (blue bars), whereas UISO cells (red bars) are relatively less sensitive and BJ cells (green bars) are resistant to YM155. (C) Dose-dependent decrease in MKL-1 cell survivin protein expression after 12 hours of YM155 treatment.

Article Snippet: Cell culture Seven MCC cell lines [MKL-1, MKL-2, MS-1, UISO, MCC13, MCC26, and WaGa (gift of J. Becker) ( 29 , 54 , 55 )], NCI-H69 small-cell lung cancer cell line [American Type Culture Collection (ATCC)], 293 human embryonic kidney cells (ATCC), U2OS osteosarcoma cell line (gift of O. Gjoerup), BJhTERT immortalized foreskin fibroblast cell line (gift of O. Gjoerup), and BJ primary foreskin fibroblasts (ATCC) were used to screen and evaluate the small molecules examined in this study ( 29 , 54 , 55 ).

Techniques: Exclusion Assay, Expressing

Journal: Science translational medicine

Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

doi: 10.1126/scitranslmed.3003713

Figure Lengend Snippet: Cell death phenotype of YM155-treated MCV-positive MCC. (A) Cell cycle analysis reveals that YM155-treated cells do not undergo mitotic catastrophe. MKL-1 cells were treated with DMSO, YM155 (100 nM), camptothecin (CPT, 1 μM), or 5-Gy γ-irradiation (IR). They were stained for propidium iodide (PI) and BrdU and harvested 12 hours later. Upper panel shows cell cycle profiles, in which no evidence of G2-M pileup is seen (a marker for mitotic catastrophe) after YM155 treatment. Bottom panel shows the corresponding propidium iodide versus BrdU uptake for these same cells showing new DNA synthesis for cells in G1, S, and G2-M. Mock-treated cells have an inverted U-shaped curve showing BrdU incorporation during S-phase DNA synthesis. Both camptothecin and γ-irradiation primarily reduce S-phase BrdU incorporation, consistent with checkpoint activation. In contrast, YM155 reduces all early DNA synthesis as measured by BrdU incorporation. (B) YM155 induces nonapoptotic cell death associated with autophagy in MKL-1 cells. MKL-1 cells were treated with DMSO, YM155 (100 nM), or bortezomib (100 nM) and immunoblotted for cleaved PARP (cPARP), cleaved caspase 3 (cCasp3), LC3-II, and α-tubulin. In contrast to YM155, the proteasome inhibitor bortezomib activates MKL-1 cell apoptosis (also see fig. S2). (C) YM155 treatment initiates programmed cell death within 12 to 24 hours after treatment. MKL-1 cells were costained with CFDA (green, live) and propidium iodide (red, dead). Column graphs (right panel) represent mean and range of percent CFDA-positive (green) and percent propidium iodide–positive cells (red).

Article Snippet: Cell culture Seven MCC cell lines [MKL-1, MKL-2, MS-1, UISO, MCC13, MCC26, and WaGa (gift of J. Becker) ( 29 , 54 , 55 )], NCI-H69 small-cell lung cancer cell line [American Type Culture Collection (ATCC)], 293 human embryonic kidney cells (ATCC), U2OS osteosarcoma cell line (gift of O. Gjoerup), BJhTERT immortalized foreskin fibroblast cell line (gift of O. Gjoerup), and BJ primary foreskin fibroblasts (ATCC) were used to screen and evaluate the small molecules examined in this study ( 29 , 54 , 55 ).

Techniques: Cell Cycle Assay, Irradiation, Staining, Marker, DNA Synthesis, BrdU Incorporation Assay, Activation Assay

Journal: Science translational medicine

Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

doi: 10.1126/scitranslmed.3003713

Figure Lengend Snippet: EC 50 (μM) concentrations for MCC cell lines.

Article Snippet: Cell culture Seven MCC cell lines [MKL-1, MKL-2, MS-1, UISO, MCC13, MCC26, and WaGa (gift of J. Becker) ( 29 , 54 , 55 )], NCI-H69 small-cell lung cancer cell line [American Type Culture Collection (ATCC)], 293 human embryonic kidney cells (ATCC), U2OS osteosarcoma cell line (gift of O. Gjoerup), BJhTERT immortalized foreskin fibroblast cell line (gift of O. Gjoerup), and BJ primary foreskin fibroblasts (ATCC) were used to screen and evaluate the small molecules examined in this study ( 29 , 54 , 55 ).

Techniques:

Journal: Science translational medicine

Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

doi: 10.1126/scitranslmed.3003713

Figure Lengend Snippet: YM155 inhibits growth of human MKL-1 MCC xenografts in NOD-SCIDγ mice. (A) MKL-1 xenograft tumors were stained with hematoxylin and eosin (H&E), and for MCV large T antigen and cytokeratin 20 (CK20) expression (magnification, ×40). (B) MKL-1 xenograft survival curves after drug treatment. Mice were subcutaneously injected with 20 million MKL-1 cells and assigned to a 3-week drug treatment after tumors became palpable. No significant difference was found between saline and bortezomib treatment. Tumor progression was significantly delayed by YM155, with none of the YM155-treated mice dying during treatment (up to day 19) compared to 23 of 31 (74%) saline-treated and 14 of 21 (67%) bortezomib-treated mice (P < 0.0001, log-rank test). Tumor progression recurred for all YM155-treated mice once treatment was stopped. (C) Piecewise linear hierarchical Bayesian model for tumor volumes in treated mice (59) (see the Supplementary Materials for details). Colored lines show estimated central population tumor volumes, with shaded regions representing 95% credible intervals. Actual tumor volumes (gray lines) for each mouse are shown for comparison. YM155 treatment retards tumor growth compared to saline or bortezomib treatment. (D) Table showing day of termination and tumor volumes for MS-1 and UISO xenograft mice treated with YM155 or saline.

Article Snippet: Cell culture Seven MCC cell lines [MKL-1, MKL-2, MS-1, UISO, MCC13, MCC26, and WaGa (gift of J. Becker) ( 29 , 54 , 55 )], NCI-H69 small-cell lung cancer cell line [American Type Culture Collection (ATCC)], 293 human embryonic kidney cells (ATCC), U2OS osteosarcoma cell line (gift of O. Gjoerup), BJhTERT immortalized foreskin fibroblast cell line (gift of O. Gjoerup), and BJ primary foreskin fibroblasts (ATCC) were used to screen and evaluate the small molecules examined in this study ( 29 , 54 , 55 ).

Techniques: Staining, Expressing, Injection, Saline, Comparison

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Heatmap of differentially expressed Atoh1 and its downstream target genes in PBS and rFGF1-treated UC mice ( n = 4). The mRNA level of Atoh1 in PBS or rFGF1-treated UC mice (upper panel, n = 4) and CD mice (lower panel, n = 5) was detected by qRT-PCR. B , C IF staining of ATOH1 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( B ) or TNBS ( C ), and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). D , E IF staining of ATOH1 in distal colon sections of DSS ( D ) or TNBS ( E )-induced two IBD mouse models, followed by PBS or rFGF1 treatment and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). F Representative images (upper panel) and IF staining of Muc2 (lower panel) in sh Atoh1 or shNC-transfected colonic organoids stimulated with vehicle or rFGF1 ( n = 6). G , H Deletion of Atoh1 in VilCre ERT2 Atoh1 fl/fl mice was confirmed by qRT-PCR ( G ) and IF staining of colon tissues ( H ) ( n = 4). I – M VilCre ERT2 Atoh1 fl/fl mice were injected with tamoxifen for five consecutive days and then given drinking water containing 1.5% DSS to induce acute colitis, followed by vehicle or rFGF1 administration for 7 days. At end of the experiment, the distal colon tissues were harvested and examined. The schematic diagram shows the strategy of tamoxifen injection, DSS challenge and rFGF1 administration ( n = 4). J – L Weight loss ( J ), disease activity index ( K ), colonic length ( L ) were monitored (n = 4). M H&E (upper panel), PAS-AB staining (middle panel) and Muc2 IF staining (lower panel) of distal colon sections ( n = 4). Data was presented as mean ± SEM. ( A , D – E , H ) two-tailed unpaired t -test; ( B , C , F , J – M ) ordinary two-way ANOVA, followed by Sidak; ( G ) Non-parametric statistical method, two-tailed Mann-Whitney test. ns, not significance; nd, not detectable.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Quantitative RT-PCR, Staining, Quantitation Assay, Transfection, Injection, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Workflow to find the rFGF1-regulated potential transcription factors of Atoh1 , and heatmap of expression level of potential transcription factors ( n = 4). B The expression levels of potential transcription factors of Atoh1 ( Tcf4 , Nr4a1 , Nfya , Tec , Hoxa13 , Ncaph2 , Kdm1a ) in distal colon sections of PBS or rFGF1-treated UC mice were analyzed by qRT-PCR ( n = 4). C , D IF staining of TCF4 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( C ) or TNBS ( D ) and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). E , F The expression levels of TCF4 in distal colon sections of DSS-treated UC mice ( E ) and TNBS-treated CD mice ( F ) after PBS or rFGF1 administration and IF staining ( n = 8). G Predicted TCF4 binding sites on the promoter of Atoh1 by the JASPAR database (left panel). Six binding sites with the most significant prediction scores were selected for pairwise deletion mutation analysis (right panel; M1, in purple; M2, in green; M3, in red). H , I Luciferase activity in HT-29 cells transfected with a human Atoh1 promoter reporter plasmid or the predicted TCF4 binding site mutant reporter along with a Tcf4 overexpression plasmid ( n = 4). J ChIP experiments. The data were normalized to the corresponding IgG ( n = 4). K Images of murine colonic organoids transfected small hairpin RNA targeting Tcf4 (sh Tcf4 ) or negative control hairpin (shNT) followed by treated with rFGF1 or vehicle ( n = 6). L IF staining of ATOH1 (upper panel) and Muc2 (lower panel) in colonic organoids in different groups and its semi-quantitation of fluorescent intensity ( n = 6). Data was presented as mean ± SEM. ( B , right panel of E – F , J ) two-tailed unpaired t -test ( Tec and Ncaph2 in penal B using non-parametric statistical method, two-tailed Mann-Whitney Test); ( H – I ) ordinary one-way ANOVA, followed by Dunnett; (right panel of C – D and L , lower panel of K ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Expressing, Quantitative RT-PCR, Staining, Quantitation Assay, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Single-cell RNA sequencing analysis (GSM4983265) showed that Fgfr2 is highly expressed in stem cells. B Representative image of colon distal stained with Lgr5 (green), FGFR2 (red) and DAPI (blue) from the C57BL/6 J mice ( n = 3). C Deletion of Fgfr2 in Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice treated with vehicle or tamoxifen was confirmed by IF staining ( n = 5). D Murine colonic organoids derived from vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were stimulated with vehicle or rFGF1. Representative images, IF staining of TCF4, ATOH1 and Muc2 in colonic organoids were shown (n = 6). E – I Vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were challenged with drinking water containing 2.5% DSS to induce UC, followed by colon tissues were harvested and examined (n = 4 for TAM + PBS group, n = 5 for other groups). Weight loss ( E ), disease activity index ( F ) and colon length ( G ) of different groups were monitored. H H&E staining of distal colon sections and histology scores (upper panel), PAS-AB staining of distal colon sections and mucin granules-positive cells per crypt (lower panel). I IF staining of TCF4 (upper panel), ATOH1 (middle panel) and Muc2 (lower panel) in distal colon sections, and its semi-quantification of IF intensity. Data was presented as mean ± SEM. (right panel of C ) two-tailed unpaired t -test; (right panel of D – F , right panel of G – I ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: RNA Sequencing, Staining, Derivative Assay, Activity Assay, Two Tailed Test

Journal: Nature Neuroscience

Article Title: Oligodendroglial fatty acid metabolism as a central nervous system energy reserve

doi: 10.1038/s41593-024-01749-6

Figure Lengend Snippet: a , Left: silver stained gels of optic nerve lysates from 2 month old wildtype mice (both genders), prepared after 24 h incubation in 10 mM glucose or 1 mM glucose (two nerves pooled per lane; one lane is equivalent to one sample). N = n = 5 for both conditions. Right: optic nerve lysates prepared after 16 h in 10 mM glucose or 0 mM glucose. Note the lack of major protein degradation. N = n = 5 for both conditions. b , Relative abundance of selected proteins in optic nerve lysates after 24 h in 1 mM glucose (left, N = n = 5) or 16 h in 0 mM glucose (right, N = n = 5). Note that enzymes of glucose and lipid metabolism show only moderate changes in abundance. Autophagy related proteins are increased in the presence of 1 mM glucose only, indicating a requirement of glucose for RNA synthesis and protein expression (N = n = 5, two technical replicates each; moderated t-statistics (more details in methods section)); Statistical significance (q-value) depicted on the right side of each panel. c, d , Western blots of lysates from wildtype optic nerves, incubated in 10 mM or 0 mM glucose for 16 h (age 8-12 weeks old, N = n = 5 for each condition) ( c ) and quantification of ACAT1 and BDH1 ( d ). Normalized to protein input (mean ± SEM, unpaired two-tailed t-test). e , Cell survival of 24 h glucose-deprived optic nerves from TFEB cKO mice (N = n = 4) and controls (N = n = 4; age 8-12 weeks). Images from longitudinal sections were stained with PI and DAPI. f , Quantified data from ( e ). There is no difference of cell survival (mean ± SEM, unpaired two-tailed Welch’s t-test). N and n indicate the total number of independent samples for each condition and the total number of independent experiments, respectively.

Article Snippet: Western blotting and Fast Green staining were performed as previously described using the following primary and secondary antibodies: ACAT1 (1:3,000, cat. no. 16215-1-AP, Proteintech), BDH1 (1:500, cat. no. 15417-1-AP, Proteintech), LC3B (1:2,000, cat. no. NB100-2220, Novusbio), Na + /K + ATPase α1 (1:1,000, cat. no. ab7671, Abcam), GLUT1 (1:1,000) , GLUT2 (1:1,000, cat. no. ab54460, abcam), GLUT3 (1:1,000, cat. no. ab191071, abcam), GLUT4 (1:1,000, cat. no. 07-1404, Millipore), MCT1 (1:1,000) , CA2 (1:1,000) and α-tubulin (1:1,000, cat. no. T5168, Sigma-Aldrich), mouse immunoglobulin G heavy and light (IgG H&L) Antibody Dylight 680 Conjugated (1:10,000, cat. no. 610-144-002); rabbit IgG H&L Antibody DyLight 800 Conjugated (1:10,000, cat. no. 611-145-002, Rockland); horseradish peroxidase-conjugated secondary antibodies (1:5,000, cat. nos.

Techniques: Staining, Incubation, Expressing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a Western blots show the time course of protein expression of FOXM1, MATα2, and MAT2β in liver tissues after BDL ( n = 3 independent experiments). b Immunofluorescence (IF) of FOXM1, MATα2, and MAT2β in primary hepatic stellate cells (HSCs) isolated from sham and BDL mice at day 5. The top row shows DAPI staining. The second and third rows show the antibody (AB) staining. The fourth row shows merged images of DAPI and FOXM1, MATα2 or MAT2β, and the fifth row shows high magnification (HM) from the merged image ( n = 3 independent experiments). c Expression of mRNA (top) and protein (bottom) of FOXM1, MATα2, MAT2β, α-SMA, and COL1A1 in HSCs isolated from WT mice and cultured for up to 5 days and FDI-6 treatment for 24 h starting at day 4. Data presented as mean ± SEM ( n = 3 per group), mRNA levels of Foxm1, Mat2a, Mat2b , Acta2 and Col1a1 in HSCs at day 5 vs. day 1, p = 0.0044, p = 0.0029, p = 0.0056, p = 0.0010 and p = 0.00002, respectively. mRNA levels of Foxm1, Mat2a, Mat2b , Acta2 and Col1a1 in HSCs at day 5 + FDI-6 vs. day 1, p = 0.0431, p = 0.0152, p = 0.0245, p = 0.0083, and p = 0.0082, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See Supplementary Fig. for densitometric values of the western blots. d Expression of mRNA and protein of FOXM1, MATα2, MAT2β, α-SMA, and COL1A1 after FDI-6 treatment in LX-2 cells. Data presented as mean ± SEM ( n = 3 per group), mRNA levels of Foxm1, Mat2a, Mat2b , Acta2 and Col1a1 in LX2 cells with DMSO treatment vs. FDI-6, p = 0.0187, p = 0.0023, p = 0.0122, p = 0.0108 and p = 0.0124, respectively. * p < 0.05, ** p < 0.01 vs. DMSO. See Supplementary Fig. for densitometric values of the western blots. e IF of LX-2 cells after treatment with FDI-6. HM, high magnification from the merged images ( n = 3 independent experiments). f FOXM1, MATα2, and MAT2β in cytoplasm and nucleus from HSCs isolated from sham and BDL mice with or without FDI-6 treatment ( n = 3 independent experiments). Densitometry for cytoplasmic protein levels is summarized in Supplementary Fig. and nuclear protein levels is summarized in Supplementary Fig. . Proliferation ( g ) and migration ( h ) of LX-2 cells in vitro after FDI-6 treatment for 24 h. Data presented as mean ± SEM ( n = 3 per group). p = 0.00016, p = 0.00002 vs.DMSO. Statistical significance was determined by using two-tailed unpaired Student’s t -test. *** p < 0.001, **** p < 0.0001 vs. DMSO ( n = 3). Abbreviations: BDL bile duct ligation, DMSO dimethylsulfoxide. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Western Blot, Expressing, Immunofluorescence, Isolation, Staining, Cell Culture, Migration, In Vitro, Two Tailed Test, Ligation

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a Liver sections from prevention groups of the corn oil (Oil) + DMSO, Oil + FDI-6, CCl 4 + DMSO, and CCl 4 + FDI-6 for three weeks, and treatment groups of Oil + DMSO, Oil + FDI-6, CCl 4 + DMSO, and CCl 4 + FDI-6 treated for two weeks after CCl 4 treatment for three weeks. IHC stained with antibodies of COL1A1, F4/80, α–SMA, FOXM1, MATα2, and MAT2β. H&E is shown in the top row. b , c show changes in ALT ( n = 6 per group) ( b ) and AST ( n = 6 in prevention group of Oil + DMSO, n = 5 in prevention group of Oil + FDI-6, n = 3 in prevention group of CCl 4 + DMSO and treatment group of Oil + FDI-6, n = 4 in prevention group of CCl 4 + FDI-6, and treatment groups of Oil + DMSO, CCl 4 + DMSO, and CCl 4 + FDI-6) ( c ) levels after FDI-6 administration in the prevention and treatment groups. Data presented as mean ± SEM, **p < 0.01, **** p < 0.0001. d Hydroxyproline content was measured in the livers from prevention and treatment groups with or without FDI-6 administration. Data presented as mean ± SEM, **p < 0.01, **** p < 0.0001 ( n = 5 per group). e mRNA levels of Foxm1 , Mat2a , and Mat2b in the livers after FDI-6 administration in the prevention and treatment groups. Data presented as mean ± SEM ( n = 4), **p < 0.01, **** p < 0.0001 ( n = 4 in prevention and treatment groups of Oil + DMSO for Foxm1 mRNA; n = 6 in prevention and treatment groups of Oil + DMSO for Mat2α and Mat2b mRNA; n = 4 in prevention and treatment groups of Oil+ FDI-6 for Foxm1 mRNA; n = 6 in prevention and treatment groups of Oil + FDI-6 for Mat2α and Mat2b mRNA level; n = 4 in prevention group of CCl 4 + DMSO for Foxm1 mRNA level; n = 6 in prevention and treatment groups of CCl 4 + DMSO for Mat2α and Mat2b mRNA and treatment group of CCl 4 + DMSO for Foxm1 mRNA; n = 5 in treatment group of CCl 4 + FDI-6 for Foxm1 mRNA level; n = 6 in prevention and treatment groups of CCl 4 + FDI-6 for Mat2α and Mat2b mRNA and prevention group of CCl4+FDI-6 for Foxm1 mRNA. f Protein levels of FOXM1, MATα2, MAT2β, α–SMA, F4/80, and COL1A1 from livers after prevention and treatment with FDI-6 ( n = 3 independent experiments). Densitometry values for protein levels are summarized in Supplementary Fig. . Statistical significance was determined by using two-tailed, unpaired Student’s t -test. Source data are provided as a Source Data file. Abbreviations: ALT alanine transaminase, AST aspartate aminotransferase.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Staining, Two Tailed Test

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: MAT2A ( a ), MAT2B ( b ) and FOXM1 ( c ) promoter activities in LX-2 cells and primary cholangiocytes ± FDI-6 or siRNA treatment as described in Methods. Effects of mutating FOX elements in LX-2 cells are shown in ( d ) MAT2A , ( e ) MAT2B , and ( f ) FOXM1 . Cells transfected with WT and mutant constructs were treated with siRNA against MAT2A, MAT2B, and FOXM1 and reporter activities were measured. Data presented as mean ± SEM ( n = 3 per group). For a, Mat2a promoter activities of D-271/ + 60, D-671/ + 60 and D-1329/ + 60 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.0002, p = 0.0052 and p = 0.0948, respectively; SC + DMASO vs. SC + FOXM1 si, p = 0.0001, p = 0.0022 and p = 0.0003, respectively; Mat2a promoter activities of D-271/ + 60 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC+Foxm1si, p = 0.0286 and 0.0149, respectively. For b , Mat2b promoter activities of D-25−/+3, D-713/+3, D-990/+3 and D-1319/+3 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.0051, p = 0.0316, p = 0.016 and p = 0.0034, respectively; SC + DMSO vs. SC + FOXM1 si, p = 0.0106, p = 0.0233, p = 0.0019 and p = 0.0049 respectively; Mat2b promoter activities of D-250/+3 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC + Foxm1 si, p = 0.0002 and 0.023, respectively. For c , Foxm1 promoter activities of D-312/+107 and D-1333/+107 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.5799 and p = 0.0020, respectively; SC + DMSO vs. SC + FOXM1 si, p = 0.7684 and p = 0.0047 respectively; Foxm1 promoter activities of D-1333/ + 107 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC + Foxm1 si, p = 0.0163 and 0.0131, respectively. For d , MAT2A promoter activities (−270/+60) of WT and MU, SC vs. SC, p = 0.015 and p = 0.28 respectively; FOXM1si vs. SC, p = 0.0032 and p = 0.042 respectively; MAT2Asi vs. SC, p = 0.0033 and p = 0.066 respectively. For e , MAT2B promoter activities (−250/+3) of WT and MU, SC vs. SC, p = 0.0017 and p = 0.11 respectively; FOXM1si vs. SC, p = 0.0018 and p = 0.022 respectively; MAT2Asi vs. SC, p = 0.0015 and p = 0.026 respectively. For f , FOXM1 promoter activities (−1333/+107) of WT and MU, SC vs. SC, p = 0.000017 and p = 0.0078 respectively; FOXM1si vs. SC, p = 0.000014 and p = 0.0063 respectively; MAT2Asi vs. SC, p = 0.00020 and p = 0.019 respectively. g ChIP assay was performed by spanning two FOX regions of the FOXM1 promoter in LX-2 cells using FOXM1, MATα2 and MAT2β antibodies after treatments that varied the expression of FOXM1, MAT2A or MAT2B in the top three rows. Seq-ChIP with anti-MATα2 and MAT2β antibodies after FOXM1 ChIP was performed as described in Methods. Representative results from three experiments are shown. h qPCR analysis of the ChIP assay from ( g ). For h , ChIP and Seq-ChIP percentage of input DNA, FOXM1 si vs. SC, MAT2A si vs. SC, MAT2B si vs. SC, FOXM1 OV vs. EV, MAT2A OV vs. EV, and MAT2B OV vs. EV, for FOXM1 ChIP, p = 0.00089, p = 0.011, p = 0.0045, p = 0.0000053, p = 0.00024, and p = 0.000039; for MAT2A seq-ChIP p = 0.0015, p = 0.0023, p = 0.0037, p = 0.000037, p = 0.00076, and p = 0.00032; for MAT2B seq-ChIP p = 0.0036, p = 0.018, p = 0.0092, p = 0.0036, p = 0.0000098, p = 0.0013 respectively. Data presented as mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. SC or EV ( n = 3 independent experiments). i EMSA was done using labeled probes containing two FOX binding motifs of the FOXM1 promoter as shown in ( f ) and 100 ug of nuclear protein from LX-2 cells after treatments that varied FOXM1/MAT2A/MAT2B ( n = 3 independent experiments). j Super shifts were done using 100 ng of recombinant proteins of FOXM1, MATα2, MAT2β alone or combined, and antibodies to FOXM1, MATα2 and MAT2β. Probe and IgG only served as negative controls. Results represent three independent experiments. k In vitro pull-down shows direct interaction between MATα2, MAT2β and FOXM1 using recombinant MATα2, MAT2β and FOXM1 proteins ( n = 3 independent experiments). l MATα2, MAT2β and FOXM1 interaction in Flox control (WT), Foxm1 Hep−/− , with or without BDL was detected by Co-IP and western blotting ( n = 3 independent experiments). Statistical significance was determined by using two-tailed, unpaired Student’s t -test. Source data are provided as a Source Data file. Abbreviations: AB antibody, EV empty vector, IP immunoprecipitation, OV overexpression, si siRNA, WT wild type, MU mutants.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Transfection, Mutagenesis, Construct, Expressing, Labeling, Binding Assay, Recombinant, In Vitro, Control, Co-Immunoprecipitation Assay, Western Blot, Two Tailed Test, Plasmid Preparation, Immunoprecipitation, Over Expression

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a H&E, Sirius red, CK19, α-SMA, and F4/80 staining in Flox control and Foxm1 Hep−/− mice after BDL as compared to sham surgery. Liver fibrosis was measured by Sirius red staining ( n = 7 per group) ( b ) and hydroxyproline assay ( n = 5 per group) ( c ), liver injury by ALT ( n = 6 per group) ( d ) and AST ( n = 6 per group) ( e ) levels, macrophage number by F4/80 ( n = 6 per group) ( f ), and ductular proliferation by CK19 ( n = 4 per group) and myofibroblast differentiation by α-SMA staining ( n = 3 per group) ( g ). For b , Sirus red area/total area for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.00000000011 and p = 0.0000015, respectively. For c , hydroxyproline (ug/g, liver) for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.000058 and p = 0.0034, respectively. For d , ALT level (ug/L) for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.000000000014 and p = 0.00000000097, respectively. For e , AST level (ug/L) for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.0000000000001 and p = 0.00000025, respectively. For f , F4/80 positive number for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.0000000020 and p = 0.00080, respectively. For g , CK19/total area for Flox BDL vs. Sham and Hep−/− BDL vs. Flox BDL, p = 0.00000078 and p = 0.00026, respectively. Data are shown as mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001. h Protein expression of FOXM1, MATα2, and MAT2β in hepatocytes, cholangiocytes, HSCs, and KCs isolated from Flox control and Foxm1 Hep−/− mice ± BDL, Densitometry values for protein levels are summarized in Supplementary Fig. . mRNA levels of Foxm1 , Mat2a , and Mat2b in hepatocytes ( n = 6 animals in Flox + Sham, Foxm1 hep−/− + Sham, Flox + BDL groups and Foxm1 hep−/− + BDL group for foxm1 and mat2b mRNA; n = 4 in Flox + Sham, Foxm1 hep−/− + Sham, Flox + BDL groups and n = 6 in foxm1 hep−/− +BDL group for mat2a mRNA) ( i ), cholangiocytes ( n = 4 animals in Flox + Sham , foxm1 hep−/− + Sham, Flox + BDL groups and foxm1hep−/− + BDL groups for foxm1 mRNA; n = 5 in Flox + Sham and foxm1 hep−/− + Sham groups and n = 4 in Flox + BDL and Foxm1 hep-−/− + BDL groups for mat2a mRNA; n = 6 in Flox + Sham, Foxm1 hep−/− + Sham, Flox + BDL groups and foxm1 hep−/− + BDL group for mat2b mRNA) ( j ), HSCs ( n = 6 animals) ( k ), and KCs ( n = 4 animals in Flox + Sham, Flox + BDL groups, foxm1 hep−/− + BDL groups and n = 5 in foxm1 hep−/− + Sham for foxm1 mRNA; n = 5 in Flox + Sham and foxm1 hep−/− + Sham groups, Flox + BDL and Foxm1 hep−/− + BDL groups for mat2a mRNA; n = 5 in Flox + Sham, Flox + BDL and Foxm1 hep−/− + BDL groups and n = 6 in foxm1 hep−/− + Sham group for mat2b mRNA). l isolated from Flox control, Foxm1 Hep−/− mice ± BDL. Data are shown as mean fold of Flox control ± SEM, * p < 0.05, *** p < 0.001, **** p < 0.0001. p values obtained via two-tailed unpaired Student’s t tests. For i , mRNA levels in hepatocytes, fold of Flox con of FOXM1, MAT2A, and MAT2B of Flox BDL vs. Sham p = 0.0000045, p = 0.000030, and p = 0.000028 respectively; of Hep −/− BDL vs. Flox BDL p = 0.0000000099, p = 0.00024, p = 0.000099 respectively. For j , mRNA levels in cholangiocytes, fold of Flox con of FOXM1, MAT2A, and MAT2B of Flox BDL vs. Sham p = 0.00023, p = 0.00000026, and p = 0.000000011, respectively; of Hep −/− BDL vs. Flox BDL p = 0.000041, p = 0.013, and p = 0.0000031, respectively. For k , mRNA levels in HSCs, fold of Flox con of FOXM1, MAT2A, and MAT2B of Flox BDL vs. Sham, p = 0.0000000000005, p = 0.000000067, and p = 0.0000000000 respectively; of Hep−/− BDL vs. Flox BDL p = 0.00000000050, p = 0.00025, and p = 0.0000000011 respectively. For l , mRNA levels in KCs, fold of Flox con of FOXM1, MAT2A, and MAT2B of Flox BDL vs. Sham, p = 0.000041, p = 0.000010, and p = 0.00000035 respectively; of Hep−/− BDL vs. Flox BDL p = 0.84, p = 0.16, and p = 0.62 respectively. Statistical significance was determined by using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file. Abbreviations: ALT alanine transaminase, AST aspartate aminotransferase, BDL bile duct ligation, Cho cholangiocytes, HSCs hepatic stellate cells, Hep hepatocytes, KCs Kupffer cells.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Staining, Control, Hydroxyproline Assay, Expressing, Isolation, Two Tailed Test, Ligation

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a H&E, Sirius red, CK19, α-SMA, and F4/80 staining in Flox control and Foxm1 HSC−/− mice after BDL as compared to sham surgery. Liver fibrosis was measured by Sirius red staining ( n = 8 animals per group) ( b ) and hydroxyproline assay ( n = 6 animals per group) ( c ), liver injury by ALT ( n = 6 animals in Flox + Sham, Flox + BDL and Foxm1 HSC−/− + BDL groups and n = 5 in Foxm1 HSC−/− + Sham) ( d ) and AST ( n = 7 in Flox + Sham, Flox + BDL groups and n = 6 in Flox + BDL group and n = 5 in foxm1 HSC−/− + BDL) ( e ) levels, macrophage number by F4/80 ( n = 4 animals per group) ( f ), and ductular proliferation by CK19 staining ( n = 6 animals per group) and myofibroblast differentiation by α-SMA staining ( n = 3 animals per group) ( g ). For b , Sirius red area/total area of Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL, p = 0.0000000015 and p = 0.0000060 respectively. For c , hydroxyproline (ug/g, liver) for Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL, p = 0.00000012 and p = 0.000094 respectively. For d , ALT level (ug/L) for Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL, p = 0.000000041 and p = 0.0000013 respectively. For e , AST level (ug/L) for Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL, p = 0.00000000066 and p = 0.00044 respectively. For f , F4/80 positive number for Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL, p = 0.0000000071 and p = 0.0073 respectively. For g , Flox BDL vs. Sham and HSC−/− BDL vs. Flox BDL for CK19/total area, p = 0.000000000075 and p = 0.080 respectively; for α-SMA area/total area, p = 0.000026 and p = 0.00011 respectively. Data are shown as mean ± SEM, *** p < 0.001, **** p < 0.0001. h Protein expression of FOXM1, MATα2, and MAT2β in hepatocytes, cholangiocytes, HSCs, and KCs isolated from Flox control and Foxm1 HSC−/− mice ± BDL, Densitometry values for protein levels are summarized in Supplementary Fig. . mRNA levels of Foxm1 , Mat2a , and Mat2b in hepatocytes ( n = 6 per group) ( i ), cholangiocytes ( n = 6 per group) ( j ), HSCs ( n = 6 per group) ( k ), and KCs ( n = 6 per group) ( l ) isolated from Flox control and Foxm1 HSC−/− mice ± BDL. Data are shown as mean fold of Flox control ± SEM, **** p < 0.0001, and ns not significant. p values obtained via two-tailed unpaired Student’s t tests. For i , mRNA levels in Hepatocytes, fold of Flox con of FOXM1, MAT2A, MAT2B for Flox BDL vs. Sham, p = 0.0000000000004, p = 0.0000000000001 and p = 0.0000000000072 respectively; for HSC−/− BDL vs Flox BDL, p = 0.000000000022, p = 0.000000000020, and p = 0.000000054 respectively. For j , mRNA levels in Cholangiocytes, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.0000000000000, p = 0.000000028, and p = 0.0000000085 respectively; for HSC−/− BDL vs Flox BDL, p = 0.17, p = 0.42, and p = 0.46 respectively. For k , mRNA levels in HSCs, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.0000052, p = 0.0000000000, and p = 0.0000000000 respectively; for HSC−/− BDL vs Flox BDL, p = 0.00000056, p = 0.00018, and p = 0.00000000002 respectively. For l , mRNA levels in KCs, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.000000000091, p = 0.00036, and p = 0.00000010 respectively; for HSC−/− BDL vs Flox BDL, p = 0.086, p = 0.61, and p = 0.15 respectively. Statistical significance was determined by using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file. Abbreviations: ALT alanine transaminase, AST aspartate aminotransferase, BDL bile duct ligation, Cho cholangiocytes, HSCs hepatic stellate cells; Hep hepatocytes, KCs Kupffer cells.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Staining, Control, Hydroxyproline Assay, Expressing, Isolation, Two Tailed Test, Ligation

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a H&E, Sirius red, CK19, α-SMA, and F4/80 staining in Flox control and Foxm1 KC−/− mice after BDL as compared to sham surgery. Liver fibrosis was measured by Sirius red staining ( n = 8 animals per group), b and hydroxyproline assay ( n = 5 animals in Flox + Sham group and n = 6 in foxm1 KC−/− + Sham, Flox + BDL and foxm1 KC−/− + BDL groups) ( c ), liver injury by ALT ( n = 6 animals per group) ( d ) and AST ( n = 6 animals per group) ( e ) levels, macrophage number by F4/80 ( n = 6 animals per group) ( f ), and ductular proliferation by CK19 ( n = 4 animals per group) and myofibroblast differentiation by α-SMA staining ( n = 3 animals per group) ( g ). For b , Sirius red area/total area of Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL, p = 0.00000000049 and p = 0.00014 respectively. For c , hydroxyproline (ug/g, liver) for Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL, p = 0.00030 and p = 0.012 respectively. For d , ALT level (ug/L) for Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL, p = 0.0000000000000 and p = 0.000000000081 respectively. For e , AST level (ug/L) for Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL, p = 0.0000000000001 and p = 0.00000025 respectively. For f , F4/80 positive number for Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL, p = 0.0000000067 and p = 0.0000094 respectively. For g , Flox BDL vs. Sham and KC−/− BDL vs. Flox BDL for CK19/total area, p = 0.00000010 and p = 0.039 respectively; for α-SMA area/total area, p = 0.000026 and p = 0.00033 respectively. Data are shown as mean ± SEM, * p < 0.05, *** p < 0.001, **** p < 0.0001. h Protein levels of FOXM1, MATα2, and MAT2β in hepatocytes, cholangiocytes, HSCs, and KCs isolated from Flox and Foxm1 KC−/− mice ± BDL, Densitometry values for protein levels are summarized in Supplementary Fig. . mRNA levels of Foxm1 , Mat2a , and Mat2b in hepatocytes ( n = 6 per group) ( i ), cholangiocytes ( n = 6 per group) ( j ), HSCs ( n = 6 per group) ( k ), and KCs ( n = 6 per group) ( l ) isolated from Flox control and Foxm1 KC−/− mice ± BDL. Data are shown as mean fold of Flox control ± SEM, *** p < 0.001, **** p < 0.0001, ns not significant. For i , mRNA levels in hepatocytes, fold of Flox con of FOXM1, MAT2A, MAT2B for Flox BDL vs. Sham, p = 0.000000012, p = 0.0000019 and p = 0.50 respectively; for KC −/− BDL vs Flox BDL, p = 0.00000086, p = 0.00060, and p = 0.0000000036 respectively. For j , mRNA levels in cholangiocytes, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.0000000000001, p = 0.0000000000005, and p = 0.00000000046 respectively; for KC −/− BDL vs Flox BDL, p = 0.071, p = 0.064, and p = 0.061 respectively. For k , mRNA levels in HSCs, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.0000000086, p = 0.0000000002, and p = 0.000000000 respectively; for KC −/− BDL vs Flox BDL, p = 0.0000032, p = 0.0000095, and p = 0.0000078 respectively. For l , mRNA levels in KCs, fold of Flox con of FOXM1, MAT2A, and MAT2B for Flox BDL vs. Sham p = 0.000000040, p = 0.000000033, and p = 0.00000075 respectively; for KC −/− BDL vs Flox BDL, p = 0.00000000071, p = 0.00000097, and p = 0.0000059, respectively. Statistical significance was determined by using two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file. Abbreviations: ALT alanine transaminase, AST aspartate aminotransferase, BDL bile duct ligation, Cho cholangiocytes, HSCs hepatic stellate cells, Hep hepatocytes, KCs Kupffer cells.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Staining, Control, Hydroxyproline Assay, Isolation, Two Tailed Test, Ligation

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a Time courses of protein expression of FOXM1, MATα2, MAT2β, SMAD3, α–SMA, and COL1A1 after TGF-β1 treatment in LX-2 cells. b FOXM1, MATα2, MAT2β, SMAD3, α-SMA and COL1A1 protein levels after siRNA knockdown of FOXM1, MAT2A, or MAT2B in LX-2 cells after 24 h. c Protein levels of FOXM1, MATα2, MAT2β, SMAD3, α-SMA, and COL1A1 after FOXM1 overexpression with or without MAT2A or MAT2B siRNA knockdown and TGF–β1 treatment (20 ng/ml) for 24 h in LX-2 cells. d Effect of LPS on protein expression of FOXM1, MATα2, MAT2β, TNF-α, and IL-6 with or without siRNA knockdown of FOXM1, MAT2A or MAT2B in RAW 264.7 cells for 24 h. e Effects of LPS on protein expression of FOXM1, MATα2, MAT2β, TNF-α, and IL-6 with FOXM1 overexpression and MAT2A or MAT2B siRNA treatment for 24 h in RAW 264.7 cells (left panel) and KCs isolated from Flox control mice (right panel). Densitometry values for protein levels are summarized in Supplementary Fig. and Supplementary Fig. . n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Expressing, Knockdown, Over Expression, Isolation, Control

Journal: Nature Communications

Article Title: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

doi: 10.1038/s41467-024-52527-8

Figure Lengend Snippet: a Following BDL and culture of hepatocytes from flox mice and Foxm1 Hep−/− , EVs were extracted from the medium and used to treat HSCs from Foxm1 HSC−/− . b Following BDL and culture of HSCs from flox mice and Foxm1 HSC−/− , EVs were extracted from the medium and used to treat hepatocytes from Foxm1 Hep−/− . c , d Following BDL and culture of KCs from flox mice and Foxm1 KC−/− , EVs were extracted from the medium and used to treat ( c ) HSCs from Foxm1 HSC−/− or ( d ) hepatocytes from Foxm1 Hep−/− . e EVs extracted from hepatocytes of Flox and Foxm1 Hep −/− after BDL were used to treat KCs from Foxm1 KC −/− and ( f ) EVs extracted from HSCs of Flox and Foxm1 HSC −/− after BDL were used to treat KCs from Foxm1 KC−/− . All EV treatments were for 24 h after which protein expression of FOXM1, MATα2 and MAT2β were measured in cell lysates by western blotting. Densitometry values for protein levels are summarized in Supplementary Fig. , n = 3 independent experiments ( g ) Summary of key findings showing the FOXM1/MAT2A/MAT2B axis in the different liver cell types driving liver inflammation and fibrosis. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was done with antibodies against FOXM1 (ab207298; CAT#: 13147-1-AP, Proteintech), MATα2 (55309-1-AP), MAT2β (BNP1-82797), α-SMA (ab5831), COL1A1 (ab270993), β-actin (ab8226), F4/80 (ab300421), SMAD3 (ab52903), tumor necrosis factor alpha (TNF-α, ab183218), tubulin (ab18251) and interleukin 6 (IL-6, ab259341).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

doi: 10.7150/thno.68667

Figure Lengend Snippet: Antibody conjugated RBCEVs show increased accumulation in target cells. RBCEVs were conjugated with biotinylated monoclonal antibodies or nanobodies via a biotinylated linker peptide and streptavidin . (A) Single EV flow cytometric analysis of monoclonal antibody conjugation on RBCEVs using the streptavidin-mediated conjugation method. (B) Copy number of isotype control monoclonal antibody (mAb) and nanobodies (EGFR VHH) per RBCEV quantified using ELISA by comparison to a standard curve of antibodies/nanobodies (n = 6-9 replicates). (C) Flow cytometry analysis of CFSE in EGFR-positive CA1a cells or EGFR-negative MOLM13 cells treated with CFSE-labeled RBCEVs coated with control or EGFR-targeting nanobodies. (D) Representative immunofluorescent images of EV uptake in the co-culture of 4T1 and 4T1-tdTomato-hEGFR cells incubated with different RBCEV treatments. RBCEVs were tracked using CFSE (green), tdTomato was shown in red while nuclei were co-stained with Hoechst (blue). Scale bar is 20 µm. (E) Percentage difference in RBCEV uptake between 4T1-tdTomato-hEGFR cells and parental 4T1 cells, expressed as a fraction of mean CFSE intensity for each RBCEV treatment. (F) Uptake of EpCAM-targeted or control CFSE-labelled RBCEVs by EpCAM-positive H358 cells or EpCAM-negative MOLM13 cells. (G) Representative immunofluorescent images of EpCAM-targeting and non-targeting RCBEV uptake by H358 cells as in (F) . RBCEV uptake was observed using CFSE (green). CellMask was used to label the cell membrane (red) and the nucleus was visualized using Hoechst (blue). Scale bar is 50 µm. (H) RT-qPCR quantification of miR-125b ASOs loaded per EV obtained via comparison of the total RNA extract from miR-125b ASO-loaded EVs to a standard curve of miR-125b ASO (n = 3 biological replicates). (I) Quantification of miR-125b ASOs loaded per individual EV obtained via native PAGE analysis of miR-125b ASO-loaded RBCEVs electrophoresed alongside a serial dilution of unloaded miR-125b ASO (n = 6 biological replicates) . (J) Representative native PAGE analysis used to assess the loading efficiency of ASOs into EVs. Each EV lane denotes a separate biological replicate prepared using EVs from 3 blood donors (D1-D3). Graphs A, C, E and F represent data from 3 biological replicates prepared from RBCEVs from independent donors. For quantification of RNA per EV in H & I , NTA was used to obtain the number of input EVs, thereby providing an estimate of ASO copy number per EV. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, ***P < 0.001.

Article Snippet: For the quantification of miR-125b ASO per EV, a Taqman real-time PCR assay (ThermoFisher ID #007655_mat) was used.

Techniques: Bioprocessing, Conjugation Assay, Control, Enzyme-linked Immunosorbent Assay, Comparison, Flow Cytometry, Labeling, Co-Culture Assay, Incubation, Staining, Membrane, Quantitative RT-PCR, Clear Native PAGE, Serial Dilution, One-tailed Test

Journal: Theranostics

Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

doi: 10.7150/thno.68667

Figure Lengend Snippet: Targeted delivery of DNA/RNA payloads to target cells using antibody functionalized RBCEVs. (A) Flow cytometry analysis reflecting the delivery of a FAM-conjugated non-targeting ASO (FAM-NC-ASO) to CA1a cells by EGFR-targeting or non-targeting RBCEVs. (B) Flow cytometry analysis demonstrating the delivery of FAM-ASO to H358 cells using EpCAM-mAb-conjugated RBCEVs or non-targeting RBCEVs. (C) Mean dGFP fluorescence of CA1a-dGFP cells determined using flow cytometry following incubation with EGFR-targeted or non-targeted RBCEVs loaded with GFP-siRNA. (D) Representative immunofluorescent images of CA1a-dGFP cells treated with dGFP-siRNA-loaded RBCEVs. Cells were stained with CellMask dye (red) while dGFP expression is shown in green. Scale bar is 20 µm. (E) Knockdown of miR-125b in CA1a cells using EVs loaded with NC-ASO or 125b-ASO loaded RBCEVs, with or without EGFR targeting. miR-125b was quantified using TaqMan RT-qPCR, normalized to U6b RNA and presented as average log 10 fold change relative to the untreated control. (F) Effect of NC-ASO or 125b-ASO loaded RBCEVs on the viability of CA1a cells, assessed using CCK8 assay. (G) miR-125b knockdown (normalized to snoRNA234) and (H) viability assays in 4T1-hEGFR cells performed similarly as in E & F . Graphs A , B , C , E , F , G and H represent data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the quantification of miR-125b ASO per EV, a Taqman real-time PCR assay (ThermoFisher ID #007655_mat) was used.

Techniques: Flow Cytometry, Fluorescence, Incubation, Staining, Expressing, Knockdown, Quantitative RT-PCR, Control, CCK-8 Assay, One-tailed Test

Journal: Theranostics

Article Title: Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

doi: 10.7150/thno.68667

Figure Lengend Snippet: EGFR-targeted miR-125b ASO loaded EVs efficiently suppress tumor progression. (A) Overview of the in vivo study used to evaluate the efficacy of antibody-conjugated EVs for targeted delivery of RNA therapeutics. (B) Evaluation of EV uptake by tumor cells in the lung for EGFR-targeted and non-targeted CFSE-labeled EVs determined based on the colocalization of CFSE and tdTomato signals using immunofluorescent imaging of lung sections. Each data point corresponds to a single tile scan of the lung acquired at random. (C) Relative miR-125b knockdown in mouse lung cells and tumor cells following administration with different EV treatments. RNA levels were normalized to snoRNA (n = 3-4 mice). (C) Percentage of tumor cells in the lung at the endpoint of the experiment for each treatment condition, obtained using flow cytometric analysis of tdTomato and hEGFR double positive cells in lung homogenates (n = 5-6 mice). (D) Percentage of lung area occupied by tumor tissue, obtained via quantification of tdTomato fluorescence of intact lung sections. (E) Representative immunofluorescent images of intact lung sections from each treatment condition stained for α-SMA and CD31. Tumor tissue was identified by the tdTomato fluorescence and shown in red. Scale bar, 1 mm. Inset shows selected areas at 4 × magnification. The graphs present the mean ± SEM. Student's one-tailed t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the quantification of miR-125b ASO per EV, a Taqman real-time PCR assay (ThermoFisher ID #007655_mat) was used.

Techniques: In Vivo, Labeling, Imaging, Knockdown, Fluorescence, Staining, One-tailed Test