Journal: STAR Protocols

Article Title: Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.xpro.2020.100028

Figure Lengend Snippet: FACS Plots Showing Lin − Sca1 + c-Kit + (LSK) Cell Fractions after MACS Purification and 2 Days of Culture Bone marrow cells were stained with α-mouse lineage cocktail, Sca1, and c-Kit antibodies. (A) The negative fraction of the LS column should be almost completely depleted of Sca1 + cells. (B) The positive fraction eluted from the LS or MS separation column is enriched in LSK cells and progenitor cells, which all express Sca1. The LSK population should comprise 6 to 8% of the Sca1 + cell fraction. Lymphoid progenitor cells require IL-7 to survive and will be depleted upon culturing with 50 ng/mL mTPO, mSCF, mFLT-3, and hIL-11. (C) Purified LSK cell fraction after 2 days of culture in HSPC medium.

Article Snippet: Anti-mouse Sca1 MicroBead Kit (VioBrightTM FITC) , Miltenyi Biotec , Cat#130-123-124.

Techniques: Purification, Staining

Journal: STAR Protocols

Article Title: Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.xpro.2020.100028

Figure Lengend Snippet:

Article Snippet: Anti-mouse Sca1 MicroBead Kit (VioBrightTM FITC) , Miltenyi Biotec , Cat#130-123-124.

Techniques: Virus, Recombinant, DNA Extraction, Cloning, Modification, Plasmid Preparation, Software, Adhesive, Real-time Polymerase Chain Reaction, Sterility, Centrifugation

Journal: Translational Stroke Research

Article Title: Remote Ischemic Preconditioning Exerts Neuroprotective Effects Via the PGC-1α/FNDC5/BDNF Pathway in Focal Brain Ischemia of Rats

doi: 10.1007/s12975-026-01422-z

Figure Lengend Snippet: Effects of RIpreC on the expression of Bcl-2 ( A , B , F , G ), Bax ( A , C , F , H ), caspase-3 ( A , D , F , J ), and TUNEL-positive neurons ( A , E ) in the ischemic brain. Bcl-2 immunoreactivity in the perilesional cortex and its protein level were significantly increased in the RIpreC group compared the IR group. By contrast, Bax immunoreactivity and its protein level were significantly reduced in the RIpreC group compared with the IR group. The Bax/Bcl-2 ratios were also significantly reduced in the RIpreC group compared with the IR group ( I ). Subsequently, the number of TUNEL-positive neurons (arrow) and the ratios of caspase-3 positive to NeuN-positive neuron in the perilesional cortex were significantly decreased in the RIpreC group compared with the IR group ( E , K , L ). Mean ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar = 50 μm ( A ) and 25 μm ( K ). ( n = 4–11 in each group)

Article Snippet: # p < 0.05 (Mann–Whitney U test), # p < 0.01 (Student’s t-test), Scale bar = 1 mm ( B ). ( n = 5–11 in each group) Co-localization of mouse anti-neuronal nuclei (NeuN) antibody (1:200; a marker of neurons; ab104224; Abcam plc, Cambridge, UK) with rabbit anti-caspase-3 antibody (1:200; 19677-1-AP; Proteintech, USA) immunoreactivities was examined using immunofluorescence staining.

Techniques: Expressing, TUNEL Assay

Journal: Translational Stroke Research

Article Title: Remote Ischemic Preconditioning Exerts Neuroprotective Effects Via the PGC-1α/FNDC5/BDNF Pathway in Focal Brain Ischemia of Rats

doi: 10.1007/s12975-026-01422-z

Figure Lengend Snippet: Effects of PGC-1αInhibition by SR-18,292. Inhibition of PGC-1α blunted the neuroprotective and neurorestorative effects of RIpreC ( A – F ). No changes in brain infarctions, neurological scores, and sensorimotor functions were observed in the RIpreC-treated SR-18,292 group compared with the IR group. Immunohistochemical analysis of the rectangular areas in A showed that immunoreactivities of PGC-1α, FNDC5, BDNF, and caspase-3 surrounding the lesion in the RIpreC-treated SR-18,292 group were similar to those in the IR group ( G – K ). Scale bar = 50 μm ( G ). ( n = 5–11 in each group)

Article Snippet: # p < 0.05 (Mann–Whitney U test), # p < 0.01 (Student’s t-test), Scale bar = 1 mm ( B ). ( n = 5–11 in each group) Co-localization of mouse anti-neuronal nuclei (NeuN) antibody (1:200; a marker of neurons; ab104224; Abcam plc, Cambridge, UK) with rabbit anti-caspase-3 antibody (1:200; 19677-1-AP; Proteintech, USA) immunoreactivities was examined using immunofluorescence staining.

Techniques: Inhibition, Immunohistochemical staining

Journal: JCI Insight

Article Title: Mindin regulates fibroblast subpopulations through distinct Src family kinases during fibrogenesis

doi: 10.1172/jci.insight.173071

Figure Lengend Snippet: Representative contour plot showing quadrants for ( A ) α-SMA + SCA1 + /CD26 – VIM hi , α-SMA + SCA1 - /CD26 – VIM hi , α-SMA - SCA1 + /CD26 – VIM hi and α-SMA – SCA1 – /CD26 – VIM hi and ( B ) α-SMA + CD26 + /SCA1 – VIM hi , α-SMA + CD26 – /SCA1 – VIM hi , α-SMA – CD26 + /SCA1 – VIM hi and α-SMA – CD26 – /SCA1 – VIM hi cells from P9 WT (left) and Snail -transgenic ( SnTg ) (right) mice. Individual value plots (mean ± SEM) of ( C ) the percentage of α-SMA + SCA1 + /CD26 – VIM hi and ( D ) the percentage α-SMA + CD26 + /SCA1 – VIM hi cells ( n = 6; P values were calculated by Welch’s t test; * P < 0.05, *** P < 0.001). ( E ) SCA1 + fibroblasts (green) and nuclear staining with DAPI (blue) in WT and SnTg skin sections in P3, P5, P7, and P9 pups. The white boxes mark the insets shown in . Note that the green stain at the bottom of the skin section is the autofluorescence of the paper used to keep the tissue uncurled during the embedding process. ( F ) Heatmap showing the probability of SCA1 + cells at a given distance below the epidermis in WT (top) and SnTg (bottom) mice. P3 ( n =3 WT and Snail Tg ), P5 ( n = 2 WT and n = 4 Snail Tg ), P7 ( n = 3 WT and n = 4 Snail Tg ), and P9 ( n = 6 WT and n = 8 Snail Tg ). ( G ) CD26 + fibroblasts (red) and nuclear staining with DAPI (blue) in WT and SnTg skin sections from P3, P5, P7, and P9 pups. The white boxes mark the insets shown in as magnified areas. The boxed areas are shown at higher magnification in . Note that the red stain at the bottom of the skin section is the autofluorescence of the paper used to keep the tissue uncurled during the embedding process. ( H ) Heatmap showing the probability of CD26 + cells at a given distance below the epidermis in WT (top) and SnTg (bottom) at P3 ( n = 2 WT and n = 3 Snail Tg ), P5 ( n = 2 WT and n = 3 Snail Tg ), P7 ( n = 3 WT and n = 3 Snail Tg ), and P9 ( n = 4 WT and n = 6 Snail Tg ).

Article Snippet: Skin tissues were fixed and sectioned as previously reported ( ) and probed with the following antibodies diluted 1:200: K5 (as previously described by CJ’s lab in Rana et al., 2023, ref. ; Badarinath et al., 2022, ref. ; Pincha et al., 2018, ref. ; Nakasaki et al., 2015, ref. ); SCA1 (R&D Systems, AF1226); CD26 (R&D Systems, AF954); CD11b (Abcam, ab8878); CD3 (ebiosciences, 14-0032-85), and F4/80 (ebiosciences, 14-4801-81). α-SMA (Abcam ab5694) was used at 1:50 dilution.

Techniques: Transgenic Assay, Staining

Journal: JCI Insight

Article Title: Mindin regulates fibroblast subpopulations through distinct Src family kinases during fibrogenesis

doi: 10.1172/jci.insight.173071

Figure Lengend Snippet: ( A ) IF staining for SCA1 in P9 WT, SnTg , and SnTg/Mindin- KO ( SnTg/Min -KO) skin (scale bar: 50 μm). ( B ) Heatmap showing the probability of SCA1 + cells at a given distance below the epidermis in WT ( n = 6), SnTg ( n = 8), and SnTg/Min -KO ( n = 4) skin. Data for WT and SnTg are the same as in F. ( C ) Transwell assay to measure migration of mixed, SCA1 + , and CD26 + fibroblasts with either buffer or Mindin as a potential chemoattractant ( n ≥ 4). ( D ) Amount of phosphorylated SRC (pSRC) and total SRC (tSRC) proteins in fibroblasts treated with either buffer or Mindin for 15 minutes. ( E ) Transwell assay with SCA1 + fibroblasts stimulated with buffer or Mindin in the presence of DMSO, PP2 (10 μM), or KbSrc4 (10 μM) ( n = 3). ( F ) Transwell assay with SCA1 + fibroblasts transduced with nontargeting (NT), Src , Fyn , or Yes shRNA with buffer or Mindin as a chemoattractant ( n = 3). ( G ) IF for SCA1 in WT and Min -KO day 7 and day 9 skin wounds. (The images were stitched using FIJI ImageJ stitching tool, ref. ; scale bar: 50 μm.) White boxes denote regions shown at higher magnification on the right-hand side of each image. ( H ) Quantification of SCA1 + cells in the wound beds day 7 and day 9 after wounding of WT and Min -KO mice ( n = 3 mice) ( I ) Percentage wound closure in WT and Min -KO mice with regard to wound size on day 1 ( n = 3 mice, 2 wounds per mice). Data represent the mean ± SEM. P values were calculated by Welch’s t test ( C and I ), 1-way ANOVA followed by Tukey’s post hoc analysis ( E ), and 2-way ANOVA followed by post hoc Šídák’s multiple comparisons test ( F and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, P > 0.05.

Article Snippet: Skin tissues were fixed and sectioned as previously reported ( ) and probed with the following antibodies diluted 1:200: K5 (as previously described by CJ’s lab in Rana et al., 2023, ref. ; Badarinath et al., 2022, ref. ; Pincha et al., 2018, ref. ; Nakasaki et al., 2015, ref. ); SCA1 (R&D Systems, AF1226); CD26 (R&D Systems, AF954); CD11b (Abcam, ab8878); CD3 (ebiosciences, 14-0032-85), and F4/80 (ebiosciences, 14-4801-81). α-SMA (Abcam ab5694) was used at 1:50 dilution.

Techniques: Staining, Transwell Assay, Migration, Transduction, shRNA

Journal: JCI Insight

Article Title: Mindin regulates fibroblast subpopulations through distinct Src family kinases during fibrogenesis

doi: 10.1172/jci.insight.173071

Figure Lengend Snippet: qPCR for expression of inflammatory cytokines from ( A ) CD26 + fibroblasts or ( B ) SCA1 + fibroblasts treated with either buffer or Mindin ( n ≥ 4). Staining for NF-κB (green) and DAPI (blue) in ( C ) CD26 + fibroblasts or ( D ) SCA1 + fibroblasts treated for 1 hour with either buffer or Mindin (scale bar: 50 μm) and ( E ) the percentage of cells with NF-κB + nuclei per field in CD26 + ( n = 3) or SCA1 + ( n = 5) fibroblast treated with either buffer or Mindin. ( F ) IF staining for K5 (red) and CD11b (top; green; macrophages) and CD3 (bottom; green; T cells) in WT and Min -KO skin sections after wounded day 7 (scale bar: 50 μm) and quantification of ( G ) CD11b + and ( H ) CD3 + cells found in the wound bed ( n = 3 mice). Data represent the mean ± SEM. P values were calculated by ratio paired t test ( A and B ) and Welch’s t test ( E , G , and H ). * P < 0.05, ** P < 0.01, **** P < 0.0001; NS, P > 0.05. nd, not detected.

Article Snippet: Skin tissues were fixed and sectioned as previously reported ( ) and probed with the following antibodies diluted 1:200: K5 (as previously described by CJ’s lab in Rana et al., 2023, ref. ; Badarinath et al., 2022, ref. ; Pincha et al., 2018, ref. ; Nakasaki et al., 2015, ref. ); SCA1 (R&D Systems, AF1226); CD26 (R&D Systems, AF954); CD11b (Abcam, ab8878); CD3 (ebiosciences, 14-0032-85), and F4/80 (ebiosciences, 14-4801-81). α-SMA (Abcam ab5694) was used at 1:50 dilution.

Techniques: Expressing, Staining

Journal: JCI Insight

Article Title: Mindin regulates fibroblast subpopulations through distinct Src family kinases during fibrogenesis

doi: 10.1172/jci.insight.173071

Figure Lengend Snippet: ( A ) Measurement of intracellular distance between 2 nearest CD26 + nuclei ( x axis) as a function of distance below the epidermis ( y axis, bin number below the epidermis; bin size = 5 μm) in WT, SnTg , and SnTg/Min -KO skin ( n = 3). (The number of CD26 + cells counted >80 in each section. The region shaded in gray marks the bins where P < 0.05, calculated using Welch’s t test.) ( B ) Collagen contraction assay, showing percentage of contraction of collagen gels seeded with mixed, CD26 + , or SCA1 + fibroblasts and treated with either buffer control or Mindin ( n ≥ 4). ( C ) Effect of SFK inhibition on Mindin-induced collagen contraction. CD26 + fibroblasts were treated with either buffer control or Mindin along with DMSO, PP2, or KbSrc4 ( n ≥ 3). ( D ) Effect of nontargeting (NT), Src , Fyn , or Yes shRNA on collagen contraction with CD26 + fibroblasts treated with either buffer control or Mindin ( n ≥ 3). ( E ) Measurement of the rate of closure (slope) in WT and Min -KO mice. The slope was calculated as the percentage of closure of a given day – the percentage of closure on the previous day ( n = 3 mice, 2 wounds per mouse). ( F ) Quantification of COL1 in buffer control or Mindin-treated CD26 + and SCA1 + fibroblasts, normalized to Lamin B1 (LAM) ( n = 4). Data represent the mean ± SEM. P values were calculated by Welch’s t test ( B and E ), ratio-paired t test ( F ), 1-way ANOVA followed by Tukey’s post hoc analysis ( C ), and 2-way ANOVA followed by post hoc Šídák’s multiple comparisons test ( D ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, P > 0.05.

Article Snippet: Skin tissues were fixed and sectioned as previously reported ( ) and probed with the following antibodies diluted 1:200: K5 (as previously described by CJ’s lab in Rana et al., 2023, ref. ; Badarinath et al., 2022, ref. ; Pincha et al., 2018, ref. ; Nakasaki et al., 2015, ref. ); SCA1 (R&D Systems, AF1226); CD26 (R&D Systems, AF954); CD11b (Abcam, ab8878); CD3 (ebiosciences, 14-0032-85), and F4/80 (ebiosciences, 14-4801-81). α-SMA (Abcam ab5694) was used at 1:50 dilution.

Techniques: Contraction Assay, Control, Inhibition, shRNA

Journal: JCI Insight

Article Title: Mindin regulates fibroblast subpopulations through distinct Src family kinases during fibrogenesis

doi: 10.1172/jci.insight.173071

Figure Lengend Snippet: qPCR for expression of signature genes of myCAFs in ( A ) CD26 + fibroblasts or ( B ) SCA1 + fibroblasts treated with either buffer or Mindin ( n ≥ 4). Expression of genes that are associated with stem cell renewing CAFs in ( C ) CD26 + ( n = 6) and ( D ) SCA1 + ( n = 4) fibroblasts treated with either buffer or Mindin measured by qPCR. ( E ) Colony formation assay of primary mouse keratinocytes (mKT) cocultured with CD26 + or SCA1 + fibroblasts pretreated with either buffer of Mindin for 24 hours ( n = 3). ( F ) Colony formation assay of primary mouse keratinocytes cultured with conditioned media (CM) collected from CD26 + fibroblasts treated with either buffer or Mindin ( n = 4). Data represent the mean ± SEM. P values were calculated by ratio paired t test ( A – D ) and Welch’s t test ( E and F ). * P < 0.05, ** P < 0.01; NS, P > 0.05. ( G ) Model of differential effects of Mindin on distinct subpopulations of dermal fibroblasts.

Article Snippet: Skin tissues were fixed and sectioned as previously reported ( ) and probed with the following antibodies diluted 1:200: K5 (as previously described by CJ’s lab in Rana et al., 2023, ref. ; Badarinath et al., 2022, ref. ; Pincha et al., 2018, ref. ; Nakasaki et al., 2015, ref. ); SCA1 (R&D Systems, AF1226); CD26 (R&D Systems, AF954); CD11b (Abcam, ab8878); CD3 (ebiosciences, 14-0032-85), and F4/80 (ebiosciences, 14-4801-81). α-SMA (Abcam ab5694) was used at 1:50 dilution.

Techniques: Expressing, Colony Assay, Cell Culture