Journal: Materials today. Bio

Article Title: Precise delivery of doxorubicin and imiquimod through pH-responsive tumor microenvironment-active targeting micelles for chemo- and immunotherapy.

doi: 10.1016/j.mtbio.2022.100482

Figure Lengend Snippet: Fig. 10. Immunostaining of tumor tissues in tumor-bearing mice after treatments. (A) Immunohistochemistry images of tumor sections stained with CD3, CD8, and TNF-α antibodies. The scale bar is 100 μm. (B) Immunofluorescence images of tumor tissues after treatments for 12 days. The scale bar is 50 μm. Blue fluorescence represents the cell nucleus stained with DAPI. Green fluorescence represents the iNOS stained with the iNOS antibody conjugated with FITC.

Article Snippet: After 30 min, the tissue slice was stained with diluted iNOS antibody (Miltenyi Biotec, catalog: 130-116-357) at 4 C overnight.

Techniques: Immunostaining, Immunohistochemistry, Staining

Journal: Neurotherapeutics

Article Title: Down-regulation of lipocalin-2 alleviates depressive-like behaviors in mice through modulation of microglial activation

doi: 10.1016/j.neurot.2026.e00862

Figure Lengend Snippet: LCN2 neutralizing antibodies alleviated the depressive-like behaviors in CUS mice. ( A ) Schematic of the experimental procedure and behavioral studies. ( B ) Levels of LCN2 measured by ELISA in the mouse hippocampus treated with IgG or anti-LCN2 (n = 6/group). ∗∗∗ P < 0.001 versus the IgG group using Student's t -test. ( C – H ) LCN2 neutralizing antibodies relieved depressive-like behaviors in CUS mice (n = 12/group) as measured by the SPT ( C ), FST ( D) , TST ( E ) and OFT ( F – H ). ∗∗∗ P < 0.001 versus the Control + IgG group; ### P < 0.001 versus the CUS + anti-IgG group using two-way ANOVA followed by the Holm-Sidak post hoc multiple comparison test.

Article Snippet: After incubation in blocking buffer, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-LCN2 (1:1000, abs134038, Absin), anti-iNOS (1:1000, 18985-1-AP, Proteintech Group), anti-p(Ser616)DRP1 (1:1000, ab314755, Abcam), anti-DRP1 (1:1000, 12957-1-AP, Proteintech Group), anti-MFN2(1:1000, 12186-1-AP, Proteintech Group), and anti-GAPDH (1:3000, 60004-1-AP, Proteintech Group).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure4. nanos1mRNAisaSmaug2targetinembryoniccorticalprecursors.A,RT-PCRfornanos1,nanos2,andnanos3mRNAsinmurinecorticesfromE11tobirth(P0).nanos1mRNAexpression wasdetectedusingtwodifferentprimersets.PCRproductsweresequencedtoconfirmspecificity.ve,SamplewithknownexpressionoftargetmRNAandusedasapositivecontrolforthereaction; -ve,samplegeneratedintheabsenceofreversetranscriptase.B,SchematicofSREsinthenanos1mRNAtranscript.YellowarrowlabeledCDSrepresentstheprotein-codingregion.C,Westernblot analysisforNanos1inE11.5to2-month-oldcortices.TheblotwasreprobedforERK1/2asaloadingcontrol.D,WesternblotofHEK-293TcellstransfectedwithaFlag-taggedmouseSmaug2construct andimmunoprecipitatedwithanti-Smaug2orwithcontrolnonspecificrabbitIgG,probedwithantibodiesforSmaug2.Asacontrol,10%oftheinputhomogenatewasloaded.E,Westernblot(top) of E12.5 cortical lysates immunoprecipitated with the same Smaug2 antibody as in D or with control, nonspecific rabbit IgG and probed with anti-Smaug2. As a positive control, 10% of the input homogenatewasloaded.Similarimmunoprecipitatesweregeneratedinparallel,mRNAwasextracted,andthesampleswereanalyzedfornanos1,nanos2,andnanos3mRNAsusingRT-PCR(second tobottompanels).F,ConfocalimagesofFISHfornanos1(left),nanos2(center),andnanos3(right)mRNAs(blackgranules)incoronalsectionsoftheE12.5cortex.v,Ventricle.Scalebar,10m.G, Higher-magnificationconfocalimagesoftheVZ/SVZofanE13.5corticalsectionshowingFISHfornanos1mRNA(red)andimmunostainingforSmaug2(green).Top,Merge.Boxedregionsareshown at higher magnification in the right panels, which also show colocalization of Smaug2 and nanos1 mRNA on the z-axis (XZ and YZ), as indicated by the hatched (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Immunoprecipitation, Control, Positive Control

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure 5. Nanos1 is necessary and sufficient to promote neurogenesis in vivo. A, Western blots of HEK-293T cell lysates cotransfected with murine Nanos1 or Flag-tagged murine Nanos2 or Nanos3 expression constructs and a control shRNA (Con) or a Nanos1 shRNA (shNos1) and probed with anti-Nanos1 or anti-Flag, as indicated. The blots were reprobed with ERK1/2 as a loading control. B–H, E13/E14 murine cortices were coelectroporated with a nuclear EGFP construct, and either a control (con) or Nanos1 shRNA (shNos1) and coronal sections were analyzed 3 d later at E16/E17.B,ImagesofelectroporatedsectionsimmunostainedforEGFP(green).v,Ventricle.Scalebar,10m.C,QuantificationofsectionssimilartothoseinBforthepercentageofEGFP-positive cells located in the different cortical regions. **p 0.01. n 3 embryos each, at least 3 sections per embryo. D, Confocal micrographs of the VZ/SVZ (three top rows) or CP (bottom row) of electroporatedsectionsimmunostainedforEGFP(green)andPax6,Ki67,Tbr2,orSatb2(allred).Arrowsindicatedouble-labeledcells.v,Ventricle.Scalebar,10m.E–H,Quantificationofsections similartothoseinDforthepercentageofEGFP-positivecellsthatexpressedPax6(E),Ki67(F),Tbr2(G),orSatb2(H).**p0.01.***p0.001.n3embryoseach,atleast3sectionsperembryo. I–K,E13/E14corticeswerecoelectroporatedwithanuclearEGFPconstructandacontrol(con)orNanos1shRNA(shNos1) anshRNA-resistanthumanNanos1expressionvector(resc)andcoronal sections were analyzed 3 d later at E16/E17. I, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 m. J, K, Sections similar to those in I were immunostained for EGFP and Pax6 or Satb2 and the proportion of EGFP-positive cells that were also positive for the marker was quantified. **p 0.01. (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: In Vivo, Western Blot, Expressing, Construct, Control, shRNA, Marker

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure6. Smaug2andnanos1mRNAareassociatedwith4E-TinaP-Body-likegranuleinPax6-positiveapicalprecursors.A,WesternblotanalysisforSmaug2(Smg2)and4E-TinlysatesofE12.5 corticalprecursorsculturedfor3dandimmunoprecipitatedwithanti-Smaug2orwithcontrol,nonspecificrabbitIgG.Asapositivecontrol,10%oftheinputhomogenatewasloaded.B,Westernblot analysisforSmaug2and4E-TinlysatesofE12.5corticalprecursorsculturedfor3dandimmunoprecipitatedwithanti-4E-Torwithcontrol,nonspecificmouseIgG.Asapositivecontrol,10%ofthe inputhomogenatewasloaded.C,ConfocalimagesofE12.5corticalprecursorsculturedfor3dandimmunostainedforSmaug2(green)and4E-T(magenta).Cultureswerealsocounterstainedwith Hoechst(blue).Top,Boxedareasareshownathighermagnificationinthebottompanels.ArrowsindicategranulesthatarepositiveforbothSmaug2and4E-T.Scalebar,5m.D,Confocalimages ofE12.53dcorticalprecursorculturesafterthePLAwithSmaug2and4E-Tantibodies.CultureswerealsocounterstainedwithHoechst(blue).Left,Boxedareasareshownathighermagnification totheright.Scalebar,10m.E,ConfocalimagesofE12.5corticalprecursorsculturedfor3dandimmunostainedforSmaug2(red)andDcp1(green).CultureswerealsocounterstainedwithHoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are double labeled for Smaug2 and Dcp1. Scale bar, 10 m. F, Confocal images of cortical precursor cultures after PLA with Smaug2 and Dcp1 antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification on the right. Scale bar, 10 m. G, RT-PCR analysis for nanos1 mRNA in 4E-T immunoprecipitates (4E-T IP) from the E12.5 cortex. As a control, similar lysates were (Figure legend continues.)

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Control

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/jneurosci.2172-15.2015

Figure Lengend Snippet: Figure7. KnockdownofSmaug2or4E-TcausesaberrantNanos1expression,andthisisresponsiblefortheSmaug2knockdown-mediatedincreaseinneurogenesis.A,Nanos1immunoreactivity inacoronalsectionoftheE16/E17cortex.v,Ventricle.Scalebar,10m.B,ConfocalimagesofcellsattheborderoftheSVZandtheIZofE16/E17murinecorticesthatwerecoelectroporated3dearlier withanuclearEGFPconstructandcontrol(con)orSmaug2(shSmg2)shRNAs.SectionswereimmunostainedforEGFP(green)andNanos1(red).ArrowsandarrowheadsindicateEGFP-positivecells that do or do not express Nanos1, respectively. Scale bar, 10 m. C, Quantification of the proportion of EGFP-positive cells expressing detectable Nanos1 in sections similar to those in B. ***p 0.001. n 3 embryos each, at least 3 sections per embryo. D, Confocal images of cells at the border of the SVZ and the IZ of E15/E16 murine cortices that were coelectroporated 2 d earlier with a nuclearEGFPconstructandcontrol(con)or4E-T(sh4ET)shRNAs.SectionswereimmunostainedforEGFP(green)andNanos1(red).ArrowsandarrowheadsindicateEGFP-positivecellsthatdoordo notexpressNanos1,respectively.Scalebar,10m.E,QuantificationoftheproportionofEGFP-positivecellsexpressingdetectableNanos1insectionssimilartothoseinD.*p0.05.n3embryos each,atleast3sectionsperembryo.F–I,E13/E14corticeswerecoelectroporatedwithanuclearEGFPconstructandcontrol(con)orSmaug2shRNA(shSmg2) Nanos1shRNA(shNos1),andcoronal corticalsectionswereanalyzed3dlateratE16/E17.F,ImagesofelectroporatedsectionsimmunostainedforEGFP(green).v,Ventricle.Scalebar,10m.G,Quantificationofsectionssimilartothose in F for the percentage of EGFP-positive cells located in the different cortical regions. *p 0.05. **p 0.01. ns, Nonsignificant. n 3 embryos each, at least 3 sections per embryo. H, I, Quantification of EGFP-positive, marker-positive cells in sections as in F immunostained for EGFP and either Pax6 (H) or Satb2 (I). *p 0.05. **p 0.01. n 3 embryos each, at least 3 sections per embryo. J, Schematic showing the proposed repressive complex involving Smg2, 4E-T, Dcp1, and nanos1 mRNA (top). When the complex is disrupted, either by environmental signals or by knockdownofcomplexcomponentssuchasSmaug2,thiscausesaberranttranslationofNanos1,therebypromotingneurogenesis(bottom).G–I,StatisticswereperformedwithANOVAandTukey’s post hoc multiple comparisons test. Other panels, Statistics were performed with Student’s t test. Error bars indicate SEM.

Article Snippet: The Flag-tagged expression constructs for mouse and human Smaug2 and Smaug1 and mouse Nanos1, Nanos 2, and Nanos3 and human Nanos1 were obtained from OriGene. shRNA vectors were obtained from EZBiolab and had the following sequences: Smaug2 shRNA-1 5 -GAG GAG AAC ATC ACC AGT TAC T-3 , Smaug2 shRNA-2 5 -GGG CTG GAA TGA GTG TGA ACAT-3 , and Nanos1 shRNA-1 5 -GCACATACCATCAAGTATTGCT-3 .

Techniques: Expressing, Marker

Journal: Cell reports

Article Title: Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

doi: 10.1016/j.celrep.2025.116868

Figure Lengend Snippet: (A, C, and E) Representative images of whole-mount human seminiferous tubules stained with antisera against PIWIL4 (green) and the indicated markers (magenta). Nuclei were counterstained with DAPI (gray). Testicular samples from 3 fertile individuals were analyzed. Scale bars: 100 μm and 20 μm (insets). (B, D, and F) Quantification of positive cells that co-stain or not, as indicated. n , the total number of SPG counted. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. (G) IHC analysis of PIWIL4 on human testis sections. Scale bars: 50 μm. Black arrow, A dark SPG; orange arrow, A pale SPG. (H) Quantification of A pale and A dark PIWIL4+ SPG. Testicular samples from 3 fertile individuals were analyzed. (I, K, M, and O) Representative images of whole-mount human seminiferous tubules stained with antisera against NANOS3 (yellow) and the indicated markers (magenta). Nuclei were counterstained with DAPI (gray). Testicular samples from 3 fertile individuals were analyzed. Scale bars: 100 μm and 20 μm (insets). (J, L, and P) Quantification of positive cells that co-stain or not, as indicated. n , the total number of SPG counted. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. (N) tSNE plot showing PIWIL4 and NANOS3 expression across SPG clusters from scRNA-seq analysis. (Q) Schematic model of the human SPG compartment based on the expression of EGR4, PPP1R36, PIWIL4, and NANOS3. See also .

Article Snippet: Rabbit polyclonal anti Human NANOS3 , Proteintech , Cat# 21679-1-AP; RRID:AB_1085925.

Techniques: Staining, Expressing

Journal: Cell reports

Article Title: Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

doi: 10.1016/j.celrep.2025.116868

Figure Lengend Snippet: (A) Differentially expressed genes (DEGs; q < 0.01, fold change >2) from PIWIL4+ vs. NANOS3+ cells. (B)Signaling pathways associated with the DEGs defined in (A). Statistical significance (−log10 ( p value)) is indicated by the bar. The number of DEGs for a given category is indicated by an “X.”. (C) Schematic of experimental design. Fresh human testis biopsies were processed in two ways: (1) dissociation followed by magnetic-activated cell sorting (MACS) to isolate FSD1+ uSPG for feeder-free culture with or without RAS inhibitors (RASis) or (2) maintained in transwell systems, also treated with or without RASis. (D and E) RT-qPCR analysis of feeder-free human uSPG (enriched for FSD1+ cells) cultured for 1 week and treated with or without RASis: lonafarnib (D) and salirasib (E). (F) Immunofluorescence analysis of human testicular tissue fragments cultured with or without RASis, stained for PIWIL4, NANOS3, and BrdU. Cell nuclei were counterstained with DAPI (gray). Scale bars: 50 μm. (G and H) Quantification of PIWIL4+ ( n = 328) and NANOS3+ ( n = 431) per cross-section across four samples. (I and J) BrdU index of PIWIL4+ (I) and NANOS3+ (J) cells in cultured testicular fragments, treated with or without RASis. Data are represented as the mean ± SEM. Four biological replications were performed. For (D), (E), and (G)–(J), data are represented as the mean ± SEM. Four biological replications were performed. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not statistically significant, as determined by unpaired t test (D and E) or one-way ANOVA (G–J).

Article Snippet: Rabbit polyclonal anti Human NANOS3 , Proteintech , Cat# 21679-1-AP; RRID:AB_1085925.

Techniques: Protein-Protein interactions, FACS, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining