Journal: Oncotarget

Article Title: Modulation of estrogen related receptor alpha activity by the kinesin KIF17

doi: 10.18632/oncotarget.18104

Figure Lengend Snippet: A. Sequence of the KIF17-Tail. The 12 amino acid NR box peptide containing the LXXLL motif is underlined. B. Co-immunoprecipitation of GFP-ERR1 with myc-EV control, myc-KIF17-T or myc-KIF17T ΔNR expressed in HEK293 cells. Immunoprecipitates and total lysates were analyzed by immunoblot using anti-GFP and anti-myc IgG. C. , D. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in MCF7 cells expressing ERRE-Luc and myc-EV, myc-KIF17-T, myc-KIF17 ΔNR (panel C) or myc-KIF17 NR (panel D). E. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in parental LM2 and LM2 NR cells transfected with ERRE-Luc. F. Same as in panel E, but comparing cells transfected with control or ERR1 siRNAs. Graphs show normalized luminescence values pooled from ≥ 3 experiments performed in triplicate. Error = SEM. *** p < 0.05.

Article Snippet: Lysates were cleared using Protein-G sepharose beads (GE Healthcare) and incubated overnight with 6-8μg rabbit anti-GFP antibody (Novus Biologicals, NB 600-303) or mouse anti-myc antibody (Sigma M4439).

Techniques: Sequencing, Immunoprecipitation, Control, Western Blot, Luciferase, Activity Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) Schematic overview of ROCK1 construct transfection used to assess entosis induction. (B) Representative immunoblots showing ROCK1, phosphorylated MLC2 (pMLC2), and tubulin (loading control) in MCF7 cells transfected with GFP, GFP–ROCK1, or constitutively active GFP–ROCK1 Δ3. Immunoblot analysis of pMLC2 was performed 6 h post-transfection, whereas ROCK1 expression was assessed at 24 h. (C) Representative 3D confocal images of live MCF7 cells expressing GFP–ROCK1 Δ3 and stained with SiR-Actin (red) and Hoechst (blue). White arrows indicate entotic structures. Boxed regions showing representative entotic structures are magnified, with corresponding orthogonal z-stack views shown alongside each image. (D) Quantification of entotic events in MCF7 cells expressing GFP–ROCK1 or GFP–ROCK1 Δ3. (E) Distribution percentages of GFP-positive cells participating in CIC structures as outer cells, inner cells, or both. (F) Representative time-lapse imaging (phase contrast, Hoechst, and GFP) capturing an entotic event in MCF7 cells expressing GFP–ROCK1 Δ3. Yellow dotted lines outline inner cells, and red dotted lines outline host cells. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Statistical analysis in panel D was performed using a paired two-tailed Student’s t-test (**p < 0.01), whereas panel E was analyzed using two-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Construct, Transfection, Western Blot, Control, Expressing, Staining, Imaging, Two Tailed Test

Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Blimp1 expression in CD4 + effector and Foxp3 + Treg cells under homeostatic conditions. ( A ) Quantitative Real time-PCR (qRT-PCR) analysis Prdm1 ( Blimp1 ) mRNA (relative to β2-microglobulin) (left) and Western blotting (right) of CD4 + Foxp3 GFP + Treg (nTreg) sorted from Ctrl Foxp3 GFP mice or in vitro differentiated Treg (iTreg,), Th1, Th17 or pathogenic (p) Th17 cells differentiated from naïve cells from the same mice (C57BL/6). (N = 3 mice/group, qPCR and N = 2 mice/sample, Western blotting). ( B ) FACS plot shows Prdm1 mRNA expression (as reported by YFP, Blimp1 YFP ) among peripheral Treg (Foxp3 + as determined by intracellular staining of Foxp3 protein), effector (Foxp3 − CD44 high ) and naïve (Foxp3 − CD44 low ) TCRβ + CD4 + cells in mesenteric lymph nodes (MLN, left) and spleen (SP, right). Bar graph shows the average percentage of Blimp1 YFP + cells among TCRβ + CD4 + Foxp3 + , TCRβ + CD4 + Foxp3 − CD44 high or TCRβ + CD4 + Foxp3 − CD44 low cells. ( C ) FACS plots and histograms overlay show percent of Blimp1 YFP + cells in gated TCRβ + CD4 + Foxp3 + cells from thymus (THY), spleen (SP), mesenteric lymph nodes (MLN) and large (LI) intestines lamina propria (LP) from control (open histogram) and Blimp1 YFP (filled histogram) mice. Gating of Foxp3 + cells (as determined by intracellular staining of Foxp3 protein) is shown in FACS plots on the left. Cumulative data from several mice is shown on graph (right). ( D ) FACS histograms show analysis of Blimp1 YFP expression in gated TCRβ + CD4 + Foxp3 + Neuropilin-1 (Nrp-1) + (full line, empty histograms) and TCRβ + CD4 + Foxp3 + Nrp-1 − (dashed line, filled histograms) cells in THY, SP, MLN and LI-LP from Blimp1 YFP mice. Lower panel shows percent of Blimp1 YFP + cells in CD4 + Foxp3 + Nrp-1 + (filled circles) and CD4 + Foxp3 + Nrp-1 − (open circles) cells iin different organs. For all graphs, each symbol represents one mouse. (N = 2–11 mice/group). * P < 0.05 and ** P < 0.01, one-way ANOVA ( B ) and paired t - test ( C ). Error bars indicate SEM.

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, In Vitro, Staining, Control

Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Increased Treg numbers in Blimp1 CKO mice is a cell-intrinsic effect associated with increased proliferation and decreased cell death. ( A ) Expression of Prdm1 (Blimp) mRNA, (qRT-PCR, relative to β2Microglobulin) in sorted Treg (CD4 + Foxp3 GFP+ ) and Teff (CD4 + Foxp3 − CD44 high ) cells from control ( Prdm1 F/F ), Foxp3 CRE CKO ( Prdm1 F/F Foxp3 cre+ ), and CD4 CRE CKO ( Prdm1 F/F CD4 CRE+ ) mice ( B ) Left panel; representative FACS plots of TCRβ + CD4 + Foxp3 + cells (as determined by intracellular staining of Foxp3 protein) in large intestine lamina propria (LI-LP), mesenteric lymph nodes (MLN), spleen (SP) and thymus (THY) from control (Ctrl) or Blimp1 CKO (CD4 cre CKO or Foxp3 cre CKO) mice. Right graphs; percentage of Foxp3 + cells among TCRβ + CD4 + cells (top) and absolute numbers of Foxp3 + cells (bottom) in LI-LP, MLN, SP and THY. Each symbol represents one mouse (N ≥ 4mice/group). Bars indicate average ± SEM. * P < 0.05, ** P < 0.01, one-way ANOVA. ( C ) Top: graphs showing the average percentage ± SEM of Ki-67 + or Zombie (fixable viability dye) + cells among CD4 + Foxp3 + (as determined by intracellular staining of Foxp3 protein) Foxp3 YFP-cre+ or CD4 + Foxp3 + Foxp3 YFP-cre- cells from Foxp3 cre CKO female mice. Each circle represents one mouse. Bottom: representative FACS plots showing analysis of Ki-67 + or Zombie + cells (N = 5 mice). ** P < 0.01, paired t -test. In ( A ) results representative of two different experiments; In ( B ) cell numbers were calculated based on the total number of cells obtained from each organ and population frequency determined by FACS analysis.

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: Expressing, Quantitative RT-PCR, Control, Staining

Journal: Scientific Reports

Article Title: Differential regulation of Effector and Regulatory T cell function by Blimp1

doi: 10.1038/s41598-017-12171-3

Figure Lengend Snippet: Blimp1 regulates unique and commonly shared genetic programs in CD4 + Treg and Teff cells. ( A ) Schematic representation of experimental approach used to obtain allotype-marked Blimp1-sufficient (Ctrl, Thy1.1 + ) and deficient (CKO, Thy1.2 + ) Teff (CD4 + Foxp3 − ) and Treg (CD4 + Foxp 3+ ) cells differentiated in the same environment in vivo . ( B ) Venn diagrams representing the number of deferentially expressed (DE) genes upregulated (top) and downregulated (bottom) in pTreg and Teff cells (CD4 cre CKO versus Ctrl) generated from Ctrl and CD4 cre CKO CD4 + naive T cells co-injected into RAG1 −/− mice and used for mRNA microarray analysis. Three biological replicates were used for each sample. Bar graphs under Venn diagram show fold change for selected genes in each group. ( C ) IPA analysis of DE genes (1.5-fold at P < 0.05): shown are comparison analysis of diseases and bio functions between common DE genes in both Foxp3 GFP + pTreg (pTreg ) and Foxp3 GFP − CD44 high (CD4 + Teff) cells, DE genes in CD4 + Teff cells only and in pTreg cells only (−log 10 p value cut-off; 1.3).

Article Snippet: APC-conjugated goat anti-mouse Neuropilin-1 (catalog SAB566A) was obtained from R&D systems and APC-conjugated anti-CD25 (PC61), PE or APC-conjugated anti-Foxp3 (FJK-16s), eFluor 450- conjugated antiKi-67 (SolA15), PE–conjugated anti-IL10 (JES5-16E3) from eBiosicences, Inc. Rabbit anti-GFP antibody (catalog 600-402-215 Rockland Immunochemicals) was used to stain Blimp1 YFP and Foxp3 GFP reporter cells when intracellular staining for Foxp3 was performed.

Techniques: In Vivo, Generated, Injection, Microarray, Comparison