Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Nest construction score for 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Age-matched Cgas +/+ and Cgas −/− mice were used as control. Mean ± SD; n = 12; **, p < 0.01, ***, p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. b-e , Quantification of the indicated Aβ40 ( b ) and Aβ42 ( c ) in TBS fraction, and Aβ40 ( d ) and Aβ42 ( e ) in guanidine fraction of cortical tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice by ELISA. Mean ± SD; n = 6; n . s ., not significant; *, p < 0.05, **, p < 0.01, Student’s t -test. f , Thioflavin S staining of hippocampal tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 50 μm. g , Quantification of Thioflavin S-labeled amyloid core numbers per mm 2 in f . Mean ± SD; n = 6; ***, p < 0.001, Student’s t -test. h , Immunostaining of Aβ and Iba1 in hippocampal dentate gyrus (DG) of 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 40 μm. i , Quantification of Aβ plaque-associated Iba1 + microglia (indicated by white arrow) in h . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. j , Western immunoblotting analysis of the expression of the indicated proteins involved in cGAS-SITNG pathway of cortical tissues in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. n = 3. k , Quantification of the expression of p-STING), p-TBK1, p-p65, and p-IRF3 relative to β-actin in j . Mean ± SD; n = 3; *, p < 0.05, ***, p < 0.001, Student’s t -test. l , Transcriptional analysis of a panel of A1 astrocyte-inducing, disease-associated microglial markers, and interferon (IFN)-stimulated genes in hippocampal tissues of 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice compared to WT mice. n = 4.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Immunostaining, Western Blot, Expressing

Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Schematic diagram showing primary cultured microglia treated with oligomeric Aβ42 (5 μM) for 24 h, followed by immunoblotting and ELISA analysis. b , Immunoblotting analysis of the expression of the indicated proteins involved in cGAS-SITNG pathway of primary WT microglia 24 h after the Aβ treatment. The non-treatment of the Aβ was used as control. n = 3. c , Quantification of the expression of p-TBK1), p-p65, and p-IRF3 relative to β-actin in b . Mean ± SD; n = 3; n . s ., not significant; *, p < 0.05, Student’s t -test. d-f , ELISA analysis for IL-6 ( d ), TNF-α ( e ) and IL-1α ( f ) in the supernatants of primary Cgas +/+ or Cgas −/− microglia 24 h after Aβ42 treatment. The non-treatment of Aβ42 was used as control. Mean ± SD; n = 4; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. g , Transcriptional analysis for activated astrocytic genes in 6-month-old Cgas +/+ ;5×FAD ( n = 5) and Cgas −/− ;5×FAD ( n = 5) mice. A1-specific: genes activated only by LPS; A2-specific: genes activated only by ischaemia; Pan-reactive: genes activated by either LPS or ischaemia. h , GFAP + astrocytic staining of cortical region in 6-month-old Cgas +/+ ;5×FAD and Cgas −/− ;5×FAD mice. Scale bar, 50 μm. i , Quantification of percentage of GFAP + area in h . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. j , Schematic diagram showing WT, Cgas +/+ , and Cgas −/− primary microglia treated with oligomeric Aβ42 (5 μM) for 24 h, the resulted microglia conditioned medium (WT MCM, Cgas +/+ MCM, and Cgas −/− MCM, respectively) individually added to Cgas +/+ primary astrocytes for inoculation for 24 h, and the concentrated astrocyte conditioned medium (WT ACM, Cgas +/+ ACM, and Cgas −/− ACM, respectively)) individually inoculated with Cgas +/+ primary neurons treated in parallel with oligomeric Aβ (750 nM) for 24 h. Primary neurons were pre-treated with adenovirus-associated virus (AAV) expressing a GFP with CaMKII promotor. k , Fluorescent signals in primary neuron cultures treated with oligomeric Aβ, together with WT, Cgas +/+ , or Cgas −/− ACM. Scale bar, 50 μm. l, m , Percentage of GFP-labeled primary neurons ( l ) and numbers of primary neurons ( m ) in k . Mean ± SD; n = 4; n . s ., not significant; *, p < 0.05, **, p < 0.01, one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Control, Staining, Virus, Labeling

Journal: bioRxiv

Article Title: Activation of innate immune cGAS-STING pathway contributes to Alzheimer’s pathogenesis in 5×FAD mice

doi: 10.1101/2022.10.30.514314

Figure Lengend Snippet: a , Schematic diagram showing Cgas +/+ primary microglia treated with oligomeric Aβ42 (5 μM) for 24 h, together with cGAS inhibitor RU.521 (50 μM) or STING inhibitor H-151 (15 μM), the resulted microglia conditioned medium (MCM) added to Cgas +/+ primary astrocytes for inoculation for 24 h, and the concentrated astrocyte conditioned medium (ACM) inoculated with cGAS +/+ primary neurons treated in parallel with oligomeric Aβ (750 nM) for 24 h. Primary neurons were pre-treated with adenovirus-associated virus (AAV) expression a GFP with CaMKII promotor. b , Fluorescent signals in primary neuron cultures treated with oligomeric Aβ42, together with the RU.521 or H-151 ACM. Scale bar, 50 μm. c, d , Percentage of GFP-labeled primary neurons ( c ) and numbers of primary neurons ( d ) in b . Mean ± SD; n = 4; n . s ., not significant; *, p < 0.05, **, p < 0.01, one-way ANOVA with Bonferroni’s post hoc test. e , Schematic diagram of STING inhibitor H-151 treatment on 3-month-old 5×FAD mice (see Methods). f-i , Quantification of the indicated Aβ40 ( f ) and Aβ42 ( g ) in TBS fraction, and Aβ40 ( h ) and Aβ42 ( i ) in guanidine fraction of cortical tissues in H-151-treated 5-month-old 5×FAD mice by ELISA. Mean ± SD; n = 6; n . s ., not significant; *, p < 0.05, **, p < 0.01, Student’s t -test. j , Thioflavin S staining of DG and cortical tissues in H-151-treated 5-month-old 5×FAD mice. Scale bar, 50 μm. k, l , Quantification of Thioflavin S-labeled amyloid core numbers per mm 2 in DG ( k ) and cortical ( l ) regions in j . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. m, n , Quantification of percentage of Thioflavin S-labeled Aβ plaque area in DG ( m ) and cortical ( n ) regions in j . Mean ± SD; n = 6; **, p < 0.01, Student’s t -test. o , Immunostaining of Aβ (6E10), Iba1 + microglial and GFAP + astrocytes of DG region in H-151-treated 5-month-old 5×FAD mice. Scale bar, 50 μm. p-r , Quantification of Aβ plaque numbers per mm 2 ( p ) and percentage of Iba1 + ( q ) and GFAP + ( r ) area in o . Mean ± SD; n = 6; **, p < 0.01, ***, p < 0.001, Student’s t -test.

Article Snippet: The secreted cytokines from primary neural cells after oligomeric Aβ42 treatment were quantified by mouse cytokine ELISA kits, including mouse IL-6 ELISA kit (E-EL-M0044c, Elabscience), mouse TNF-α ELISA kit (E-MSEL-M0002, Elabscience), mouse IL-1α ELISA kit (E-EL-M3059, Elabscience), and moue IFN-β ELISA kit (42410-1, PBL Assay Science) accordingly to the manufacture’s instruction.

Techniques: Virus, Expressing, Labeling, Enzyme-linked Immunosorbent Assay, Staining, Immunostaining

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet: TDP-43 Regulates APA of Genes Important for Pluripotency, Including Sox2 (A) Scatterplot displaying relative changes of pA sites upon differentiation of hESCs ( A and S3B) and changes accruing by knocking down TDP-43 in undifferentiated hESCs for 48 h (n = 4 control short hairpin [shCTRL] and n = 8 sh TDP-43 using 2 TDP-43 -targeting short hairpin RNAs [shRNAs] with 4 replicates for each; adjusted p < 0.05, Fisher’s exact test). Linear regression (gray line) and the 90% confidence interval region (light blue) are shown (Pearson’s correlation coefficient [r] = 0.62). Increased use of the proximal pA site has positive values, and decreased use has negative values, based on genes passing filtering and statistical analysis as outlined in C–S3E. (B and C) Non-redundant Gene Ontology (GO) terms (B) and ground-state and general pluripotency factors (C), characterized by ( Kalkan et al., 2017 ), of genes exhibiting APA upon cTdp-43 KO in mESCs (n = 10 independent sample replicates per group; genes passing filtering and statistical analysis as outlined in Figure S3 C–S3E). (D) Representative diagrams displaying the transcript isoforms of Sox2 upon cTdp-43 KO in mESCs and frequencies of the pA sites in Sox2 and SOX2 upon cTdp-43 KO in mESCs or KD of TDP-43 and 72 h CHIR99021 treatment of hESCs; samples as above. (E) The positions of TDP-43 crosslinking (red bars) in Sox2 transcript from mESCs, as defined by iCLIP, surrounding the proximal and distal pA sites; n = 2 biological replicates ( Figure S5 C) combined into a single track. (F) Western blot analysis of SOX2, TDP-43, and histone H3 following tamoxifen treatment (3 days) of cTdp-43 KO mESCs; independent replicates are shown. (G) An illustration of the EGFP - SOX2 3′ UTR reporter minigene displaying endogenous positions of the proximal and distal pA sites and the miR-21 binding site. (H–J) Representative flow cytometry analyses of HEK293T cells with the doxycycline-inducible TDP-43 gene cassette co-transfected with miR-21 (transfected cells were gated using tdTomato as described in ), and the respective EGFP-SOX2 3′ UTR constructs: unmodified (H), harboring deletion of the proximal pA site (I), or with deletion of the miR-21 binding site (J). Control (CTR) indicates cells that were not treated with doxycycline. (K) Boxplot depicting relative levels of miR-21 in undifferentiated mESCs and in primitive streak (PS)-like progenitors generated by 3 d CHIR99021 treatment (n = 6 independent replicates, two sided t test; p value ∗∗∗ ≤ 0.001). (L) Boxplot analyses depicting levels of the Sox2 transcript in WT mESCs and cells lacking the endogenous miR-21 binding site in Sox2 ( Sox2ΔmiR21 ) under pluripotency conditions (top) and upon PS differentiation (bottom) (n = 5 and 6 independent replicates, two-sided t test; ∗ p ≤ 0.01).

Article Snippet: Sox2 monoclonal Antibody , Cell Signaling , Cat no. 2748S; RRID: 823640.

Techniques: Control, Western Blot, Binding Assay, Flow Cytometry, Transfection, Construct, Generated

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet: Efficient Dissolution of Pluripotency upon Depletion of TDP-43 Requires Paraspeckles (A) Representative photomicrographs demonstrating the downregulation of Neat1_2 paraspeckles in spontaneously differentiating mESCs by deletion of the triple helix ( ΔTH ) in the 3′ region (further results in Figure S6 A). Red, Neat1_1 and _2 probes; blue, DAPI (nuclear stain). Scale bars, 10 μm. (B and C) Pluripotency assessment by SSEA-1 (B, right, quantified gated positive cells) and intracellular NANOG (C) flow cytometry of spontaneously differentiating Neat1ΔTH and WT mESCs (duration indicated). IgG-treated samples were used for gating positive cells (red line in B). Error bars, SD; two-sided t test; biological replicates, n = 3 per time point; ∗ p < 0.05, ∗∗ p < 0.01. (D–F) In vivo analysis of the developmental potency of mESCs exhibiting downregulation of paraspeckles using a 2n mESC - 4n aggregated mouse embryo complementation assay, giving rise, respectively and exclusively, to embryonic and extraembryonic tissues (D). Shown are mouse embryos (E7.75–E8.0) resulting from aggregations of Neat1ΔTH and Neat1ΔpA mESCs with 4n 2- to 4-cell-stage embryos and representative analysis of FOXA2 (E) and BRACHYURY (F) by immunostaining (n = 7 for Neat1ΔTH [5 are shown in B and S6C], n = 7 for Neat1ΔpA [2 are shown in Figure S6 D], and n = 3 for parental control mESC [2 are shown in Figure S6 E]). Non-manipulated embryos are shown on the right. Blue, DAPI (nuclear stain). Scale bars, 100 μm. A, anterior; P, posterior; p, proximal; d, distal. (G–L) Pluripotency assessment by intracellular immunostaining flow cytometry (G–J; error bars, SD, two-sided t test in I, n = 3), RNA-seq (K; n = 3/group, Fisher’s exact test), and growth kinetics (L; Mann-Whitney U test, n ≥ 8/group as indicated by dots) in differentiating parental cTdp-43 KO mESCs or the same line harboring Neat1ΔTH , treated with tamoxifen during 2.5 days of spontaneous differentiation or left untreated (all consisting of independent replicates). Also shown are representative flow cytometry plots of SOX2 (G) and quantification of gated positive cells according to the IgG control (H) and of NANOG (I and J). IgG-treated samples were used to gate the positive cells (dotted red line) and to quantify the enrichment of these cells. In (K), shown are up- and downregulated representative differentiation and pluripotency genes according to the ScoreCard panel ( Tsankov et al., 2015 , and Kalkan et al., 2017 , respectively), comparing the impact of Tdp-43 KO in mESCs lacking Neat1_2 ( Neat1ΔTH ) with control mESCs harboring WT Neat1 . In (L), the width of colored intervals represents the interquartile range of the growth kinetics measurements. (M) Histogram depicting rearrangements (direction and degree) of pA sites following cTdp-43 KO in Neat1ΔTH mESCs compared with the respective parental WT cells (genes passing filtering and statistical analysis as outlined in C–S3E; APA in the range of ±5-fold change, p < 0.01). mESCs were treated with tamoxifen for 2.5 days or left untreated during spontaneous differentiation (n = 3 independent replicates/group).

Article Snippet: Sox2 monoclonal Antibody , Cell Signaling , Cat no. 2748S; RRID: 823640.

Techniques: Dissolution, Staining, Flow Cytometry, In Vivo, Immunostaining, Control, RNA Sequencing, MANN-WHITNEY

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet:

Article Snippet: Sox2 monoclonal Antibody , Cell Signaling , Cat no. 2748S; RRID: 823640.

Techniques: Recombinant, Produced, Gentle, Knock-Out, Reverse Transcription, TaqMan Assay, Labeling, Staining, Flow Cytometry, Western Blot, Clarification Assay, Membrane, Sample Prep, Sequencing, Derivative Assay, Next-Generation Sequencing, Gene Expression, Fractionation, Activation Assay, Nucleic Acid Electrophoresis, CRISPR, Plasmid Preparation, Modification, shRNA, Software

Journal: bioRxiv

Article Title: The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

doi: 10.1101/2024.12.02.626379

Figure Lengend Snippet: A, Left, purified recombinant HA-GluA1 ATD and HA-GluA2 ATD produced in 293T cells, then used as bait in pull-downs. Center, schematic of pull-down assay after incubation of recombinant ATDs with whole mouse brain lysates. Right, silver stain of proteins eluted after pull-down and PAGE-SDS. B, Subcellular localization of specific and unique GluA1 ATD and GluA2 ATD interactors. C, Partial list of GluA1 ATD (left column, blue) and GluA2 ATD (right column, yellow)-interacting proteins in the mouse brain identified in proteomic screen. D, co-IP analysis of the interaction between recombinant α2δ1 and GluA1 (n≥3). E, co-IP analysis of the interaction between recombinant α2δ1 and GluA2 (n≥3). F, co-IP analysis of the interaction between α2δ1 and HA-GluA1 ATD (n≥3). G, GluA1 / α2δ1 interaction in mouse brain homogenates after pull-down with GluA1 CTD antibody. GluA2 is used as positive control (n=3). H, Left, Molecular model of the ATD of AMPAR (pdb: 6njl) docked to α2δ1 (pdb: 7vfv) in complex with the VGCC allowing a trans interaction obtained in ClusPro docking server. GluA1 subunits are show in light blue, GluA2 subunits are shown in yellow. α2δ1 is shown in dark green, the rest of the VGCC complex in light green. Right, the region boxed in the H is depicted at higher magnification, highlighting some of the residues involved in the interaction between GluA1 ATD and α2δ1. Potential intramolecular H-bonds between the selected residues are indicated in magenta. Rendering of the molecular complexes was performed in ChimeraX.

Article Snippet: After blocking tissue with 5% swine serum (Jackson Immuno Research, # 014-000-121) and 2% BSA (Cell Signaling, #9998S) in permeabilizing conditions (0.1% Triton X-100, Sigma-Aldrich, # T8787), samples were incubated overnight at 4° C with the following primary antibodies: GluA1 (rabbit, Cell signaling, #13185, 1:500 dilution), PSD-95 (mouse, Synaptic Systems, #124 011, 1:500), and VGLUT1 (guinea pig, Synaptic Systems, #135 304, 1:500) followed by incubation with Alexa 488 goat anti-mouse (Life Technologies, #A-11001, 1:500), Alexa 647 goat anti-rabbit (Life Technologies, #A21245, 1:500) and Alexa 568 goat anti-guinea pig (Life Technologies, #A11075, 1:500) secondary antibodies for 1.5 hour at RT.

Techniques: Purification, Recombinant, Produced, Pull Down Assay, Incubation, Silver Staining, Co-Immunoprecipitation Assay, Positive Control

Journal: bioRxiv

Article Title: The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

doi: 10.1101/2024.12.02.626379

Figure Lengend Snippet: A, Schematic of the presynaptic α2δ1 deletion at CA3➔CA1 synapses, comparing α2δ1 f/f (left) with α2δ1 ΔCA3 (right) mice. B, α2δ1 mRNA ISH in the hippocampus of α2δ1 f/f (top) and α2δ1 ΔCA3 (bottom) mice, showing low magnification (left); CA3 (center) and CA1 (right) photomicrographs. Asterisks identify putative interneurons preserving α2δ1 expression in field CA3 in α2δ1 ΔCA3 mice. C, Representative immunostaining of GluA1 (red) and PSD-95 (green) and VGLUT1 (blue) in hippocampal field CA1 in α2δ1 f/f (top) and α2δ1 ΔCA3 mice (bottom) samples. Scale bar, 25 µm (10 µm insets). D, Representative Structured Illumination Microscopy (SIM) images of GluA1 (red), PSD-95 (green) and VGLUT1 (blue) in hippocampal area CA1 SR in α2δ1 f/f (left) and α2δ1 ΔCA3 (right) samples. Scale bar, 1 µm. E, F, average density of VGLUT1 and PSD-95 positive puncta, respectively, in CA1 SR. G, Proportion of PSD-95 colocalizing with VGLUT1. H, average density of GluA1-positive puncta. I, J, Proportion of GluA1 colocalizing with VGLUT1 and PSD-95, respectively. K, Representative mEPSC traces for α2δ1 f/f (top) and α2δ1 ΔCA3 (bottom) CA1 PNs. L,M, mEPSC amplitude and frequency, respectively, in α2δ1 f/f and α2δ1 ΔCA3 PNs. N, Representative individual mEPSC traces. O, P, mEPSC 10-90% rise time and decay tau, respectively, in α2δ1 f/f and α2δ1 ΔCA3 CA1 PNs. Q, Schematic of the preparation used for evoked EPSC recordings in R-T. R, S, Paired-pulse ratios (PPR) and AMPAR/NMDAR EPSC ratios in α2δ1 f/f and α2δ1 ΔCA3 CA1 PNs, respectively. T, AMPAR EPSC normalized to the mean AMPAR EPSC amplitude before LTP induction (arrow). AMPAR EPSC current traces from α2δ1 f/f (black) and α2δ1 ΔCA3 (teal) neurons shown to the right of R-T. n=3-8 mice/genotype (C-J), n=5-14 cells/genotype (K-T). Scale bars: 5 pA, 200 ms (K), 2 pA, 50 ms (N), 50 pA, 50 ms (R-T). *, p≤0.05; n.s., not statistically significant, unpaired t-test (E-J), Mann-Whitney U test (L-T). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Article Snippet: After blocking tissue with 5% swine serum (Jackson Immuno Research, # 014-000-121) and 2% BSA (Cell Signaling, #9998S) in permeabilizing conditions (0.1% Triton X-100, Sigma-Aldrich, # T8787), samples were incubated overnight at 4° C with the following primary antibodies: GluA1 (rabbit, Cell signaling, #13185, 1:500 dilution), PSD-95 (mouse, Synaptic Systems, #124 011, 1:500), and VGLUT1 (guinea pig, Synaptic Systems, #135 304, 1:500) followed by incubation with Alexa 488 goat anti-mouse (Life Technologies, #A-11001, 1:500), Alexa 647 goat anti-rabbit (Life Technologies, #A21245, 1:500) and Alexa 568 goat anti-guinea pig (Life Technologies, #A11075, 1:500) secondary antibodies for 1.5 hour at RT.

Techniques: Preserving, Expressing, Immunostaining, Microscopy, MANN-WHITNEY

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet: TDP-43 Maintains Pluripotency and Enhances Somatic Cell Reprogramming (A) Volcano plot displaying gene expression fold changes and their respective statistical score (adjusted p value, Fisher’s exact test), comparing untreated and tamoxifen-treated cTdp-43 KO undifferentiated mESCs (n = 10 biological replicates per condition). Known pluripotency and trophectoderm markers are labeled red and blue, respectively (marked factors fulfilled p < 0.01). (B) Representative photomicrographs of untreated cTdp-43 KO mESCs, following tamoxifen treatment in 2iLIF medium and following 2-day spontaneous differentiation. (C and D) Representative flow cytometry analyses of spontaneously differentiating (2 days) iTDP-43-EGFP mESCs that were treated with doxycycline (overexpression [OE]) or left untreated and immunostained for SSEA-1 (C) and NANOG (D). Non-treated cells were used for immunoglobulin G (IgG) control staining. The mean of three independent experiments is depicted on the right (error bars, SD); two-sided t test, ∗∗∗ p < 0.001. (E–I) Reprogramming of hiF-T secondary fibroblasts with or without (empty vector) transduction of TDP-43, cells harboring doxycycline-inducible reprogramming factors ( Cacchiarelli et al., 2015 ) (E), number of alkaline phosphatase (AP)-positive human induced pluripotent stem cell (hiPSC) colonies emerging on day 10 and 14 of reprogramming (F and G; dots represent replicates from three independent experiments, Mann-Whitney U test, ∗∗∗ p < 0.001), representative photomicrographs (H), and TRA-1-60 flow cytometry analysis (I); n = 3 independent experiments, gating was based on an IgG control. (J and K) Reprogramming of primary human fibroblasts (J) and the number of AP-positive colonies on day 30 (dots represent replicates from three independent experiments) with or without (empty vector) transfection of TDP-43 (K); statistical analysis as in (F) and (G).

Article Snippet: Nanog monoclonal Antibody , Cell Signaling , Cat no. 8822; RRID: AB_11217637.

Techniques: Gene Expression, Labeling, Flow Cytometry, Over Expression, Control, Staining, Plasmid Preparation, Transduction, MANN-WHITNEY, Transfection

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet: Efficient Dissolution of Pluripotency upon Depletion of TDP-43 Requires Paraspeckles (A) Representative photomicrographs demonstrating the downregulation of Neat1_2 paraspeckles in spontaneously differentiating mESCs by deletion of the triple helix ( ΔTH ) in the 3′ region (further results in Figure S6 A). Red, Neat1_1 and _2 probes; blue, DAPI (nuclear stain). Scale bars, 10 μm. (B and C) Pluripotency assessment by SSEA-1 (B, right, quantified gated positive cells) and intracellular NANOG (C) flow cytometry of spontaneously differentiating Neat1ΔTH and WT mESCs (duration indicated). IgG-treated samples were used for gating positive cells (red line in B). Error bars, SD; two-sided t test; biological replicates, n = 3 per time point; ∗ p < 0.05, ∗∗ p < 0.01. (D–F) In vivo analysis of the developmental potency of mESCs exhibiting downregulation of paraspeckles using a 2n mESC - 4n aggregated mouse embryo complementation assay, giving rise, respectively and exclusively, to embryonic and extraembryonic tissues (D). Shown are mouse embryos (E7.75–E8.0) resulting from aggregations of Neat1ΔTH and Neat1ΔpA mESCs with 4n 2- to 4-cell-stage embryos and representative analysis of FOXA2 (E) and BRACHYURY (F) by immunostaining (n = 7 for Neat1ΔTH [5 are shown in B and S6C], n = 7 for Neat1ΔpA [2 are shown in Figure S6 D], and n = 3 for parental control mESC [2 are shown in Figure S6 E]). Non-manipulated embryos are shown on the right. Blue, DAPI (nuclear stain). Scale bars, 100 μm. A, anterior; P, posterior; p, proximal; d, distal. (G–L) Pluripotency assessment by intracellular immunostaining flow cytometry (G–J; error bars, SD, two-sided t test in I, n = 3), RNA-seq (K; n = 3/group, Fisher’s exact test), and growth kinetics (L; Mann-Whitney U test, n ≥ 8/group as indicated by dots) in differentiating parental cTdp-43 KO mESCs or the same line harboring Neat1ΔTH , treated with tamoxifen during 2.5 days of spontaneous differentiation or left untreated (all consisting of independent replicates). Also shown are representative flow cytometry plots of SOX2 (G) and quantification of gated positive cells according to the IgG control (H) and of NANOG (I and J). IgG-treated samples were used to gate the positive cells (dotted red line) and to quantify the enrichment of these cells. In (K), shown are up- and downregulated representative differentiation and pluripotency genes according to the ScoreCard panel ( Tsankov et al., 2015 , and Kalkan et al., 2017 , respectively), comparing the impact of Tdp-43 KO in mESCs lacking Neat1_2 ( Neat1ΔTH ) with control mESCs harboring WT Neat1 . In (L), the width of colored intervals represents the interquartile range of the growth kinetics measurements. (M) Histogram depicting rearrangements (direction and degree) of pA sites following cTdp-43 KO in Neat1ΔTH mESCs compared with the respective parental WT cells (genes passing filtering and statistical analysis as outlined in C–S3E; APA in the range of ±5-fold change, p < 0.01). mESCs were treated with tamoxifen for 2.5 days or left untreated during spontaneous differentiation (n = 3 independent replicates/group).

Article Snippet: Nanog monoclonal Antibody , Cell Signaling , Cat no. 8822; RRID: AB_11217637.

Techniques: Dissolution, Staining, Flow Cytometry, In Vivo, Immunostaining, Control, RNA Sequencing, MANN-WHITNEY

Journal: Molecular Cell

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

doi: 10.1016/j.molcel.2019.03.041

Figure Lengend Snippet:

Article Snippet: Nanog monoclonal Antibody , Cell Signaling , Cat no. 8822; RRID: AB_11217637.

Techniques: Recombinant, Produced, Gentle, Knock-Out, Reverse Transcription, TaqMan Assay, Labeling, Staining, Flow Cytometry, Western Blot, Clarification Assay, Membrane, Sample Prep, Sequencing, Derivative Assay, Next-Generation Sequencing, Gene Expression, Fractionation, Activation Assay, Nucleic Acid Electrophoresis, CRISPR, Plasmid Preparation, Modification, shRNA, Software

Journal: Nature Cell Biology

Article Title: Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

doi: 10.1038/s41556-024-01387-x

Figure Lengend Snippet: a , Schematic of the pulsed SILAC experiment to evaluate protein synthesis. Pulse treatment for 24 h allowed for metabolic labelling of newly translated proteins. Protein synthesis was determined by the heavy to unlabelled ratio quantified by MS, as described in refs. , . Two independent experiments were performed. b , Pulsed SILAC was performed and protein extracts from control or PPIA knockdown (Kd) cell lines were analysed to measure newly synthetized proteins. The heatmap represents the relative degree of protein synthesis ( n = 345 overlapping proteins compared between the different cell types). c , Uptake of heavy amino acids by control or PPIA Kd 293T cells was quantified following a pulsed SILAC experiment. A value of 0.5 indicates the equal presence of light and heavy labelled peptides. * P < 0.05, ** P < 0.01 and **** P < 0.0001; two-sided Wilcoxon rank-sum test (comparing 160 PPIA target proteins to 1,280 non-targets; similar findings were observed for HeLa cells). d , List of PPIA client proteins involved in protein phase separation based on PhaSepDB2.0. Proteins are listed by their official gene name. Nuclear bodies include nucleoli, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukemia (PML) nuclear bodies and histone locus bodies. e , Immunoprecipitation of endogenous PPIA protein in HeLa cells followed by a western blot to detect the PPIA protein partners (poly(A)-binding protein 1 (PABPC1), DEAD-box helicase 6 (DDX6), Ras GTPase-activating protein-binding protein 1 (G3BP1) and nucleophosmin 1 (NPM1)). Data are representative of two independent experiments, confirmed by unbiased MS. f , PPIA activity is required for substrate binding. PPIA wild type, but not the G104A mutant, binds to PABPC1, DDX6 and NPM1 in IP–western experiments in HeLa cells. Data are representative of two independent experiments, confirmed by unbiased MS. g , Reduced expression of PPIA substrates following knockdown of the chaperone. HeLa cells were stably transduced with negative control or PPIA knockdown construct Kd1. Shown are representative immunoblots of n = 3 independent biological replicates. Right: a pairwise comparison shows significant reduction of PABPC1, DDX6 and NPM1 in PPIA knockdown cells by densitometry. GAPDH expression was used as a reference. Data are means ± s.d.; * P < 0.05, ** P < 0.01; two-sided paired Student’s t -test. DMEM, Dulbecco’s modified Eagle medium.

Article Snippet: Control and PPIA Kd1 Hela cells were transfected with pcDNA3.1-PPIA vector or corresponding empty pcDNA3.1 control vector for 48 h. Following stress induction with sodium arsenite (50 μM, Sigma-Aldrich) for 1 h, immunostaining for G3BP1 protein, a marker of stress granule assembly, was performed using a rabbit polyclonal anti-G3BP1 antibody (cat. no. 13057-2-AP, Proteintech).

Techniques: Multiplex sample analysis, Metabolic Labelling, Control, Knockdown, Immunoprecipitation, Western Blot, Binding Assay, Protein Binding, Activity Assay, Mutagenesis, Expressing, Stable Transfection, Transduction, Negative Control, Construct, Comparison, Modification

Journal: Nature Cell Biology

Article Title: Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

doi: 10.1038/s41556-024-01387-x

Figure Lengend Snippet: a , Impaired translation in haematopoietic stem and progenitor cells following pharmacological PPIA inhibition. Haematopoietic stem cells (lin − /cKit + /Sca1 + /CD34 − /CD135 − ) were isolated and expanded in vitro as previously published . Translation rates were measured through bio-orthogonal labeling with a fluorescently labeled puromycin analog for two hours in cells that were pre-treated with DMSO or PPIA inhibitor TMN355 (10 μM, 24 h). Shown is a representative of three independent biological replicates, in which n = 1,880 (DMSO) and n = 1,303 (TMN355) cells were analysed and fluorescence was measured per cell. Scale bar, 100 μm (**** P < 0.0001, two-sided Wilcoxon rank-sum test). Bottom panel depicts DAPI counterstain. b , Reduced expression of PPIA substrates in OCI-AML3 cells following PPIA knockdown. Western blot analyses to detect protein expression of PPIA and PPIA protein partners PABPC1, DDX6, and NPM1 in the OCI-AML3 cell line. GAPDH was used as a loading control for protein normalization and densitometry was measured relative to GAPDH expression. The images represent results of two independent experiments. c , Protein expression of PPIA client proteins is decreased in Ppia −/− HSPCs. Western blot analyses to detect protein expression of PPIA and PPIA protein partners PABPC1, DDX6, G3BP1, and NPM1 in mouse lineage-depleted bone marrow cells. The images represent results of Ppia +/− and Ppia −/− animals ( n = 1 each). β-tubulin was used as a loading control for protein normalization. This experiment was performed once. d , Ppia knockout versus heterozygous haematopoietic stem and progenitor cells (lin − /c-Kit + ) upregulate genes involved in translation. Volcano plot shows comparable up- and down − regulation of genes. Gene set enrichment analysis of haematopoietic stem and progenitor cells of Ppia knockout or heterozygous animals. N = 3 independent animals were analysed per group. Genes encoding the entire mouse chaperome or the ubiquitin-proteasome system (UPS) were not significantly increased in knockout cells. However, the gene ontology ‘cytoplasmic translation’ was significantly upregulated in knockout cells. e , Ppia knockout cells show the transcriptional signature of aging. Haematopoietic stem and progenitor cells (lin − /cKit + ) of three Ppia knockout animals compared to three heterozygous animals show significant upregulation of the aging marker gene P-selectin . In addition, we observed a strong gene set enrichment resembling aged haematopoietic stem cells . Statistics derived using two-sided Wilcoxon rank-sum test ( n = 3 mice per group). f , Genes encoding PPIA substrates are upregulated in Ppia knockout haematopoietic stem and progenitor cells. Relative FPKM changes shown in cumulative violin plots representing three animals per genotype ( Ppia knockout versus heterozygous); y-axis represented in log2. Gene set enrichment analysis of haematopoietic stem and progenitor cells (lin − /c-Kit + ) of Ppia knockout or heterozygous animals shows significant upregulation of PPIA substrates ( n = 307) compared to the overall proteome ( n = 6,114) in knockout animals. Statistics derived using two-sided Wilcoxon rank-sum test. g , No increased spontaneous aggregation of misfolded proteins in absence of PPIA. Left panel: Protein misfolding was quantified using the molecular rotor ProteoStat with affinity for aggregated proteins. Increased protein aggregation causes the dye to stop spinning and emit fluorescence. We analysed misfolding in HeLa cells transduced with either scramble lentivirus or following PPIA knockdown, and used proteasome inhibition as positive control (MG132, 10 μM, 16 h). Scale bar, 80 μm. Right panel: Relative mean intensity per cell was plotted and calculated after blinding. A total of 8 independently treated replicates were analysed per group in this assessment. Statistics calculated using two-sided Wilcoxon rank-sum test ( n = 154 control cells, n = 147 PPIA knockdown cells for, n = 75 for MG132-treated control cells, and n = 86 for MG132-treated PPIA knockdown cells; the experiment was performed once).

Article Snippet: Control and PPIA Kd1 Hela cells were transfected with pcDNA3.1-PPIA vector or corresponding empty pcDNA3.1 control vector for 48 h. Following stress induction with sodium arsenite (50 μM, Sigma-Aldrich) for 1 h, immunostaining for G3BP1 protein, a marker of stress granule assembly, was performed using a rabbit polyclonal anti-G3BP1 antibody (cat. no. 13057-2-AP, Proteintech).

Techniques: Inhibition, Isolation, In Vitro, Labeling, Fluorescence, Expressing, Knockdown, Western Blot, Control, Knock-Out, Ubiquitin Proteomics, Marker, Derivative Assay, Transduction, Positive Control

Journal: Nature Cell Biology

Article Title: Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

doi: 10.1038/s41556-024-01387-x

Figure Lengend Snippet: a , Stress-granule formation was visualized and quantified with G3BP1 staining after stress induction with sodium arsenite in HeLa control or PPIA Kd cells. DAPI, blue; G3BP1, green. Scale bars, 50 µm. PPIA knockdown was partially rescued by the reintroduction of knockdown-resistant PPIA . Cell viability was measured on an automated cell counter with acridine orange/propidium iodide staining solution using n = 6 independently treated replicates per group. Data are means ± s.d.; ** P < 0.01, **** P < 0.0001; two-sided Wilcoxon rank-sum test; n = 616 (control), n = 656 (control + PPIA ), n = 254 ( PPIA knockdown) and n = 293 ( PPIA knockdown + PPIA ) cells were analysed following blinding. Data are representative of three independent experiments. b , Staining for DDX6 revealed significantly fewer P-bodies in HeLa cells following PPIA knockdown. Scale bars, 20 μm. The arrowhead indicates a representative P-body. **** P < 0.0001; two-sided Wilcoxon rank-sum test; n = 457 (control) and n = 284 ( PPIA knockdown) cells were analysed following blinding. Data are representative of three independent experiments. c , OCI-AML3 cells that express a stable, gene-edited NPM1-mCherry fusion, exhibited smaller, more fragmented nucleoli following PPIA knockdown. Cell designation was blinded for the analyst. Scale bars, 20 μm. *** P < 0.001, **** P < 0.0001; two-sided Wilcoxon rank-sum test; n = 564 (control) and n = 617 ( PPIA knockdown) cells were analysed following blinding. Data are representative of three independent experiments. d , PLAs in primary haematopoietic stem cells (lin − /c-Kit + /Sca1 + /CD34 − /CD135 − ) between PPIA and PABPC1, DDX6 and NPM1. Shown is the signal quantification using single-antibody staining as a control. Scale bar, 5 μm. **** P < 0.0001; two-sided Wilcoxon rank-sum test. Cells were analysed following blinding. Box plots indicate minima, maxima and quartiles; n = 20 randomly chosen cells per group. Data are representative of two independent experiments. .

Article Snippet: Control and PPIA Kd1 Hela cells were transfected with pcDNA3.1-PPIA vector or corresponding empty pcDNA3.1 control vector for 48 h. Following stress induction with sodium arsenite (50 μM, Sigma-Aldrich) for 1 h, immunostaining for G3BP1 protein, a marker of stress granule assembly, was performed using a rabbit polyclonal anti-G3BP1 antibody (cat. no. 13057-2-AP, Proteintech).

Techniques: Staining, Control, Knockdown

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 1. CDH1 was expressed in a small subset of cells in the seminiferous tubules of mouse testes. a) Immunohistochemical analyses of seminiferous tubules in mature mouse testes showed that CDH1 was expressed in a small fraction of germ cells (arrows). The section was counterstained with hematoxylin. Bar ¼ 20 lm. b) Double staining with CDH1 (green) and GATA4 (red). GATA4 was expressed in the nuclei of Sertoli cells and Leydig cells. CDH1-posi- tive cells (arrows) were located on the basement membrane in seminiferous tu- bules and were negative for GATA4. The strong green fluorescence shown in the cytoplasm of Leydig cells was autofluores- cence. Bar ¼ 20 lm. c) Western blot analysis of CDH1 (arrow, about 124 kDa) from mouse testes. Lysate from testes (lane 1) and the COS7 cells transfected with the CDH1 cDNA expression vector (lane 2) and control COS7 (lane 3) were electropho- resed. Left panel shows the Western blot with the monoclonal antibody (ECCD-2), and right panel shows the Western blot with the polyclonal antibody. The extra band of about 90 kDa shown in lane 3 of the right panel was probably the degradation product of ectopically expressed CDH1. d) Double staining of CDH1 with polyclonal antibody (left), monoclonal antibody (middle), and merged images (right). e) CDH1-positive cells had oval-shaped nuclei displaying no heterochromatin with DAPI staining (mid- dle, arrowhead). f–h) Double staining of CDH1 (left) and ITGA6 (f, middle), ITGB1 (g, middle), or TACSTD1 (h, middle) and merged images (right). CDH1-positive cells also were positive for ITGA6, ITGB1, and TACSTD1. Bar ¼ 10 lm.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Immunohistochemical staining, Double Staining, Membrane, Fluorescence, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Staining

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 2. CDH1 was expressed continuously in seminiferous tubules of the mouse testis from the postnatal stage to old age: P1 (a), P4 (b), 1 wk (c), 2 wk (d), 4 wk (e), 8 wk (f), and 480 days (g) of age, respectively. h–i) CDH1 expression was observed in all spermatogonia of both KitlSl/KitlSl-d (h) and KitW/KitW-v (i) mice. Bar ¼ 50 lm. A higher magnification of the cells indicated by the arrows is shown in each inset. Bar ¼ 10 lm.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Expressing

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 3. CDH1 was expressed in undiffer- entiated type A spermatogonia. a) Whole- mount immunohistochemistry revealed the cell shape and the topographical arrange- ment of CDH1-positive cells. b) A higher magnification of some clusters comprising one, two, or eight cells outlined in a CDH1- positive spermatogonia were connected to each other via intercellular bridges in the cluster, whereas some cells adhered to neighboring cells via extensive cell bound- aries where CDH1 staining was concen- trated (arrowheads). c) Paired cells extending long filopodia resembling leading processes. d) Paired cells connected with a long, fine cellular process. e) Frequency of the clusters with different numbers of CDH1-positive cells. The vertical axis shows the number of colonies of different sizes. The horizontal axis shows the number of cells comprising each cluster. Bars ¼ 20 lm.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Immunohistochemistry, Staining

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 4. Whole-mount double immuno- staining of CDH1 and KIT in long seminif- erous tubules. a) Panoramic view of a CDH1-stained seminiferous tubule. Roman numerals show the stages of spermatogenic epithelia. Bar ¼ 5 mm. b–d) Higher magnification of panel a indicated by arrows with each character. Left panels show KIT staining, middle panels show CDH1 staining, and right panels show merged images of the left and middle panels. b) KIT staining showed IN sper- matogonia (larger cells in the left half of panel) and type B spermatogonia (smaller cells in the right half of panel). No Aal cells were stained with anti-KIT antibody. c) KIT staining showed preleptotene spermato- cytes (smaller cells) and differentiating Aal spermatogonia (larger cells). Many Aal cells were stained with the anti-KIT antibody at this stage. Note that some cells expressed CDH1 only (arrowheads). d) KIT staining showed A1 spermatogonia. Most A1 cells lost CDH1 expression, whereas some A1 cells retained a weak CDH1 expression (arrowheads). e–g) Double staining of CDH1 and KIT (e–f) or CDH1 and TACSTD1 (g) showed heterogeneity of KIT or TACSTD1 expression in a cluster. e) Two of four cells in an Aal cluster were KIT negative (arrowheads), whereas the other two cells were KIT positive (arrows). f) One cell of an Apr cluster was KIT negative (arrowhead), and the other was KIT positive (arrow). g) Only two cells (arrow and arrowhead) showed strong CDH1 expression in a TACSTD1-positive cluster. One showed strong TACSTD1 expression (arrow), whereas the other showed weak TACSTD1 expression (arrowhead). Bars ¼ 20 lm.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Immunostaining, Staining, Expressing, Double Staining

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 5. Stem cell markers were expressed in a subset of CDH1-positive cells. a) Double staining of CDH1 and POU5F1. The nuclei of all CDH1-positive cells were stained with POU5F1, but the expression level of POU5F1 was low in some As spermatogonia (arrowheads). b) Double staining of CDH1 and RET. RET was expressed in almost all As and Apr cells but rarely expressed in Aal cells. c) Double staining of CDH1 and GFRA1. The staining pattern of GFRA1 was similar to that of RET. d) Double staining of CDH1 and ZBTB16. The nuclear staining of ZBTB16 was mosaic in CDH1-positive cell clusters. Nonspecific binding of the secondary antibody stained the peritubular fibroblast cells (asterisks). Bar ¼ 20 lm. e) Schematic view of the expression level of several surface antigens and POU5F1 through the early develop- ment of spermatogonia. The expression of CDH1 is reduced during the transition from undifferentiated type A spermatogonia to A1 spermatogonia. By contrast, the expression of KIT and TACSTD1 is upregulated. RET and GFRA1 are expressed mainly in As and Apr cells and expressed rarely in Aal cells.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Double Staining, Staining, Expressing, Binding Assay

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 6. Continuous BrdU labeling showed that the cell cycle of CDH1- positive spermatogonia was much longer than that of other spermatogo- nia. a–b) After 1 wk of labeling, a CDH1-positive cell (green) labeled with no BrdU (a), and a CDH1-positive cell labeled with BrdU (red; b). c) After 3 wk of labeling, all the CDH1-positive and CDH1-negative cells were labeled with BrdU. The insets show a higher magnification of the cells indicated by arrows in each panel. Bars ¼ 10 lm. d) Ratios of BrdU- positive cells among CDH1-negative spermatogonia (red line) and CDH1- positive spermatogonia (blue line) are shown after 1, 2, and 3 wk of continuous BrdU labeling.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Labeling

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 7. Kinetics of CDH1-positive cells in seminiferous tubules after busulfan treatment. a) Two weeks postinjection, most spermatogonia had disappeared, but CDH1-positive cells were not so affected. b) Three weeks postinjection, most spermatocytes had disappeared, but Sertoli cells, spermatids, and CDH1-positive cells survived. c) Some CDH1-positive cells started to proliferate after 4 wk. d) Six weeks postinjection, many spermatocytes appeared in some seminiferous tubules, and many spermatogonia were regenerated, but most of them lost CDH1 expression. Left panels show CDH1 staining, and right panels show DAPI staining. Arrowheads indicate CDH1-positive cells. Bar ¼ 10 lm.

Article Snippet: For double staining with CDH1 and POU5F1, the samples were incubated with 2 lg/ml goat anti-mouse CDH1 antibody and 4 lg/ml rabbit anti-human POU5F1 antibody (sc-9081; Santa Cruz Biotechnology) in ‘‘Can Get Signal Immunostain Solution A’’ overnight at 48C.

Techniques: Expressing, Staining

Journal: Cancer Research

Article Title: A Novel Peptide Specifically Binding to Interleukin-6 Receptor (gp80) Inhibits Angiogenesis and Tumor Growth

doi: 10.1158/0008-5472.can-05-0188

Figure Lengend Snippet: Figure 1. Characterization of binding activity of selected phage display clones. A, individual sIL-6Ra-binding phage clones were tested for their ability to compete with IL-6 protein for binding to immobilized sIL-6Ra. Phages were added to sIL-6Ra-coated wells in a 96-well plate at a concentration of 1012 pfu/mL. After 1 hour of incubation with phage, IL-6 protein was added to the wells, and bound IL-6 was then measured using biotin-conjugated anti–IL-6 mAb, HRP-conjugated streptavidin, and TMB substrate as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, statistically significant increase compared with the corresponding control value. B, phage clone 1 and phage clone 7 (1012 pfu/mL; which had been selected based on their ability to compete with IL-6 for binding to immobilized sIL-6Ra) were added to various cell monolayers combined with IL-6 protein and incubated for 2 hours at room temperature. After washing, the cell-bound phages were detected using HRP-conjugated anti-M13 mAb and TMB substrate as described in Materials and Methods. Columns, means of at least three independent experiments in triplicate; bars, SD. C, phage clone 1 and phage clone 7 were tested for their binding activity and location in C33A cervical carcinoma cells by immunofluorescence staining. C33A cells (4 105) were seeded onto coverslips. After treatment with phage clone 1 and phage clone 7 for 2 hours, immunostaining was done using anti-M13 antibody followed by FITC-conjugated antimouse IgG. Membrane localization of phage clone 7 was then observed by fluorescence microscopy. The position of the cell nucleus was confirmed by staining with Hoechst 33258 fluorescent dye. Original magnification, 400. Representative of three independent experiments. D, immunofluorescence staining of phage clone 7–treated C33A cell monolayer shows colocalization of IL-6Ra and phage clone 7. Polyclonal goat antihuman IL-6Ra antibody and monoclonal anti-M13 antibody were applied to the phage clone 7–treated C33A cells and incubated at 4jC overnight. After washes in PBS, the samples were treated with donkey antigoat IgG rhodamine-conjugated secondary antibody and FITC-conjugated goat antimouse secondary antibody for 1 hour at room temperature. The immunofluorescence-labeled cells were then analyzed by fluorescence microscopy as described in Materials and Methods.

Article Snippet: After washes in PBS, the samples were treated with donkey antigoat IgG rhodamine-conjugated secondary antibody (R&D Systems) and FITC-conjugated goat antimouse secondary antibody (R&D Systems) at a dilution of 1:200 for 1 hour at room temperature.

Techniques: Binding Assay, Activity Assay, Clone Assay, Concentration Assay, Incubation, Control, Immunofluorescence, Staining, Immunostaining, Membrane, Fluorescence, Microscopy, Labeling