Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 and its receptor CXCR4 were expressed and localized in the ovary, with their binding activating the JAK/STAT signaling pathway. (A) Immunofluorescence staining of ovarian follicles revealed that CXCL12 was co-localized with its receptor CXCR4 in ovarian follicles. (B) CXCL12 overexpression significantly promoted the expression of CXCR4 , JAK2 and STAT1 . (C) Knocking down CXCL12 significantly inhibited the expression of CXCR4 , JAK2 and STAT1 . (D) CXCL12 overexpression in GCs promoted the protein expression of CXCR4, JAK2 and STAT1 as well as the phosphorylation of JAK2 and STAT1. When CXCL12 was knocked down, the results were reversed. (E) MSX-122 inhibitor-based treatment further confirmed that CXCL12 promoted the total protein and phosphorylation levels of JAK2 and STAT1 (The t test was used for the above analyses comparing two individual samples. * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Protein detection was achieved using the following antibodies: anti-CCND1 mouse monoclonal antibody (1:250, Proteintech), anti-PCNA rabbit polyclonal antibody (1:250, Proteintech), anti-Bcl2 rabbit polyclonal antibody (1:250, Proteintech), anti-Bax rabbit polyclonal antibody (1:250, Proteintech), anti-CITED1 rabbit polyclonal antibody (1:50, Proteintech), anti-WNT10B mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-CXCR4 mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-phospho-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam, Cambridge, UK), anti-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam), anti-phospho-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-GAPDH mouse monoclonal antibody (1:2,500, Proteintech), 1:1,000 goat anti-rabbit secondary antibody IgG (Proteintech) and 1:1,000 goat anti-mouse secondary antibody IgG (Proteintech).

Techniques: Binding Assay, Immunofluorescence, Staining, Over Expression, Expressing, Phospho-proteomics

Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 and its receptor CXCR4 were expressed and localized in the ovary, with their binding activating the JAK/STAT signaling pathway. (A) Immunofluorescence staining of ovarian follicles revealed that CXCL12 was co-localized with its receptor CXCR4 in ovarian follicles. (B) CXCL12 overexpression significantly promoted the expression of CXCR4 , JAK2 and STAT1 . (C) Knocking down CXCL12 significantly inhibited the expression of CXCR4 , JAK2 and STAT1 . (D) CXCL12 overexpression in GCs promoted the protein expression of CXCR4, JAK2 and STAT1 as well as the phosphorylation of JAK2 and STAT1. When CXCL12 was knocked down, the results were reversed. (E) MSX-122 inhibitor-based treatment further confirmed that CXCL12 promoted the total protein and phosphorylation levels of JAK2 and STAT1 (The t test was used for the above analyses comparing two individual samples. * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Protein detection was achieved using the following antibodies: anti-CCND1 mouse monoclonal antibody (1:250, Proteintech), anti-PCNA rabbit polyclonal antibody (1:250, Proteintech), anti-Bcl2 rabbit polyclonal antibody (1:250, Proteintech), anti-Bax rabbit polyclonal antibody (1:250, Proteintech), anti-CITED1 rabbit polyclonal antibody (1:50, Proteintech), anti-WNT10B mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-CXCR4 mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-phospho-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam, Cambridge, UK), anti-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam), anti-phospho-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-GAPDH mouse monoclonal antibody (1:2,500, Proteintech), 1:1,000 goat anti-rabbit secondary antibody IgG (Proteintech) and 1:1,000 goat anti-mouse secondary antibody IgG (Proteintech).

Techniques: Binding Assay, Immunofluorescence, Staining, Over Expression, Expressing, Phospho-proteomics

Journal: Blood Advances

Article Title: STAT1-mediated epigenetic regulation of LIN28A controls iPSC-derived platelet production through the let-7–RALB axis ∗

doi: 10.1182/bloodadvances.2024015557

Figure Lengend Snippet: STAT1 is the upstream regulator of LIN28A in imMKCLs. The siRNA SMARTpool designed against human STAT1 (siSTAT1) and a siNT were transfected into imMKCLs. (A) A schematic illustration of the experimental procedure of siRNA-mediated STAT1 knockdown in imMKCLs. (B) STAT1 knockdown significantly decreased STAT1 and LIN28A mRNA expression and increased let-7a-5p expression. (C) Intracellular protein levels of STAT1 (mean fluorescence intensity detected in siNT or siSTAT1 imMKCLs by intracellular flow cytometry. STAT1 expression was compared using a histogram overlay in the left panel. (D) STAT1 knockdown improved iPSC-PLT generation by imMKCLs (clone 7). (E) Representative flow cytometry plots of iPSC-PLTs generated from siNT or siSTAT1 imMKCLs in static or turbulent flow conditions. CD41 + CD42b + platelets were identified from a platelet-sized gate. (F) STAT1 knockdown decreased mRNA expression of representative immune-related genes in imMKCLs. (G) The secretion of IL-8 from clone 7 and clone 7-3 during the maturation stage (DOX-OFF day 6). Data are expressed as the mean ± SEM from 3 to 4 independent experiments. Unpaired 2-tailed Student t tests were used to assess statistical significance. IL-8, interleukin 8; MK, megakaryocyte; ns, not significant.

Article Snippet: After 2 washes with phosphate-buffered saline, cells were incubated with an anti-STAT1 polyclonal antibody (Proteintech; 10144-2-AP; 0.4 μg/100 μL) for 1 hour on ice.

Techniques: Transfection, Knockdown, Expressing, Fluorescence, Flow Cytometry, Generated

Journal: Blood Advances

Article Title: STAT1-mediated epigenetic regulation of LIN28A controls iPSC-derived platelet production through the let-7–RALB axis ∗

doi: 10.1182/bloodadvances.2024015557

Figure Lengend Snippet: Pharmacological inhibition of STAT1 enhances iPSC-PLT production under turbulent flow conditions. (A) Chemical structures of fludarabine and flavopiridol, the 2 established inhibitors for STAT1 phosphorylation. (B) Dose optimization on iPSC-PLT production of fludarabine (0 to ∼5 μM) and flavopiridol (0 to ∼100 nM). (C) iPSC-PLT generation by imMKCLs (clone 7) in the absence and presence of fludarabine (1 μM) or flavopiridol (10 nM) under static or turbulent flow conditions. (D) Representative flow cytometry plots of iPSC-PLTs generated in the absence and presence of fludarabine (1 μM) or flavopiridol (10 nM) in static or turbulent flow conditions. CD41 + CD42b + platelets were identified from a platelet-sized gate. (E) Fludarabine (1 μM) or flavopiridol (10 nM) treatments elevated let-7a-5p expression. (F) Flavopiridol (10 nM) treatment reduced mRNA expression of representative immune-related genes in imMKCLs at the maturation stage. Data are expressed as the mean ± SEM from 4 to 5 independent experiments. Statistical significance was assed using 1-way analysis of variance or unpaired 2-tailed Student t tests. DMSO, dimethyl sulfoxide.

Article Snippet: After 2 washes with phosphate-buffered saline, cells were incubated with an anti-STAT1 polyclonal antibody (Proteintech; 10144-2-AP; 0.4 μg/100 μL) for 1 hour on ice.

Techniques: Inhibition, Phospho-proteomics, Flow Cytometry, Generated, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Estradiol (E2) concentration shapes the chromatin binding landscape of estrogen receptor alpha

doi: 10.1016/j.jbc.2025.108499

Figure Lengend Snippet: STAT1 influences ER binding at low 10 −12 M E2 concentration . A , heatmap based on the K-means clustering of TF fractions in each CUT&RUN cluster. B , top negatively and positively correlated TFs identified from FIMO motif discovery. C , immunoblot analysis of STAT1 protein expression from the shSTAT1 #1 and shSTAT1 #2 versus shCTRL MCF7 transduction. D , profile of the spike-in normalized read density of the STAT1 knockdown cells in the 10 −12 M E2 and 10 −8 M E2 against the shCTRL MCF7 cells. E , immunoprecipitation of ER and STAT1 with the 10 −12 M E2 and 10 −8 M E2 induced MCF7 cells. F , TCGA data of STAT1 expression based on premenopausal and postmenopausal patients in the breast cancer cohort. G , Kaplan–Meier survival plot for STAT1 expression in patients with ER + breast cancer (from a database of 55 independent transcriptomic datasets ). CUT&RUN, cleavage under targets and release using nuclease; ER, estrogen receptor; TCGA, The Cancer Genome Atlas Program; TF, transcription factor.

Article Snippet: Antibodies: Estrogen receptor alpha polyclonal (abcam, ab3575, RRID: AB_303921 ); estrogen receptor alpha, 6F11 (Santa Cruz, sc-56836, RRID: AB_1122654 ); estrogen receptor alpha, D6R2W (Cell Signaling Technologies (CST), 13258, RRID: AB_2632959 ); phospho-estrogen receptor alpha, Ser118, 16J4 (Cell Signaling Technologies, 2511, RRID: AB_331289 ); STAT1 monoclonal STAT1-79 (Invitrogen, AH00832); STAT1 rabbit polyclonal (Proteintech, 10144-2-AP, RRID: AB_2286875 ); beta-actin (abcam, ab8226, RRID: AB_306371 ); normal rabbit IgG (Cell Signaling Technologies, 2729, RRID: AB_1031062 ); tri-methyl-histone H3 rabbit monoclonal, lys27, C36B11 (CST, 9733S, RRID: AB_2616029 ); Alexa Fluor 488-conjugated AffiniPure Goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, 111-545-144, RRID: AB_2338052 ); IRDye 680RD Goat anti-mouse IgG (LI-COR, 925-68070, RRID: AB_2651128 ); IRDye 800CW Goat anti-rabbit IgG (LI-COR, 925-32211, RRID: AB_2651127 ); IRDye 800CW Goat anti-mouse IgG2b (LI-COR, 926-32352, RRID: AB_2782999 ).

Techniques: Binding Assay, Concentration Assay, Western Blot, Expressing, Transduction, Knockdown, Immunoprecipitation