Journal:

Article Title: Identification of a New Hybrid Serum Response Factor and Myocyte Enhancer Factor 2-binding Element in MyoD Enhancer Required for MyoD Expression during Myogenesis

doi: 10.1091/mbc.E06-09-0867

Figure Lengend Snippet: The CArG element in MyoD gene DRR is an hybrid sequence composed of binding sites for both SRF and MEF2 factors. (A) Comparison of the classical CArG sequence to the divergent CArG elements present in MyoD-DRR and MLC1A promoters. (B) MyoD-wtCArG and MyoD-MLC1A sequence-containing oligonucleotides were radiolabeled and used as probes in EMSA with nuclear extracts from growing (Pro) and differentiated (Dif) C2 myoblasts. Rabbit polyclonal antibodies raised against SRF or MEF2 were added or not before the DNA binding reaction. Two complexes A and B, and anti-SRF or anti-MAF2 supershifted (complex sA and sB), are indicated by arrowheads. An asterisk marks the complex specifically formed with differentiated cell extracts and not supershifted by anti-SRF antibodies. (C) Single point mutations of the MyoDwt-CArG probes as shown were used in EMSA with nuclear extracts from differentiated C2 cells. Antibodies to SRF, MEF2, or both were added or not before DNA binding. The complexes formed are indicated with arrows in B.

Article Snippet: Labeled probe was added to the mixture and incubated at 30°C for 10 min. For supershift and in vitro competition analysis, 0.2 μg of rabbit polyclonal antibody (SRF G20, sc-335X; MEF2 H300, sc-10794X; Santa Cruz Biotechnology, Santa Cruz, CA) or 10–100 ng of His-tagged proteins was included before probe addition and incubated with the mixture for 1 h on ice.

Techniques: Sequencing, Binding Assay, Comparison

Journal: Journal of Immunology Research

Article Title: Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs

doi: 10.1155/2022/6061746

Figure Lengend Snippet: A peripheral population can migrate into the thymus and contribute to PanK- and FoxN1-expressing thymic epithelial cells. C57BL6 fetal thymi were transplanted under the kidney capsule of actin H2BGFP mice and analyzed for the presence of GFP-expressing peripheral cells 3-12 weeks after transplant using IHC. (a–d) Week 3 transplanted thymic tissue sections showing expression of PanK (a), GFP+ peripheral cells (b), FoxN1 (c), and merged image (d). (e–h) Week 6 transplanted thymic tissue section showing expression of PanK (e), GFP+ peripheral cells (f), FoxN1 (g), and merged image (h). (i–l) Week 9 transplanted thymic tissue section showing expression of PanK (i), GFP+ peripheral cells (j), FoxN1 (k), and merged image (l). (m–p) Week 12 transplanted thymic tissue section showing expression of PanK (m), GFP+ peripheral cells (n), FoxN1 (o), and merged image (p). GFP-, PanK-, and FoxN1-expressing cells of interest are enlarged in the insets to show colocalization of both proteins and represent the cells shown in the white boxes. PanK+ FoxN1+ GFP+ cells and PanK+ FoxN1- GFP+ cells at all time points are indicated by white and red arrows, respectively. The results presented are representative of the three to five replicates of each experiment that were performed.

Article Snippet: The following primary antibodies were used for experiments: CD45-PE Cy7, CD45-APC Cy7, and CD45 APC (clone 30-F11, BD Biosciences, BioLegend), pan-keratin (Polyclonal, Dako), EpCAM-PE (clone G8.8, eBioscience), FoxN1 (clone G-20, Santa Cruz Biotechnology), FSP1 (clone S100A4, BioLegend), Thy1.2 (clone 30-H12, BD Biosciences), CD11b (clone, BioLegend), CD11b PerCpcy5.5 (clone M1/70, BioLegend), CD4-biotinylated (clone RM4-5, BioLegend), CD8-biotinylated (clone 53-6.7, BioLegend), CD19 (clone 1D3, BioLegend), rabbit anti-GFP (Life), and CD205 (LY75/DEC-205) (clone HD30 (Millipore).

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs

doi: 10.1155/2022/6061746

Figure Lengend Snippet: Reaggregate thymic organ cultures show that bone marrow-derived CD45+ EpCAM+ cells can give rise to PanK+ FoxN1+ thymic epithelial cells. (a–d) Sections of RTOC consisting of C57BL/6 fetal thymus mixed with sorted actin H2BGFP-expressing bone marrow-derived CD45+ EpCAM+ cells. A representative RTOC section showing PanK expression (a); FoxN1 expression (b) with GFP-expressing cells derived from the CD45+ EpCAM+ cells sorted from adult GFP-expressing bone marrow (c), (d) merged image of PanK, FoxN1, and GFP. GFP-, PanK-, and FoxN1-expressing cells of interest are enlarged in the insets to show coexpression of both proteins and represent the cells shown in the white boxes. (e–g) Representative 200x image of the RTOC section showing pan-keratin expression (e); staining with DAPI (f), and the merged image of PanK, and DAPI (g). A minimum of three replicates of all experiments were performed.

Article Snippet: The following primary antibodies were used for experiments: CD45-PE Cy7, CD45-APC Cy7, and CD45 APC (clone 30-F11, BD Biosciences, BioLegend), pan-keratin (Polyclonal, Dako), EpCAM-PE (clone G8.8, eBioscience), FoxN1 (clone G-20, Santa Cruz Biotechnology), FSP1 (clone S100A4, BioLegend), Thy1.2 (clone 30-H12, BD Biosciences), CD11b (clone, BioLegend), CD11b PerCpcy5.5 (clone M1/70, BioLegend), CD4-biotinylated (clone RM4-5, BioLegend), CD8-biotinylated (clone 53-6.7, BioLegend), CD19 (clone 1D3, BioLegend), rabbit anti-GFP (Life), and CD205 (LY75/DEC-205) (clone HD30 (Millipore).

Techniques: Derivative Assay, Expressing, Staining

Journal: Oncogenesis

Article Title: Fatty acid synthase regulates estrogen receptor-α signaling in breast cancer cells

doi: 10.1038/oncsis.2017.4

Figure Lengend Snippet: FASN inhibition activates p21 WAF1/CIP1 and p27 Kip1 and simultaneously deactivates the PI3K/AKT pathway. ( a ) Left: Protein was isolated from MCF-7 cells following treatment with C75 for 48 h in the absence or presence of 10 −9 mol/l E 2 , as described in ‘Materials and methods'. p21 WAF1/CIP1 and p27 Kip1 expression was determined by immunoblotting using anti-p21 WAF1/CIP1 mouse monoclonal and anti-p27 Kip1 rabbit polyclonal antibodies, respectively. The blot was stripped and re-probed with an antibody to β-actin to assess equal loading of lysate proteins and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in three independent experiments. Right: E 2 -depleted MCF-7 cells were seeded at 1 × 10 4 cells/well in a four-well chamber slide. After 48 h incubation with 10 −9 mol/l E 2 in the absence or presence of 5 μg/ml C75, p21 WAF1/CIP1 and p27 Kip1 cellular localization was evaluated after a 2 h incubation with anti-p21 WAF1/CIP1 mouse monoclonal and p27 Kip1 rabbit polyclonal antibodies diluted 1:200 in 0.05% Triton X-100/PBS. After labeling, cells were extensively washed, and the localization of p21 WAF1/CIP1 and p27 Kip1 was detected by indirect immunofluorescence by incubating with FITC-conjugated anti-mouse (p21 WAF1/CIP1 ) or TRITC-conjugated anti-rabbit (p27 Kip1 ) IgG secondary antibodies. Figure shows a representative immunostaining analysis. Similar results were obtained in three independent experiments. ( b ) Protein was isolated from E 2 -stimulated MCF-7 cells following treatment with C75 for 6 h as described in ‘Materials and methods', and AKT activation was determined by immunoblotting using a specific phospho-AKT polyclonal antibody. The blot was stripped and re-probed with antibodies for total AKT protein and β-actin to assess equal loading of lysate proteins and transfer. Bottom: Immunoreactive bands for phospho-AKT were scanned and normalized to total AKT. After a second normalization to β-actin, the value obtained in E 2 -stimulated MCF-7 cells was set to 100% and the different treatment groups were expressed as a percentage of control levels. Data are presented as the mean±s.d. of two independent experiments and the asterisks indicate values that are significantly different (* P <0.05) from E 2 -stimulated AKT activity.

Article Snippet: The primary antibodies used in this studies were obtained from the following suppliers: FASN monoclonal antibody (clone 23) was from BD Biosciences Pharmingen (San Diego, CA, USA); β-actin goat polyclonal, ERα (G-20) rabbit polyclonal antibody, and p27 KIP1 rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Isolation, Expressing, Western Blot, Incubation, Labeling, Immunofluorescence, Immunostaining, Activation Assay, Control, Activity Assay

Journal: Journal of the American Society of Nephrology

Article Title: The Sorting Nexin 3 Retromer Pathway Regulates the Cell Surface Localization and Activity of a Wnt-Activated Polycystin Channel Complex

doi: 10.1681/asn.2016121349

Figure Lengend Snippet: Figure 3. Knockdown of SNX3 or VPS35 leads to increased surface expression of endogenous PC1 and PC2 in LLC-PK1 cells and in Xenopus pronephric tubules. (A) Surface expression of endogenous PC1 and PC2 in LLC-PK1 cells was increased after selective knockdown of SNX3 or VPS35 siRNA (20 nM) compared with a scrambled control siRNA. Surface biotinylated PC1 and PC2 was compared with total PC1/PC2. Na+-K+-ATPase and calnexin were used as positive and negative controls, respectively, for surface biotinylation. (B) The ratio of biotinylated/total PC1 calculated from four independent experiments showed a significant increase after SNX3 (P,0.05) and VPS35 (P,0.05) knockdown: Scrambled siRNA = 0.13960.047; SNX3 siRNA = 0.30660.048; VPS35 siRNA = 0.60060.171. Similarly, a significant increase in biotinylated PC2 was observed after SNX3 (P,0.05) and VPS35 (P,0.01) knockdown in triplicate experiments: Scrambled siRNA = 0.23560.035; SNX3 siRNA = 0.52760.076; VPS35 siRNA = 1.28960.038. The increase in biotinylated PC1 and PC2 was quantitatively greater after VPS35 knockdown compared with SNX3 despite a more complete inhibition of the latter. (C) EndoH digestion of surface biotinylated proteins in LLCPK1 cells. Whereas PC1 and Na+ K+ ATPase were EndoH resistant, surface labeled PC2 was EndoH sensitive. Calnexin, an ER resident protein, acts as a negative control for the biotinylation assay. Representative experiment of two shown. (D) Endogenous PC2 expression in LLC-PK1 cells detected by widefield immuno- fluorescence microscopy using a specific PC2 antibody (g20, Santa Cruz, CA) as previously described.18 A proportion of PC2 was detectable in the lateral plasma membrane as well as in primary cilia colabeled with an antibody to acetylated tubulin (arrows). Left: A clear increase in plasma membrane PC2 staining was observed after knockdown of SNX3 or VPS35 relative to untransfected controls or cells transfected with a scrambled siRNA. Right: No change in cilia PC2 expression was detectable. Images are representative of three separate experiments. Scale bars, 10 mm. (E) Surface staining intensity was calculated as a percentage relative to control untransfected cells and expressed as bar diagrams. Left: A significant increase in surface expression of PC2 was seen after SNX3 and VPS35 knockdown (P,0.01): Scrambled siRNA = 25.54%61.68, n=30; SNX3 siRNA = 14.74%64.24, n=30; VPS35 siRNA = 35.01%62.28, n=30. Right: there was no significant difference in cilia localization of PC2: control siRNA = 92.8864.52, n=50; scrambled siRNA = 92.42%67.58, n=32; SNX3 siRNA = 93.3%63.88, n=31; VPS35 siRNA = 96.43%63.57, n=39. (F) Uninjected Xenopus controls (i, ii) and embryos injected unilaterally with a total of 1.6 pmol Vps34-MO (iii, iv) were analyzed at stage 40 by PC2 immunofluorescence (red). ECL was used to visualize the apical side of the proximal tubules (green) and nuclei were counterstained with DAPI (blue). Images were analyzed by confocal microscopy and all three z planes are shown. The white lines indicate the position used for x,z and y,z planes, respectively. (G) Bar diagram to quantify the apical pixel intensity of PC2 staining. Error bars represent SD, and statistical significance was determined using paired t test (* P,0.05). Note that the pronephric tubules of Vps35 morphants are dilated, which is likely due to the effect of the retromer complex on the secretion of Wnt ligands and their role in kidney tubulogenesis.11,26 *, P,0.05; ***, P,0.001.

Article Snippet: Antibodies were purchased from the following suppliers: mouse anti-Thio (Invitrogen), mouse anti-GST (Serotec), mouse anti–Clathrin HC (BD Biosciences), rabbit anti-SNX3 (Abcam, Inc.), mouse anti-EEA1 (BD Biosciences), goat anti-PC2 (G20) (Santa Cruz Biotechnology), mouse anti–AP2 m2 subunit (AP50; BD Biosciences), rat anti-HA (Roche), rabbit anti-HA (Santa Cruz 8 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2017 Biotechnology), rabbit anti–c-Myc (Santa Cruz Biotechnology), rat anti–c-Myc (Serotec), mouse anti-Pk/V5 (Serotec), mouse anti-Flag (Sigma-Aldrich), and mouse anti-MBP (NEB).

Techniques: Knockdown, Expressing, Control, Inhibition, Labeling, Negative Control, Cell Surface Biotinylation Assay, Microscopy, Clinical Proteomics, Membrane, Staining, Transfection, Injection, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells

doi: 10.3389/fimmu.2023.1261081

Figure Lengend Snippet: Lin28a , Lin28b , and Let-7g expressed in fetal murine thymi. Immunofluorescence staining of fetal thymic sections. (A) 4% PFA-fixed E13.5 thymic sections were stained for LIN28b (green, white arrow shows cytoplasm staining) and FOXN1 (red, pink arrow shows nuclear staining). (B) The frozen thymic sections were stained for LIN28b (green) and MHCII (red). (C) 4% PFA-fixed E18.5 thymic sections were stained for LIN28b (green) and FOXN1 (red). (D) The frozen thymic sections were stained for LIN28b (green) and MHCII (red). The top right shows a digitally enlarged image of the area indicated in panels (A-D) . (E) Quantitative analysis of LIN28b + FOXN1 + cells in FOXN1 + TECs. (F) . Gene expression levels of Lin28a, Lin28b , and Let-7g were measured in EpCAM + MHCII - and EpCAM + MCHII lo TECs sorted from E13.5 thymi, as well as in EpCAM + MHCII lo and EpCAM + MHCII hi TECs sorted from E18.5 thymi. The mRNA level of Lin28b in EpCAM + MHCII - TECs at E13.5 was used as a reference with a value of 1, and the mRNA levels of other genes were normalized accordingly and expressed as fold changes. Data are representative of two individual experiments and at least 3-5 samples per time point. Student’s t test (E, F) results between EpCAM + MCHII - or MHCII lo and MHCII hi TEC subsets: * P < 0.05, ** P < 0.01, ****P<0.0001, Scale bar = 0.1 mm. ND, Not detected.

Article Snippet: Primary antibodies included specific polyclonal rabbit anti-LIN28a (A117, Cell Signaling Technology, Beverly, MA; 1:100); specific polyclonal rabbit anti-LIN28b (ProteinTech Group, Inc; Chicago, IL; 1:50) ( , ); goat anti-mouse FOXN1 (WHN G-20, Santa Cruz Biotechnology; Santa Cruz, CA.

Techniques: Immunofluorescence, Staining, Gene Expression

Journal: Frontiers in Immunology

Article Title: Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells

doi: 10.3389/fimmu.2023.1261081

Figure Lengend Snippet: Lin28a , Lin28b and Let-7g are expressed in postnatal murine thymi. Immunofluorescence staining of thymic sections from BL6 WT mice at 2 months of age (A-D) . (A, B) . 4% PFA-fixed thymic sections were stained for LIN28a (A, green) or LIN28b (B, green) with FOXN1 (red). (C, D). The frozen thymic sections were stained for anti-LIN28a (C, green), anti-LIN28b (D, green), and anti-MHCII (red). (E) Quantitative analysis showed the frequency of LIN28a + and LIN28b + cells in total FOXN1 + TECs. (F) Gene expression levels of Lin28a, Lin28b and Let-7g were measured by qPCR in MHCII lo and MHCII hi TEC subsets sorted from 2-month-old murine thymi. The mRNA level of Lin28a in MHCII hi subsets was used as a reference with a value of 1, and the mRNA levels of Lin28b and Let-7g were normalized accordingly and expressed as fold changes. (G-I). Gene expression levels of Lin28a, Lin28b , and Let-7g were measured by qPCR in MHCII lo and MHCII hi TEC subsets sorted from 1-, 2-, 3-, 4-, 8- and 24-week-old thymi. The mRNA levels of Lin28a and Lin28b in MHCII hi TECs and Let-7g in MHCII lo TECs at week 1 were used as references with a value of 1, and the mRNA levels of Lin28a, Lin28b , and Let-7g at other time points were normalized accordingly and expressed as fold changes. Each time point represents at least three to five individuals. (J-L). Immunofluorescence staining, 4% PFA-fixed thymic sections from BL6 white mice at 20 days (J) , 2 months (K) , and 6 months (L) of age were stained for LIN28b (green) and FOXN1 (red) antibodies. The double-positive staining is shown in yellow (purple arrows). One-way ANOVA (F-H) results are shown between MHCII lo and MHCII hi TEC subsets: * P < 0.05, *** P < 0.001, **** P < 0.0001. Bars indicate means ± SEMs. Nd, not detected; Sd, significant difference. Scale bar = 0.1 mm.

Article Snippet: Primary antibodies included specific polyclonal rabbit anti-LIN28a (A117, Cell Signaling Technology, Beverly, MA; 1:100); specific polyclonal rabbit anti-LIN28b (ProteinTech Group, Inc; Chicago, IL; 1:50) ( , ); goat anti-mouse FOXN1 (WHN G-20, Santa Cruz Biotechnology; Santa Cruz, CA.

Techniques: Immunofluorescence, Staining, Gene Expression

Journal: Frontiers in Immunology

Article Title: Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells

doi: 10.3389/fimmu.2023.1261081

Figure Lengend Snippet: Specific overexpression of LIN28a in TECs in iLin28a transgenic adult mice. (A) Gene expression of Lin28a was measured in MHCII lo and MHCII hi TECs sorted from iLin28a +/+ WT (+/+) and iLin28a OE transgenic (OE) mice at 6 weeks of age. Data are representative of two individual experiments (+/+: n= 4; OE: n=5). (B) Flow cytometry intracellular staining analysis. Representative profiles of LIN28a and MHCII staining in total CD45 - cells. (C) The histogram shows LIN28a overlapping expression on gate MHCII + cells, and the summary data of LIN28a percentage are shown on the right. (D-G) Immunofluorescence staining, 4% PFA-fixed postnatal day 21 thymic sections, the thymi of +/+ (D) and OE (E) mice were stained for LIN28a (green), FOXN1 (red) antibodies, and DAPI (blue). The frozen thymic sections of +/+ (F) and OE (G) mice were stained for anti-LIN28a (green) and MHCII (red) antibodies and UEA-1 (purple). (H, I) Gene expression of Lin28b (H) and Let-7g (I) was measured in MHCII lo and MHCII hi TECs sorted from +/+ (H) and OE (I) mice at 6 weeks of age. Data are representative of three individual experiments. (+/+: n=7, OE: =5) Student’s t test (A, C, H, I) results between +/+ and OE mice: * P < 0.05, *** P < 0.001, **** P <0.0001. Bars indicate means ± SEMs. Scale bar = 0.1 mm.

Article Snippet: Primary antibodies included specific polyclonal rabbit anti-LIN28a (A117, Cell Signaling Technology, Beverly, MA; 1:100); specific polyclonal rabbit anti-LIN28b (ProteinTech Group, Inc; Chicago, IL; 1:50) ( , ); goat anti-mouse FOXN1 (WHN G-20, Santa Cruz Biotechnology; Santa Cruz, CA.

Techniques: Over Expression, Transgenic Assay, Gene Expression, Flow Cytometry, Staining, Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells

doi: 10.3389/fimmu.2023.1261081

Figure Lengend Snippet: The gene expression of Lin28b and Let-7g was associated with the Foxn1 expression in the thymus. (A, B). Gene expression of Lin28b (A) and Let-7g (B) in total TECs from +/+, +/Z, Z/Z, and Z/N thymi of 3-week-old mice. Data are representative of three individual experiments, (+/+: n= 3; +/Z: n=5, Z/Z: n=5, Z/N: n=4). (C, D). Gene expression of Lin28b (C) and Let-7g (D) in MHCII lo and MHCII hi TEC subsets and total TECs from +/Z and Z/Z thymi. Data are representative of two individual experiments, (+/Z: n=4, Z/Z: n=5). One-way ANOVA (A, B) results between each colony of mice; Student’s t-test (C, D) results between Z/+ and Z/Z mice: *P <0.05, **P <0.01, ***P <0.001. ns: not significant. Bars indicate means ± SEM.

Article Snippet: Primary antibodies included specific polyclonal rabbit anti-LIN28a (A117, Cell Signaling Technology, Beverly, MA; 1:100); specific polyclonal rabbit anti-LIN28b (ProteinTech Group, Inc; Chicago, IL; 1:50) ( , ); goat anti-mouse FOXN1 (WHN G-20, Santa Cruz Biotechnology; Santa Cruz, CA.

Techniques: Gene Expression, Expressing