gfp  (Thermo Fisher)


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    Structured Review

    Thermo Fisher gfp
    zfTTLL11 is required for early embryonic zebrafish development. a Immunofluorescence image of a zebrafish embryo (at 4 h post fertilization [hpf]), showing PolyE (green), tubulin (red). Scale bar, 20 μm. Representative image of N = 3 independent experiments. b Semiquantitative RT-PCR showing zfTTLL11 expression in zebrafish embryos. Lanes 1, 4-cell stage; 2, 8-cell stage; 3, 64- cell stage; 4, 256-cell stage; 5, sphere; 6, shield; 7, 70% epiboly; 8, 90% epiboly; and 9, 24 hpf. Eef1a1 was amplified as a control. Representative blot of N = 4 independent experiments. c Fluorescent image of a spindle in a zebrafish embryo expressing <t>GFP-zfTTLL11</t> (green) and H2B-mCherry (red). Scale bar, 10 μm. Representative image of N = 3 independent experiments. d Zebrafish embryos (36 hpf) injected at the zygote stage with scrambled MO (control), zfTTLL11-Morpholinos (MO), MO and zfTTLL11 mRNA (WT) or MO and catalytically inactive zfTTLL11 (MO + E466G) mRNA. Scale bar, 1 mm. e Cumulative bar plot of developmental defects (severe or mild) in 36-hpf embryos of N = 5 independent experiments, representative of a total of four independent experiments (≥20 embryos scored per condition). *** P
    Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Chromosome segregation fidelity requires microtubule polyglutamylation by the cancer downregulated enzyme TTLL11"

    Article Title: Chromosome segregation fidelity requires microtubule polyglutamylation by the cancer downregulated enzyme TTLL11

    Journal: Nature Communications

    doi: 10.1038/s41467-022-34909-y

    zfTTLL11 is required for early embryonic zebrafish development. a Immunofluorescence image of a zebrafish embryo (at 4 h post fertilization [hpf]), showing PolyE (green), tubulin (red). Scale bar, 20 μm. Representative image of N = 3 independent experiments. b Semiquantitative RT-PCR showing zfTTLL11 expression in zebrafish embryos. Lanes 1, 4-cell stage; 2, 8-cell stage; 3, 64- cell stage; 4, 256-cell stage; 5, sphere; 6, shield; 7, 70% epiboly; 8, 90% epiboly; and 9, 24 hpf. Eef1a1 was amplified as a control. Representative blot of N = 4 independent experiments. c Fluorescent image of a spindle in a zebrafish embryo expressing GFP-zfTTLL11 (green) and H2B-mCherry (red). Scale bar, 10 μm. Representative image of N = 3 independent experiments. d Zebrafish embryos (36 hpf) injected at the zygote stage with scrambled MO (control), zfTTLL11-Morpholinos (MO), MO and zfTTLL11 mRNA (WT) or MO and catalytically inactive zfTTLL11 (MO + E466G) mRNA. Scale bar, 1 mm. e Cumulative bar plot of developmental defects (severe or mild) in 36-hpf embryos of N = 5 independent experiments, representative of a total of four independent experiments (≥20 embryos scored per condition). *** P
    Figure Legend Snippet: zfTTLL11 is required for early embryonic zebrafish development. a Immunofluorescence image of a zebrafish embryo (at 4 h post fertilization [hpf]), showing PolyE (green), tubulin (red). Scale bar, 20 μm. Representative image of N = 3 independent experiments. b Semiquantitative RT-PCR showing zfTTLL11 expression in zebrafish embryos. Lanes 1, 4-cell stage; 2, 8-cell stage; 3, 64- cell stage; 4, 256-cell stage; 5, sphere; 6, shield; 7, 70% epiboly; 8, 90% epiboly; and 9, 24 hpf. Eef1a1 was amplified as a control. Representative blot of N = 4 independent experiments. c Fluorescent image of a spindle in a zebrafish embryo expressing GFP-zfTTLL11 (green) and H2B-mCherry (red). Scale bar, 10 μm. Representative image of N = 3 independent experiments. d Zebrafish embryos (36 hpf) injected at the zygote stage with scrambled MO (control), zfTTLL11-Morpholinos (MO), MO and zfTTLL11 mRNA (WT) or MO and catalytically inactive zfTTLL11 (MO + E466G) mRNA. Scale bar, 1 mm. e Cumulative bar plot of developmental defects (severe or mild) in 36-hpf embryos of N = 5 independent experiments, representative of a total of four independent experiments (≥20 embryos scored per condition). *** P

    Techniques Used: Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Injection

    2) Product Images from "Amyloid-beta peptides 40 and 42 employ distinct molecular pathways for cell entry and intracellular transit at the BBB endothelium"

    Article Title: Amyloid-beta peptides 40 and 42 employ distinct molecular pathways for cell entry and intracellular transit at the BBB endothelium

    Journal: bioRxiv

    doi: 10.1101/2022.11.17.516996

    Time dependent accumulation of sulforhodamine labeled Aβ (SR-Aβ) peptides in late endosomes. hCMEC/D3 cells expressing GFP-Rab7 incubated with SR-Aβ for 15 minutes. Then the cells were washed with DMEM and imaged by live cell imaging up to 60 minutes. (A-C) Accumulation of SR-Aβ40 following incubations at (A) 17min (B) 37 min (C) 62 min; (D-E) Accumulation of SR-Aβ42 following incubations at (D) 14min (E) 36 min (F) 51 min.
    Figure Legend Snippet: Time dependent accumulation of sulforhodamine labeled Aβ (SR-Aβ) peptides in late endosomes. hCMEC/D3 cells expressing GFP-Rab7 incubated with SR-Aβ for 15 minutes. Then the cells were washed with DMEM and imaged by live cell imaging up to 60 minutes. (A-C) Accumulation of SR-Aβ40 following incubations at (A) 17min (B) 37 min (C) 62 min; (D-E) Accumulation of SR-Aβ42 following incubations at (D) 14min (E) 36 min (F) 51 min.

    Techniques Used: Labeling, Expressing, Incubation, Live Cell Imaging

    Time dependent accumulation of sulforhodamine labeled Aβ40 peptides in recycling endosomes. hCMEC/D3 cells expressing GFP-Rab11 incubated with SR-Aβ40 for 15 minutes. Then the cells were washed with DMEM and imaged by live cell imaging up to 60 minutes. (A-C) Accumulation of SR-Aβ40 following incubations at (A) 24min (B) 30 min (C) 36 min (D) 48min (E) 54 min (F) 57 min.
    Figure Legend Snippet: Time dependent accumulation of sulforhodamine labeled Aβ40 peptides in recycling endosomes. hCMEC/D3 cells expressing GFP-Rab11 incubated with SR-Aβ40 for 15 minutes. Then the cells were washed with DMEM and imaged by live cell imaging up to 60 minutes. (A-C) Accumulation of SR-Aβ40 following incubations at (A) 24min (B) 30 min (C) 36 min (D) 48min (E) 54 min (F) 57 min.

    Techniques Used: Labeling, Expressing, Incubation, Live Cell Imaging

    3) Product Images from "The UBP5 histone H2A deubiquitinase counteracts PRC2-mediated repression to regulate Arabidopsis development and stress responses"

    Article Title: The UBP5 histone H2A deubiquitinase counteracts PRC2-mediated repression to regulate Arabidopsis development and stress responses

    Journal: bioRxiv

    doi: 10.1101/2022.11.15.516593

    UBP5 is a nuclear protein that interacts with PRC2 and colocalises with PWO1. A and B, transient and inducible expression in N. benthamiana epidermal cells, bar = 10µm. A, i35S::UBP5-GFP (i, confocal; ii, bright field; iii, overlay). B, i35S::UBP5-GFP and i35S::PWO1-mCherry co-transformation (i, i35S::UBP5-GFP; ii, i35S::PWO1-mCherry; iii, overlay). Arrows indicate speckles. C, FET-APB measurements for nuclei exemplified in B, with a distinction for speckle (spec) and non-speckle localisation. CLF-GFP and PWO1-mCherry measurement was used as positive control (Mikulski et al., 2019). An average of efficiency for n = 7-19 is shown. Significance level was measured in comparison to control or as indicated using Student’s t-test and is represented by *p
    Figure Legend Snippet: UBP5 is a nuclear protein that interacts with PRC2 and colocalises with PWO1. A and B, transient and inducible expression in N. benthamiana epidermal cells, bar = 10µm. A, i35S::UBP5-GFP (i, confocal; ii, bright field; iii, overlay). B, i35S::UBP5-GFP and i35S::PWO1-mCherry co-transformation (i, i35S::UBP5-GFP; ii, i35S::PWO1-mCherry; iii, overlay). Arrows indicate speckles. C, FET-APB measurements for nuclei exemplified in B, with a distinction for speckle (spec) and non-speckle localisation. CLF-GFP and PWO1-mCherry measurement was used as positive control (Mikulski et al., 2019). An average of efficiency for n = 7-19 is shown. Significance level was measured in comparison to control or as indicated using Student’s t-test and is represented by *p

    Techniques Used: Expressing, Transformation Assay, Positive Control

    4) Product Images from "CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target"

    Article Title: CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI157101

    ECs enhance the proliferative potential and promote leukemic progression of C/G-CB cells. ( A ) Diagram of experimental design. This experiment was performed twice using 2 separate CB units. See Supplemental Figure 4 for the repeat experiment. ( B ) Growth kinetics of C/G-CB and GFP-CB cells in EC coculture or MC. ( C ) C/G-CB cells expanded in EC coculture for 9 weeks were reseeded in EC coculture either directly (direct contact) or in EC Transwells (indirect contact) or placed in liquid culture containing SFEM II (with SCF, FLT3L, and TPO). After 7 days, the number of GFP + cells was quantified by flow cytometry. Data in B and C presented as mean ± SD from 3 technical replicates. Statistical significance was determined by 1-way ANOVA. **** P
    Figure Legend Snippet: ECs enhance the proliferative potential and promote leukemic progression of C/G-CB cells. ( A ) Diagram of experimental design. This experiment was performed twice using 2 separate CB units. See Supplemental Figure 4 for the repeat experiment. ( B ) Growth kinetics of C/G-CB and GFP-CB cells in EC coculture or MC. ( C ) C/G-CB cells expanded in EC coculture for 9 weeks were reseeded in EC coculture either directly (direct contact) or in EC Transwells (indirect contact) or placed in liquid culture containing SFEM II (with SCF, FLT3L, and TPO). After 7 days, the number of GFP + cells was quantified by flow cytometry. Data in B and C presented as mean ± SD from 3 technical replicates. Statistical significance was determined by 1-way ANOVA. **** P

    Techniques Used: Flow Cytometry

    Transcriptional profile of C/G-CB cells in EC coculture recapitulates primary C/G AML. ( A ) Unsupervised clustering by uniform manifold and projection (UMAP) analysis of C/G-CB and GFP-CB cells in reference to primary AML samples. Dashed circle indicates C/G-CB cells cocultured with ECs at week 6 and 12 time points. Normal bone marrow (NBM, n = 68); KMT2A ( n = 319); RUNX1-RUNX1T1 ( n = 157); CBFB-MYH11 ( n = 120); other ( n = 444); CBFA2T3-GLIS2 ( n = 39). Primary patient data are described in Smith et al. ( 8 ). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. ( B ) Top: Expression of ERG, BMP2, and GATA1 in GFP-CB versus C/G-CB cells over weeks in EC and MC conditions as well as in C/G-fusion-positive primary versus NBM samples. Bottom: Single-sample gene set enrichment (ssGSEA) scores of Hedgehog, TGF-β, and WNT signaling pathways for GFP-CB versus C/G-CB cells and NBM samples versus primary-fusion-positive samples. CBFA2T3-GLIS2 primary samples ( n = 39); NBM samples ( n = 68). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by unpaired, 2-sided, nonparametric Wilcoxon’s rank-sum test, analyzing differences in expression between C/G-CB in EC or MC conditions and GFP controls in EC or MC conditions, and differences between primary C/G AML samples and healthy NBM. ( C ) Heatmap of differentially expressed genes in C/G-CB versus GFP-CB cells in EC coculture or MC. ( D ) GSEA plots of C/G and HSC signature genes comparing C/G-CB cells in EC coculture versus MC at week 6 of culture. ( E ) Pathways that are upregulated (left) and downregulated (right) in C/G-CB cells in EC coculture compared with MC. ( C – E ) n = 4 technical replicates for C/G-CB cells in EC coculture at week 6; n = 3 technical replicates for all other groups.
    Figure Legend Snippet: Transcriptional profile of C/G-CB cells in EC coculture recapitulates primary C/G AML. ( A ) Unsupervised clustering by uniform manifold and projection (UMAP) analysis of C/G-CB and GFP-CB cells in reference to primary AML samples. Dashed circle indicates C/G-CB cells cocultured with ECs at week 6 and 12 time points. Normal bone marrow (NBM, n = 68); KMT2A ( n = 319); RUNX1-RUNX1T1 ( n = 157); CBFB-MYH11 ( n = 120); other ( n = 444); CBFA2T3-GLIS2 ( n = 39). Primary patient data are described in Smith et al. ( 8 ). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. ( B ) Top: Expression of ERG, BMP2, and GATA1 in GFP-CB versus C/G-CB cells over weeks in EC and MC conditions as well as in C/G-fusion-positive primary versus NBM samples. Bottom: Single-sample gene set enrichment (ssGSEA) scores of Hedgehog, TGF-β, and WNT signaling pathways for GFP-CB versus C/G-CB cells and NBM samples versus primary-fusion-positive samples. CBFA2T3-GLIS2 primary samples ( n = 39); NBM samples ( n = 68). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by unpaired, 2-sided, nonparametric Wilcoxon’s rank-sum test, analyzing differences in expression between C/G-CB in EC or MC conditions and GFP controls in EC or MC conditions, and differences between primary C/G AML samples and healthy NBM. ( C ) Heatmap of differentially expressed genes in C/G-CB versus GFP-CB cells in EC coculture or MC. ( D ) GSEA plots of C/G and HSC signature genes comparing C/G-CB cells in EC coculture versus MC at week 6 of culture. ( E ) Pathways that are upregulated (left) and downregulated (right) in C/G-CB cells in EC coculture compared with MC. ( C – E ) n = 4 technical replicates for C/G-CB cells in EC coculture at week 6; n = 3 technical replicates for all other groups.

    Techniques Used: Cell Culture, Expressing

    Integrative transcriptomics of primary samples and C/G-CB identify FOLR1 therapeutic target. ( A ) Diagram of computational workflow to identify C/G-specific CAR targets. See Methods and Supplemental Figure 6 for details. Normal tissues include bulk bone marrow (BM) samples and peripheral blood (PB) CD34 + samples. ( B and C ) Expression of C/G-specific CAR targets in primary-fusion-positive patients versus normal BM (NBM) ( B ) and C/G-CB versus GFP-CB cells ( C ). CBFA2T3-GLIS2 primary samples ( n = 39); NBM samples ( n = 68). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. ( D ) Top: Gating strategies used to identify AML cells and normal lymphocytes, monocytes, and myeloid cells in 4 representative patients based on CD45 expression and SSC. Bottom: FOLR1 expression in the AML blast subpopulation versus normal cells. ( E ) Quantification of FOLR1 expression (geometric mean fluorescent intensity, MFI) among AML blasts and their normal counterparts across n = 15 patients. Autofluorescence was used as control. **** P
    Figure Legend Snippet: Integrative transcriptomics of primary samples and C/G-CB identify FOLR1 therapeutic target. ( A ) Diagram of computational workflow to identify C/G-specific CAR targets. See Methods and Supplemental Figure 6 for details. Normal tissues include bulk bone marrow (BM) samples and peripheral blood (PB) CD34 + samples. ( B and C ) Expression of C/G-specific CAR targets in primary-fusion-positive patients versus normal BM (NBM) ( B ) and C/G-CB versus GFP-CB cells ( C ). CBFA2T3-GLIS2 primary samples ( n = 39); NBM samples ( n = 68). For cultured cells, n = 4 technical replicates for C/G-CB cells in EC coculture at week; n = 3 technical replicates for all other groups. ( D ) Top: Gating strategies used to identify AML cells and normal lymphocytes, monocytes, and myeloid cells in 4 representative patients based on CD45 expression and SSC. Bottom: FOLR1 expression in the AML blast subpopulation versus normal cells. ( E ) Quantification of FOLR1 expression (geometric mean fluorescent intensity, MFI) among AML blasts and their normal counterparts across n = 15 patients. Autofluorescence was used as control. **** P

    Techniques Used: Expressing, Cell Culture

    C/G-CB cells induce leukemia, recapitulating primary disease. ( A ) Diagram of experimental design. ( B ) Kaplan-Meier survival curves of NSG-SGM3 mice transplanted with GFP-CB control and C/G-CB cells. Statistical differences in survival were evaluated using the Mantel-Cox log-rank test. n = 4 mice per group. ( C ) Representative histology of H E stain of femur taken from a mouse transplanted with C/G-CB cells (top) and cells from a C/G-positive patient sample (bottom) after development of leukemia. Original magnification, ×2.5 (left), ×40 (middle), and ×63 (right). See Supplemental Figure 1 for all H E stains from C/G-CB–transplanted mice. n = 4 mice for C/G-CB cells and n = 2 mice for PDX. ( D ) Expression of the RAM immunophenotype in C/G-CB cells harvested from the bone marrow (BM) of a representative mouse at necropsy compared to a primary patient sample and PDX marrow xenograft cells. In all 3 samples, malignant cells were gated based on human CD45 expression and SSC. n = 4 mice for C/G-CB cells, n = 2 mice for PDX derived from BM cells of patient B. ( E ) Left and middle: Representative immunohistochemistry showing high expression of ERG (×10 magnification) and CD56 (×5 magnification) in the femur of a representative mouse transplanted with C/G-CB cells. Right: Small aggregates of blasts with high CD56 expression detected in a BM biopsy of a chemotherapy-refractory C/G-fusion-positive patient, consistent with residual, adherent, patchy disease distribution (×100 magnification). n = 4 mice. ( F ) Kaplan-Meier plot showing survival in primary (1°, n = 4 mice), secondary (2°, n = 7 mice), and tertiary (3°, n = 5 mice) transplantations of C/G-CB cells. ( G ) Engraftment of C/G-CB cells in the BM at time of symptomatic leukemia, shown as percentage human CD45 + cells. Images on the right are H E stain of femurs taken from mice indicated by “a” and “b.” See Supplemental Figure 1 for all H E stains from C/G-CB–transplanted mice. ( H ) Quantification of CD56 + cells among human CD45 + cells isolated from the BM at necropsy following development of symptomatic leukemia. ( I ) Expression of AMKL markers, CD41 and CD42, in C/G-CB and PDX cells harvested from the BM at necropsy. C/G-CB cells were gated on human CD45 + cells. PDX cells were gated on human CD45 + CD56 + cells. ( J ) Quantification of CD41/CD42 subsets described in I . Bars indicate mean ± SEM. ( G – J ) 1°, n = 4 mice per group; 2°, n = 7 mice; and 3°, n = 5 mice.
    Figure Legend Snippet: C/G-CB cells induce leukemia, recapitulating primary disease. ( A ) Diagram of experimental design. ( B ) Kaplan-Meier survival curves of NSG-SGM3 mice transplanted with GFP-CB control and C/G-CB cells. Statistical differences in survival were evaluated using the Mantel-Cox log-rank test. n = 4 mice per group. ( C ) Representative histology of H E stain of femur taken from a mouse transplanted with C/G-CB cells (top) and cells from a C/G-positive patient sample (bottom) after development of leukemia. Original magnification, ×2.5 (left), ×40 (middle), and ×63 (right). See Supplemental Figure 1 for all H E stains from C/G-CB–transplanted mice. n = 4 mice for C/G-CB cells and n = 2 mice for PDX. ( D ) Expression of the RAM immunophenotype in C/G-CB cells harvested from the bone marrow (BM) of a representative mouse at necropsy compared to a primary patient sample and PDX marrow xenograft cells. In all 3 samples, malignant cells were gated based on human CD45 expression and SSC. n = 4 mice for C/G-CB cells, n = 2 mice for PDX derived from BM cells of patient B. ( E ) Left and middle: Representative immunohistochemistry showing high expression of ERG (×10 magnification) and CD56 (×5 magnification) in the femur of a representative mouse transplanted with C/G-CB cells. Right: Small aggregates of blasts with high CD56 expression detected in a BM biopsy of a chemotherapy-refractory C/G-fusion-positive patient, consistent with residual, adherent, patchy disease distribution (×100 magnification). n = 4 mice. ( F ) Kaplan-Meier plot showing survival in primary (1°, n = 4 mice), secondary (2°, n = 7 mice), and tertiary (3°, n = 5 mice) transplantations of C/G-CB cells. ( G ) Engraftment of C/G-CB cells in the BM at time of symptomatic leukemia, shown as percentage human CD45 + cells. Images on the right are H E stain of femurs taken from mice indicated by “a” and “b.” See Supplemental Figure 1 for all H E stains from C/G-CB–transplanted mice. ( H ) Quantification of CD56 + cells among human CD45 + cells isolated from the BM at necropsy following development of symptomatic leukemia. ( I ) Expression of AMKL markers, CD41 and CD42, in C/G-CB and PDX cells harvested from the BM at necropsy. C/G-CB cells were gated on human CD45 + cells. PDX cells were gated on human CD45 + CD56 + cells. ( J ) Quantification of CD41/CD42 subsets described in I . Bars indicate mean ± SEM. ( G – J ) 1°, n = 4 mice per group; 2°, n = 7 mice; and 3°, n = 5 mice.

    Techniques Used: Mouse Assay, Staining, Expressing, Derivative Assay, Immunohistochemistry, Isolation

    5) Product Images from "Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes"

    Article Title: Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

    Journal: bioRxiv

    doi: 10.1101/2022.11.14.516471

    Overexpression of PPM1H rescues AV transport in Lrrk2 -p.G2019S knock-in mouse cortical neurons. (A) Time lapse images of axonal mScarlet-LC3+ and GFP-PPM1H WT + vesicles in a p.G2019S KI mouse cortical neuron. Scale bar, 10 µm. (B) Kymographs of axonal mScarlet-LC3+ vesicles in p.G2019S KI mouse cortical neurons co-expressing GFP, GFP-PPM1H WT , or GFP-PPM1H H153D . Example AV traces are highlighted. (C-G) Pause number (C) and pause duration (D) of motile AVs, fraction of time paused (E) of all AVs, directional reversals (F) and Δ run length (G) of motile AVs in G2019S KI mouse cortical neurons transiently expressing GFP, GFP-PPM1H WT , or GFP-PPM1H H153D (mean ± SD for panel C and E; n = 66-107 motile AVs (C, D, F, G) and 68-111 total AVs (E) from 21-24 axons from 3 independent experiments; *p=0.01106; **p=0.00144; ***p
    Figure Legend Snippet: Overexpression of PPM1H rescues AV transport in Lrrk2 -p.G2019S knock-in mouse cortical neurons. (A) Time lapse images of axonal mScarlet-LC3+ and GFP-PPM1H WT + vesicles in a p.G2019S KI mouse cortical neuron. Scale bar, 10 µm. (B) Kymographs of axonal mScarlet-LC3+ vesicles in p.G2019S KI mouse cortical neurons co-expressing GFP, GFP-PPM1H WT , or GFP-PPM1H H153D . Example AV traces are highlighted. (C-G) Pause number (C) and pause duration (D) of motile AVs, fraction of time paused (E) of all AVs, directional reversals (F) and Δ run length (G) of motile AVs in G2019S KI mouse cortical neurons transiently expressing GFP, GFP-PPM1H WT , or GFP-PPM1H H153D (mean ± SD for panel C and E; n = 66-107 motile AVs (C, D, F, G) and 68-111 total AVs (E) from 21-24 axons from 3 independent experiments; *p=0.01106; **p=0.00144; ***p

    Techniques Used: Over Expression, Knock-In, Expressing

    6) Product Images from "A novel cell-based transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy"

    Article Title: A novel cell-based transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2022.101385

    Transplantation of GFP-rat SG cells to irradiated SG through the Wharton's duct. A , Establishment of irradiated SG rat models. Anesthetized rats were immobilized in a tube shielded with lead. Only the head and neck regions were exposed. The nude rats were locally irradiated in the head and neck regions with a single dose of 15 Gy. B , The catheter was inserted into Wharton's duct from the sublingual caruncle (red circle) to the submandibular gland. C , IHC of transplanted SGs was performed using anti-GFP antibody. GFP-positive cells were observed in the duct of the RT + SG cell group. D , AMY expression after RT in SGs. Low AMY expression was found in the RT group. AMY expression was recovered in the RT + SG cell group 12 weeks after the transplantation. E , SFR after the transplantation. SFR was significantly increased in the RT + SG cell group 12 weeks after the transplantation with SG cells (N = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Transplantation of GFP-rat SG cells to irradiated SG through the Wharton's duct. A , Establishment of irradiated SG rat models. Anesthetized rats were immobilized in a tube shielded with lead. Only the head and neck regions were exposed. The nude rats were locally irradiated in the head and neck regions with a single dose of 15 Gy. B , The catheter was inserted into Wharton's duct from the sublingual caruncle (red circle) to the submandibular gland. C , IHC of transplanted SGs was performed using anti-GFP antibody. GFP-positive cells were observed in the duct of the RT + SG cell group. D , AMY expression after RT in SGs. Low AMY expression was found in the RT group. AMY expression was recovered in the RT + SG cell group 12 weeks after the transplantation. E , SFR after the transplantation. SFR was significantly increased in the RT + SG cell group 12 weeks after the transplantation with SG cells (N = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Transplantation Assay, Irradiation, Immunohistochemistry, Expressing

    7) Product Images from "Survivin is a mechanosensitive cell cycle regulator in vascular smooth muscle cells"

    Article Title: Survivin is a mechanosensitive cell cycle regulator in vascular smooth muscle cells

    Journal: bioRxiv

    doi: 10.1101/2022.11.09.515885

    Survivin is required for stiffness-mediated cell cycle progression. ( A–G ) Human vascular smooth muscle cells (hVSMCs) were transfected with control siRNA or siRNAs to survivin [1 and #2], synchronized to G0 via serum starvation, and plated on soft or stiff hydrogels with 10% FBS for 24 h. Total cell lysates were analyzed by RT-qPCR ( A–C ) or immunoblotting ( D–G ) to determine mRNA and protein levels of survivin ( A and E ), cyclin D1 (CCND1; B and F ), and cyclin A (CCNA; C and G ). Expression levels were normalized to those in hVSMCs treated with control siRNA on stiff hydrogels. n = 4 (A), n = 3 (B), n = 3 (C), n = 5 (E), n = 3 (F), and n = 5 (G) independent experiments. S-phase entry with survivin knockdown and overexpression was assessed by EdU incorporation, which was normalized to hVSMCs treated with control siRNA on stiff hydrogels ( H ) or infected with GFP adenovirus (Adv) on soft hydrogels ( I ). n = 5 (H) and n = 5 (I) independent experiments. Data are means + SEMs. * p
    Figure Legend Snippet: Survivin is required for stiffness-mediated cell cycle progression. ( A–G ) Human vascular smooth muscle cells (hVSMCs) were transfected with control siRNA or siRNAs to survivin [1 and #2], synchronized to G0 via serum starvation, and plated on soft or stiff hydrogels with 10% FBS for 24 h. Total cell lysates were analyzed by RT-qPCR ( A–C ) or immunoblotting ( D–G ) to determine mRNA and protein levels of survivin ( A and E ), cyclin D1 (CCND1; B and F ), and cyclin A (CCNA; C and G ). Expression levels were normalized to those in hVSMCs treated with control siRNA on stiff hydrogels. n = 4 (A), n = 3 (B), n = 3 (C), n = 5 (E), n = 3 (F), and n = 5 (G) independent experiments. S-phase entry with survivin knockdown and overexpression was assessed by EdU incorporation, which was normalized to hVSMCs treated with control siRNA on stiff hydrogels ( H ) or infected with GFP adenovirus (Adv) on soft hydrogels ( I ). n = 5 (H) and n = 5 (I) independent experiments. Data are means + SEMs. * p

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Over Expression, Infection

    8) Product Images from "Gelatin coating enhances therapeutic cell adhesion to the infarcted myocardium via ECM binding"

    Article Title: Gelatin coating enhances therapeutic cell adhesion to the infarcted myocardium via ECM binding

    Journal: PLOS ONE

    doi: 10.1371/journal.pone.0277561

    A greater number of GelMA-coated BMCs are retained on decell heart tissue than uncoated BMC. A) Quantitative analysis of the number of BMC per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of coated versus uncoated GFP + BMC on decellularized tissue before and after rinsing. (n = 5 decell tissue per test group; p**
    Figure Legend Snippet: A greater number of GelMA-coated BMCs are retained on decell heart tissue than uncoated BMC. A) Quantitative analysis of the number of BMC per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of coated versus uncoated GFP + BMC on decellularized tissue before and after rinsing. (n = 5 decell tissue per test group; p**

    Techniques Used:

    Retention of uncoated BMC on glass substrates coated with purified ECM components. A) Mean number of BMCs per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of uncoated GFP + BMC on various ECM substrates before and after centrifugal rinsing. (n = 5 slides per test group; p*
    Figure Legend Snippet: Retention of uncoated BMC on glass substrates coated with purified ECM components. A) Mean number of BMCs per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of uncoated GFP + BMC on various ECM substrates before and after centrifugal rinsing. (n = 5 slides per test group; p*

    Techniques Used: Purification

    Retention of GelMA-coated BMC on glass substrates coated with purified ECM components. A) Mean number of BMC per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of GelMA coated GFP + BMC on various ECM substrates before and after rinsing. (n = 5 slides per test group; p*
    Figure Legend Snippet: Retention of GelMA-coated BMC on glass substrates coated with purified ECM components. A) Mean number of BMC per area determined by ImageJ before and after washing for coated versus uncoated BMC. B) Microscopic images of GelMA coated GFP + BMC on various ECM substrates before and after rinsing. (n = 5 slides per test group; p*

    Techniques Used: Purification

    BMC retention. A) Flow cytometric analysis of digested heart tissue demonstrates a higher percentage of GFP + cells in mice treated with coated cells compared to mice transplanted with uncoated cells. The majority of GFP cells were CD45 + . B) Quantitative analysis of GFP + BMC in digested heart tissue for coated versus uncoated cells by flow cytometry (signal in the PBS arm represents background). C) Immunohistochemical analysis demonstrated higher numbers of retained GFP + BMC in mice treated with coated cells. GFP expression is shown in green with DAPI nuclear staining in blue. D) Quantitative analysis of GFP + BMC in digested heart tissue for coated versus uncoated cells based on immunohistochemical analysis (signal in the PBS arm represents background). (N = 3 mice/group; * P
    Figure Legend Snippet: BMC retention. A) Flow cytometric analysis of digested heart tissue demonstrates a higher percentage of GFP + cells in mice treated with coated cells compared to mice transplanted with uncoated cells. The majority of GFP cells were CD45 + . B) Quantitative analysis of GFP + BMC in digested heart tissue for coated versus uncoated cells by flow cytometry (signal in the PBS arm represents background). C) Immunohistochemical analysis demonstrated higher numbers of retained GFP + BMC in mice treated with coated cells. GFP expression is shown in green with DAPI nuclear staining in blue. D) Quantitative analysis of GFP + BMC in digested heart tissue for coated versus uncoated cells based on immunohistochemical analysis (signal in the PBS arm represents background). (N = 3 mice/group; * P

    Techniques Used: Mouse Assay, Flow Cytometry, Immunohistochemistry, Expressing, Staining

    Expression of β1 integrins does not promote GelMA adhesion. A) Protocol for experiments examining the role of integrins in cell adhesion of GelMA coated cells. B) HUVEC and H9C2 cells analyzed by flow cytometry for β1 integrins by primary anti-β1 followed by secondary anti-AF647. C) Microscopic imagining of BMC incubated with cultured cells (HUVEC or H9C2) then subjected to rinsing by centrifugation at 800xg for 5 minutes. (n = 5 slides per test group). GFP expression of the BMC cells is shown in green and the HUVEC or H9C2 cells are shown in red using Cell Tracker Red.
    Figure Legend Snippet: Expression of β1 integrins does not promote GelMA adhesion. A) Protocol for experiments examining the role of integrins in cell adhesion of GelMA coated cells. B) HUVEC and H9C2 cells analyzed by flow cytometry for β1 integrins by primary anti-β1 followed by secondary anti-AF647. C) Microscopic imagining of BMC incubated with cultured cells (HUVEC or H9C2) then subjected to rinsing by centrifugation at 800xg for 5 minutes. (n = 5 slides per test group). GFP expression of the BMC cells is shown in green and the HUVEC or H9C2 cells are shown in red using Cell Tracker Red.

    Techniques Used: Expressing, Flow Cytometry, Incubation, Cell Culture, Centrifugation

    9) Product Images from "Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors"

    Article Title: Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors

    Journal: Genes

    doi: 10.3390/genes13112056

    In vitro transduction studies with Ad5/3(L) and Ad5/3(S) vectors. ( A ) Vector structure. Upper panel: Vector capsids. Both vectors are based on Ad5. Ad5/3(S) contains the Ad5 fiber tail, the “short” (S) Ad3 fiber shaft and the Ad3 fiber knob. (Ad fibers are homotrimers, which are depicted by three fiber knobs.) Ad5/3(L) contains the original “long” (L) Ad5 fiber shaft and Ad3 fiber knob. Lower panel: Two sets of E1/E3 deleted (first-generation) Ad5/3(S) and Ad5/3(L) vectors were generated containing either a CMV promoter-driven GFP gene (Ad5/3-GFP(S) and Ad5/3-GFP(L) or a CMV promoter-driven firefly luciferase gene (Ad5/3-luc(S) and Ad5/3-luc(L)). SV40pA: SV40 polyadenylation signal. ITR: adenoviral inverted repeat. ψ: adenoviral packaging signal. ( B ) Competition of Ad5/3-GFP(S) and Ad5/3-GFP(L) by recombinant Ad3 fiber knob (JO4) or rDSG2. Colon cancer T84 cells were incubated with 50 μg/mL of proteins for 1 h. Cells were then infected with viruses at an MOI of 100 pfu/cell. Viruses were removed 1 h after infection and GFP expression was analyzed ~20 h later by flow cytometry. Cntr: untransduced control cells.
    Figure Legend Snippet: In vitro transduction studies with Ad5/3(L) and Ad5/3(S) vectors. ( A ) Vector structure. Upper panel: Vector capsids. Both vectors are based on Ad5. Ad5/3(S) contains the Ad5 fiber tail, the “short” (S) Ad3 fiber shaft and the Ad3 fiber knob. (Ad fibers are homotrimers, which are depicted by three fiber knobs.) Ad5/3(L) contains the original “long” (L) Ad5 fiber shaft and Ad3 fiber knob. Lower panel: Two sets of E1/E3 deleted (first-generation) Ad5/3(S) and Ad5/3(L) vectors were generated containing either a CMV promoter-driven GFP gene (Ad5/3-GFP(S) and Ad5/3-GFP(L) or a CMV promoter-driven firefly luciferase gene (Ad5/3-luc(S) and Ad5/3-luc(L)). SV40pA: SV40 polyadenylation signal. ITR: adenoviral inverted repeat. ψ: adenoviral packaging signal. ( B ) Competition of Ad5/3-GFP(S) and Ad5/3-GFP(L) by recombinant Ad3 fiber knob (JO4) or rDSG2. Colon cancer T84 cells were incubated with 50 μg/mL of proteins for 1 h. Cells were then infected with viruses at an MOI of 100 pfu/cell. Viruses were removed 1 h after infection and GFP expression was analyzed ~20 h later by flow cytometry. Cntr: untransduced control cells.

    Techniques Used: In Vitro, Transduction, Plasmid Preparation, Generated, Luciferase, Recombinant, Incubation, Infection, Expressing, Flow Cytometry

    In vitro transduction studies. ( A ) T84 cells were infected with Ad5/3-GFP viruses at the indicated MOIs. GFP expression was analyzed 20 h after infection by flow cytometry. The left panel shows the percentage of GFP-positive cells; the right panel shows the mean GFP fluorescence intensity (MFI). n = 3. * p
    Figure Legend Snippet: In vitro transduction studies. ( A ) T84 cells were infected with Ad5/3-GFP viruses at the indicated MOIs. GFP expression was analyzed 20 h after infection by flow cytometry. The left panel shows the percentage of GFP-positive cells; the right panel shows the mean GFP fluorescence intensity (MFI). n = 3. * p

    Techniques Used: In Vitro, Transduction, Infection, Expressing, Flow Cytometry, Fluorescence

    Biodistribution of GFP expression after intravenous Ad5/3 injection. ( A ) In vivo GFP imaging. TC1-DSG2 + tumor-bearing non-DSG2 transgenic mice (litter mates) and DSG2-transgenic mice were intravenously injected via the tail with pfu of Ad5/3-GFP(L) or Ad5/3-GFP(S). Mice were subjected to imaging 3 days later. Shown are two mice injected with Ad5/3-GFP(L) and four mice injected with Ad5/3-GFP(S) from the font and the back. The color reflects the radian efficiency, the range of which is indicated in the bars to the right of the images. The tag numbers are indicated below the images. ( B ) GFP and DSG2 immunohistochemistry analyses of selected organs and the tumor harvested on day 3 after Ad5/3-GFP (L) injection. GFP staining appears brown. The scale bar is 20 μm. Shown are representative sections of mouse #990. DSG2 staining of tumor sections is shown in Figure 4 A.
    Figure Legend Snippet: Biodistribution of GFP expression after intravenous Ad5/3 injection. ( A ) In vivo GFP imaging. TC1-DSG2 + tumor-bearing non-DSG2 transgenic mice (litter mates) and DSG2-transgenic mice were intravenously injected via the tail with pfu of Ad5/3-GFP(L) or Ad5/3-GFP(S). Mice were subjected to imaging 3 days later. Shown are two mice injected with Ad5/3-GFP(L) and four mice injected with Ad5/3-GFP(S) from the font and the back. The color reflects the radian efficiency, the range of which is indicated in the bars to the right of the images. The tag numbers are indicated below the images. ( B ) GFP and DSG2 immunohistochemistry analyses of selected organs and the tumor harvested on day 3 after Ad5/3-GFP (L) injection. GFP staining appears brown. The scale bar is 20 μm. Shown are representative sections of mouse #990. DSG2 staining of tumor sections is shown in Figure 4 A.

    Techniques Used: Expressing, Injection, In Vivo, Imaging, Transgenic Assay, Mouse Assay, Immunohistochemistry, Staining

    10) Product Images from "Microprism-based two-photon imaging of the lateral cortex of the mouse inferior colliculus reveals novel organizational principles of the auditory midbrain"

    Article Title: Microprism-based two-photon imaging of the lateral cortex of the mouse inferior colliculus reveals novel organizational principles of the auditory midbrain

    Journal: bioRxiv

    doi: 10.1101/2022.11.05.515308

    The response of the LC vs DC to different acoustic stimulations. (A and D) The superimposed 2P images of both GFP and jRGECO1a signals from the LC imaged via a microprism and DC imaged from the surface, respectively, across different animals. (B and E) Aligned with the 2P images in a and b, the pseudocolor images show the activity of the cells on the LC and the DC, respectively, across different animals in response to the pure tone based on their best frequency as graded green circles for the responsive cells and solid red circles for the non-responsive cells. (C and F) Aligned with the 2P images in a and b, the pseudocolor images show the activity of the cells on the LC and the DC, respectively, across different animals in response to AM-noise based on their best modulation frequency as graded green circles for the responsive cells and solid red circles for the non-responsive cells. All solid irregular white lines were made at the border of the GABAergic modules.
    Figure Legend Snippet: The response of the LC vs DC to different acoustic stimulations. (A and D) The superimposed 2P images of both GFP and jRGECO1a signals from the LC imaged via a microprism and DC imaged from the surface, respectively, across different animals. (B and E) Aligned with the 2P images in a and b, the pseudocolor images show the activity of the cells on the LC and the DC, respectively, across different animals in response to the pure tone based on their best frequency as graded green circles for the responsive cells and solid red circles for the non-responsive cells. (C and F) Aligned with the 2P images in a and b, the pseudocolor images show the activity of the cells on the LC and the DC, respectively, across different animals in response to AM-noise based on their best modulation frequency as graded green circles for the responsive cells and solid red circles for the non-responsive cells. All solid irregular white lines were made at the border of the GABAergic modules.

    Techniques Used: Activity Assay

    The validation of imaging setup. All images were taken for the GFP signals exclusively expressed in GABAergic cells. (A) A low magnification image of the IC surface with the microprism showing a laser lesion spot made through the microprism to validate the position of the microprism relative to the IC. (B) A histological coronal section at the level of the IC showing that the laser lesion made by the microprism was located at the sagittal surface of the LC. (C-D) Low magnification images of the IC surface without the microprism showing a laser lesion spot made on the dorsal surface of the LC near one of the GABAergic modules. The dotted white lines represent the alignment of the laser lesion to the location of the GABAergic modules at the lateral top surface. (E) A histological coronal section at the level of the IC showing that the laser lesion made on the IC surface targeted one of the GABAergic modules at the on the dorsal portion of the LC. The solid irregular white line was made at the border of the GABAergic modules.
    Figure Legend Snippet: The validation of imaging setup. All images were taken for the GFP signals exclusively expressed in GABAergic cells. (A) A low magnification image of the IC surface with the microprism showing a laser lesion spot made through the microprism to validate the position of the microprism relative to the IC. (B) A histological coronal section at the level of the IC showing that the laser lesion made by the microprism was located at the sagittal surface of the LC. (C-D) Low magnification images of the IC surface without the microprism showing a laser lesion spot made on the dorsal surface of the LC near one of the GABAergic modules. The dotted white lines represent the alignment of the laser lesion to the location of the GABAergic modules at the lateral top surface. (E) A histological coronal section at the level of the IC showing that the laser lesion made on the IC surface targeted one of the GABAergic modules at the on the dorsal portion of the LC. The solid irregular white line was made at the border of the GABAergic modules.

    Techniques Used: Imaging

    The distribution of GABAergic cells om the LC imaged via microprism, and DC imaged from the surface. All images were taken for the GFP signals exclusively expressed in GABAergic cells. (A-B) Low magnification images for the GFP signals obtained from the IC surface with or without the microprism, respectively. The dotted white line in b represented the medial and lateral horizons of the IC. (C-D and G) The 2P images of the GABAergic cells on the LC imaged via microprism showing the GABAergic modules across multiple animals. (E and H) The 2P images of the GABAergic cells on the lateral top surface of the IC imaged from above showing the GABAergic modules across multiple animals (The red box in B). (F and I) The 2P images of the GABAergic cells on the medial dorsal surface of the IC imaged from above showing a homogenous distribution of GABAergic cells of different size with no signs of the GABAergic modules across multiple animals (The blue box in B). All solid irregular white lines were made at the border of the GABAergic modules; D: Dorsal, L: Lateral, R: Rostral.
    Figure Legend Snippet: The distribution of GABAergic cells om the LC imaged via microprism, and DC imaged from the surface. All images were taken for the GFP signals exclusively expressed in GABAergic cells. (A-B) Low magnification images for the GFP signals obtained from the IC surface with or without the microprism, respectively. The dotted white line in b represented the medial and lateral horizons of the IC. (C-D and G) The 2P images of the GABAergic cells on the LC imaged via microprism showing the GABAergic modules across multiple animals. (E and H) The 2P images of the GABAergic cells on the lateral top surface of the IC imaged from above showing the GABAergic modules across multiple animals (The red box in B). (F and I) The 2P images of the GABAergic cells on the medial dorsal surface of the IC imaged from above showing a homogenous distribution of GABAergic cells of different size with no signs of the GABAergic modules across multiple animals (The blue box in B). All solid irregular white lines were made at the border of the GABAergic modules; D: Dorsal, L: Lateral, R: Rostral.

    Techniques Used:

    11) Product Images from "Insights into cargo sorting by SNX32 in neuronal and non-neuronal cells: physiological implications in neurite outgrowth"

    Article Title: Insights into cargo sorting by SNX32 in neuronal and non-neuronal cells: physiological implications in neurite outgrowth

    Journal: bioRxiv

    doi: 10.1101/2022.11.04.515170

    Interplay of SNX32, SNX4 and Rab11 in transferrin trafficking: A-C) Followed by SMARTpool mediated gene down regulation of (A)SNX4, (B)SNX4 and SNX32 (C) SNX4KD and over expression of GFP-SNX32, the transferrin (Alexa Fluor 568 conjugated) Pulse-Chase e xperiment was carried out as described in materials and method section, the cells were fixed at specified timepoints, immunostained using early endosomal marker EEA1, DAPI was used to stain nucleus, Scale 10µm, inset 5µm (magnified regions are shown as insets).D) Quantification of percentage localization of transferrin (Alexa Fluor 568 conjugated) with EEA1 at corresponding time points, data represent mean ±SEM (N=3, Q≥15 random frames per independent experiments), P value
    Figure Legend Snippet: Interplay of SNX32, SNX4 and Rab11 in transferrin trafficking: A-C) Followed by SMARTpool mediated gene down regulation of (A)SNX4, (B)SNX4 and SNX32 (C) SNX4KD and over expression of GFP-SNX32, the transferrin (Alexa Fluor 568 conjugated) Pulse-Chase e xperiment was carried out as described in materials and method section, the cells were fixed at specified timepoints, immunostained using early endosomal marker EEA1, DAPI was used to stain nucleus, Scale 10µm, inset 5µm (magnified regions are shown as insets).D) Quantification of percentage localization of transferrin (Alexa Fluor 568 conjugated) with EEA1 at corresponding time points, data represent mean ±SEM (N=3, Q≥15 random frames per independent experiments), P value

    Techniques Used: Over Expression, Pulse Chase, Marker, Staining

    SNX32 undergoes PX domain assisted localization to PI(4)P enriched endosomal membranes in addition to early endosomes (A) GFP-SNX32 localization with early endosomal marker EEA1, (B) Quantifications showing percentage co-localization of GFP-SNX32 in HeLa cells, data represent mean ±SEM (N=3, n≥60cells per independent experiments) (C) GFP-SNX32 localization at cell periphery (D) GFP-SNX32 colocalization with peri-nuclear recycling endosomal marker mCherry-Rab11, (E) colocalization of GFP-SNX32 with Trans Golgi Network marker TGN46, Scale bar 10µm, inset 5µm (magnified regions are shown as insets). F-G) SIM image showing co-localization of F) GFP-GOLPH3 and mCherry-SNX32, G) GFP-SNX32 and mCherry-Rab11, Scale bar 10µm, inset 1µm (magnified regions are shown as insets). H) PIP Strip membrane immunoblotted using His antibody showing preferential binding of His-SNX32ΔC to PI(3)P, PI(4)P, PI(5)P, PA (representative immunoblot out of 3 biological replicates). I-N) PAO/DMSO treatment in HeLa cells overexpressing I) GFP-PH OSBP , J) RFP-PH PLCį , K) endogenous EEA1, L) HA-SNX32ΔC, M) HA-SNX32ΔN, N) HA-SNX32FL, Scale bar 10µm.
    Figure Legend Snippet: SNX32 undergoes PX domain assisted localization to PI(4)P enriched endosomal membranes in addition to early endosomes (A) GFP-SNX32 localization with early endosomal marker EEA1, (B) Quantifications showing percentage co-localization of GFP-SNX32 in HeLa cells, data represent mean ±SEM (N=3, n≥60cells per independent experiments) (C) GFP-SNX32 localization at cell periphery (D) GFP-SNX32 colocalization with peri-nuclear recycling endosomal marker mCherry-Rab11, (E) colocalization of GFP-SNX32 with Trans Golgi Network marker TGN46, Scale bar 10µm, inset 5µm (magnified regions are shown as insets). F-G) SIM image showing co-localization of F) GFP-GOLPH3 and mCherry-SNX32, G) GFP-SNX32 and mCherry-Rab11, Scale bar 10µm, inset 1µm (magnified regions are shown as insets). H) PIP Strip membrane immunoblotted using His antibody showing preferential binding of His-SNX32ΔC to PI(3)P, PI(4)P, PI(5)P, PA (representative immunoblot out of 3 biological replicates). I-N) PAO/DMSO treatment in HeLa cells overexpressing I) GFP-PH OSBP , J) RFP-PH PLCį , K) endogenous EEA1, L) HA-SNX32ΔC, M) HA-SNX32ΔN, N) HA-SNX32FL, Scale bar 10µm.

    Techniques Used: Marker, Stripping Membranes, Binding Assay

    BAR domain of SNX32 undergoes protein-protein interactions and assist in the early endosomal localization of SNX32: A) Relative amount of SNX32 transcripts analysed by quantitative PCR in different cell lines such as HeLa, U87MG and Neuro2a normalised with respect to Gapdh. B-D) Localization of HA SNX32ΔN with B) early endosomal marker EEA1 C) GFP SNX1, EEA1 G) GFP-SNX4, EEA1: Scale bar 10µm, inset 5µm (magnified regions are shown as insets). E) HA SNX32ΔC colocalizes with GFP PH OSBP , protein module showing preferential association with PI(4)P, Scale bar 10µm, inset 5µm (magnified regions are shown as insets). F) Quantifications showing percentage cRlRcali]aWiRQ Rf HA SNX32ΔN/ HA SNX32ΔC with respective compartment markers in HeLa cells, data represent mean ±SEM (N=3, n≥60cells per independent experiments). G-L) Wortmannin/DMSO treatment in HeLa cells overexpressing G) endogenous EEA1, H) RFP-PH PLCδ , I) GFP-PH OSBP , J) HA-SNX32ΔN, K) HA-SNX32ΔC, L) HA-SNX32FL, Scale bar 10µm. M)Membrane-Cytosol fractions of HeLa cells transiently transfected with HA-SNX32ΔC or GFP-PH OSBP showing PAO treatment causes delocalization of HA-SNX32ΔC and GFP-PH OSBP proteins to the cytosolic fraction, S-Cytosol, P-membrane fraction (representative immunoblot out of 3 biological replicates, values represent the ratio of S to P fractions normalised to vinculin).
    Figure Legend Snippet: BAR domain of SNX32 undergoes protein-protein interactions and assist in the early endosomal localization of SNX32: A) Relative amount of SNX32 transcripts analysed by quantitative PCR in different cell lines such as HeLa, U87MG and Neuro2a normalised with respect to Gapdh. B-D) Localization of HA SNX32ΔN with B) early endosomal marker EEA1 C) GFP SNX1, EEA1 G) GFP-SNX4, EEA1: Scale bar 10µm, inset 5µm (magnified regions are shown as insets). E) HA SNX32ΔC colocalizes with GFP PH OSBP , protein module showing preferential association with PI(4)P, Scale bar 10µm, inset 5µm (magnified regions are shown as insets). F) Quantifications showing percentage cRlRcali]aWiRQ Rf HA SNX32ΔN/ HA SNX32ΔC with respective compartment markers in HeLa cells, data represent mean ±SEM (N=3, n≥60cells per independent experiments). G-L) Wortmannin/DMSO treatment in HeLa cells overexpressing G) endogenous EEA1, H) RFP-PH PLCδ , I) GFP-PH OSBP , J) HA-SNX32ΔN, K) HA-SNX32ΔC, L) HA-SNX32FL, Scale bar 10µm. M)Membrane-Cytosol fractions of HeLa cells transiently transfected with HA-SNX32ΔC or GFP-PH OSBP showing PAO treatment causes delocalization of HA-SNX32ΔC and GFP-PH OSBP proteins to the cytosolic fraction, S-Cytosol, P-membrane fraction (representative immunoblot out of 3 biological replicates, values represent the ratio of S to P fractions normalised to vinculin).

    Techniques Used: Real-time Polymerase Chain Reaction, Marker, Transfection

    SNX32 but not SNX6 plays a significant role in surface localization of BSG: A) U87MG cells showing the colocalization of GFP-SNX32 with endogenous ARF6 and BSG on membrane, Scale 10µm, inset 5µm (magnified regions are shown as insets). B) Representative line intensity plot of U87MG cell showing intensity overlap of GFP-SNX32, ARF6 and BSG.C) Neuro2a cells showing colocalization of GFP SNX 32 with cMyc-BSG on vesicles, Scale 10µm, inset 5µm (magnified regions are shown as insets). D-F) Snap shots from live TIRF microscopic imaging of Neuro2a cells stably expressing pHluorin BSG transfected with SCR, SNX32 or SNX6 siRNA SMARTpool followed by doxycycline treatment for pHluorin BSG induction, Scale 10µm. G) Quantification of surface population of normalised of BSG vesicles, (N=3, n≥6cells per independent experiments), P value 0.0003(**** P
    Figure Legend Snippet: SNX32 but not SNX6 plays a significant role in surface localization of BSG: A) U87MG cells showing the colocalization of GFP-SNX32 with endogenous ARF6 and BSG on membrane, Scale 10µm, inset 5µm (magnified regions are shown as insets). B) Representative line intensity plot of U87MG cell showing intensity overlap of GFP-SNX32, ARF6 and BSG.C) Neuro2a cells showing colocalization of GFP SNX 32 with cMyc-BSG on vesicles, Scale 10µm, inset 5µm (magnified regions are shown as insets). D-F) Snap shots from live TIRF microscopic imaging of Neuro2a cells stably expressing pHluorin BSG transfected with SCR, SNX32 or SNX6 siRNA SMARTpool followed by doxycycline treatment for pHluorin BSG induction, Scale 10µm. G) Quantification of surface population of normalised of BSG vesicles, (N=3, n≥6cells per independent experiments), P value 0.0003(**** P

    Techniques Used: Imaging, Stable Transfection, Expressing, Transfection

    SNX32 undergoes BAR domain mediated association with SNX1: A) The domain architecture indicating the N- and C-terminal endpoints of SNX32FL, SNX32ΔC and SNX32ΔN. B) Co-immunoprecipitation of GFP /HA-tagged SNX–proteins transiently transfected in HEK293T cells showing the coprecipitation of GFP-SNX1 and HA-SNX32ΔN, GBP immunoprecipitation was carried out as described in the materials and methods section, immunoblotted using GFP and HA antibody. C)Coomassie blue-stained SDS-PAGE gel of affinity purification profile of His SNX32ΔC, samples representing each step of purification, FT: Flow-through. D) Coomassie blue-stained SDS-PAGE gel of His-SNX32 FL, His-SNX32ΔC, GSTSNX32FL, GST-SNX32 ΔC after induction, P: pellet and S: supernatant fractions. E) Coomassie blue-stained SDS-PAGE gel of affinity co-purification profile of GST-SNX1/His-SNX32ΔN, samples representing each step of purification, FT: Flow-through. F) homology model of SNX32 BAR domain(cyan) in complex with SNX1 BAR domain(green). G) Interacting amino acid residues present in the dimeric interface of SNX32(cyan)-SNX1(green). H) Snap shots of polar interactions across the dimeric interface. I)homology model of SNX32 BAR homodimer J) Interacting amino acid residues present in the dimeric interface of SNX32(cyan)-SN32(red). K) Snap shots of polar interactions across the dimeric interface
    Figure Legend Snippet: SNX32 undergoes BAR domain mediated association with SNX1: A) The domain architecture indicating the N- and C-terminal endpoints of SNX32FL, SNX32ΔC and SNX32ΔN. B) Co-immunoprecipitation of GFP /HA-tagged SNX–proteins transiently transfected in HEK293T cells showing the coprecipitation of GFP-SNX1 and HA-SNX32ΔN, GBP immunoprecipitation was carried out as described in the materials and methods section, immunoblotted using GFP and HA antibody. C)Coomassie blue-stained SDS-PAGE gel of affinity purification profile of His SNX32ΔC, samples representing each step of purification, FT: Flow-through. D) Coomassie blue-stained SDS-PAGE gel of His-SNX32 FL, His-SNX32ΔC, GSTSNX32FL, GST-SNX32 ΔC after induction, P: pellet and S: supernatant fractions. E) Coomassie blue-stained SDS-PAGE gel of affinity co-purification profile of GST-SNX1/His-SNX32ΔN, samples representing each step of purification, FT: Flow-through. F) homology model of SNX32 BAR domain(cyan) in complex with SNX1 BAR domain(green). G) Interacting amino acid residues present in the dimeric interface of SNX32(cyan)-SNX1(green). H) Snap shots of polar interactions across the dimeric interface. I)homology model of SNX32 BAR homodimer J) Interacting amino acid residues present in the dimeric interface of SNX32(cyan)-SN32(red). K) Snap shots of polar interactions across the dimeric interface

    Techniques Used: Immunoprecipitation, Transfection, Staining, SDS Page, Affinity Purification, Purification, Copurification

    SNX32 undergoes BAR domain-mediated association with SNX4 A) GBP co-immunoprecipitation of GFP /HA-tagged SNX–proteins transiently transfected in HEK293T cells showing GFP-SNX1/ GFP-SNX4/ GFP-SNX8 or GFP-SNX32 efficiently precipitating HA-SNX32 (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity). B) Co-immunoprecipitation of GFP/HA-tagged SNX–proteins transiently transfected in HEK293T cells showing GFP-SNX4 precipitating HA-SNX32ΔN (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity). C)homology model of BAR domains of SNX32-SNX4 complex. D) Schematic depiction of the amino acid residues lining the heterodimeric interface of SNX32(cyan)-SNX4(magenta). E) Polar interactions present at the dimeric interface of SNX32(cyan)-SNX4(magenta). F) GBP co-immunoprecipitation of GFP/HA-tagged SNX– proteins transiently transfected in HeLa cells showing difference in the amount of GFP-SNX4 precipitated HA-SNX32 mutants (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity).
    Figure Legend Snippet: SNX32 undergoes BAR domain-mediated association with SNX4 A) GBP co-immunoprecipitation of GFP /HA-tagged SNX–proteins transiently transfected in HEK293T cells showing GFP-SNX1/ GFP-SNX4/ GFP-SNX8 or GFP-SNX32 efficiently precipitating HA-SNX32 (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity). B) Co-immunoprecipitation of GFP/HA-tagged SNX–proteins transiently transfected in HEK293T cells showing GFP-SNX4 precipitating HA-SNX32ΔN (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity). C)homology model of BAR domains of SNX32-SNX4 complex. D) Schematic depiction of the amino acid residues lining the heterodimeric interface of SNX32(cyan)-SNX4(magenta). E) Polar interactions present at the dimeric interface of SNX32(cyan)-SNX4(magenta). F) GBP co-immunoprecipitation of GFP/HA-tagged SNX– proteins transiently transfected in HeLa cells showing difference in the amount of GFP-SNX4 precipitated HA-SNX32 mutants (representative immunoblot out of 3 biological replicates, values represent the ratio of HA to GFP band intensity).

    Techniques Used: Immunoprecipitation, Transfection

    PX domain of SNX32 participates in the interaction with CIMPR, TfR and F131 of SNX32 is critical for interaction with cargo: A) GBP co-immunoprecipitation of GFP tagged SNX4 and SNX32 transiently transfected in HEK293T cells showing GFP-SNX32 efficiently precipitating TfR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of TfR to GFP band intensity). B) GBP co-immunoprecipitation of GFP tagged SNX1 and SNX32 transiently transfected in HEK293T cells showing GFP-SNX32 efficiently precipitating CIMPR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of CIMPR to GFP band intensity).C) His affinity chromatography-based pulldown showing His-SNX32ΔC precipitating TfR from membrane enriched HeLa cell lysate fraction, His pulldown was carried out as described in material method section and immunoblotted using His and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of His to TfR band intensity). D) His affinity chromatography-based pulldown showing His-SNX32ΔC precipitating CIMPR from membrane enriched HeLa cell lysate fraction, His pulldown was carried out as described in material method section and immunoblotted using His and TfR antibody (representative immunoblot out of 3 biological replicates). E)Cargo/IncE binding site of SNX32 PX domain as observed in crystal structure (PDB ID:6E8R) reported by Chandra et.,al 16 , inset showing the stacking interaction between F131 of SNX32 and F116 of IncE F) Co-immunoprecipitation of GFP tagged SNX32 wild type (WT) and GFP trap of GFP-tagged SNX32 WT/ SNX32 F131D, showing both efficiently precipitating ESCPE-1 sub unit SNX1 whereas SNX32 F131D failed to precipitate CIMPR, each transiently transfected in HEK293T cells. The elute was resolved in SDS-PAGE and immunoblotted using GFP, SNX1 and CIMPR antibody G) GBP co-immunoprecipitation of GFP tagged SNX–proteins transiently transfected in HeLa cells showing GFP-SNX32 but not GFP-SNX32 F131D efficiently pulling down TfR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of TfR to GFP band intensity).
    Figure Legend Snippet: PX domain of SNX32 participates in the interaction with CIMPR, TfR and F131 of SNX32 is critical for interaction with cargo: A) GBP co-immunoprecipitation of GFP tagged SNX4 and SNX32 transiently transfected in HEK293T cells showing GFP-SNX32 efficiently precipitating TfR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of TfR to GFP band intensity). B) GBP co-immunoprecipitation of GFP tagged SNX1 and SNX32 transiently transfected in HEK293T cells showing GFP-SNX32 efficiently precipitating CIMPR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of CIMPR to GFP band intensity).C) His affinity chromatography-based pulldown showing His-SNX32ΔC precipitating TfR from membrane enriched HeLa cell lysate fraction, His pulldown was carried out as described in material method section and immunoblotted using His and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of His to TfR band intensity). D) His affinity chromatography-based pulldown showing His-SNX32ΔC precipitating CIMPR from membrane enriched HeLa cell lysate fraction, His pulldown was carried out as described in material method section and immunoblotted using His and TfR antibody (representative immunoblot out of 3 biological replicates). E)Cargo/IncE binding site of SNX32 PX domain as observed in crystal structure (PDB ID:6E8R) reported by Chandra et.,al 16 , inset showing the stacking interaction between F131 of SNX32 and F116 of IncE F) Co-immunoprecipitation of GFP tagged SNX32 wild type (WT) and GFP trap of GFP-tagged SNX32 WT/ SNX32 F131D, showing both efficiently precipitating ESCPE-1 sub unit SNX1 whereas SNX32 F131D failed to precipitate CIMPR, each transiently transfected in HEK293T cells. The elute was resolved in SDS-PAGE and immunoblotted using GFP, SNX1 and CIMPR antibody G) GBP co-immunoprecipitation of GFP tagged SNX–proteins transiently transfected in HeLa cells showing GFP-SNX32 but not GFP-SNX32 F131D efficiently pulling down TfR, GBP immunoprecipitation was carried out as described in materials and methods section and immunoblotted using GFP and TfR antibody (representative immunoblot out of 3 biological replicates, values represent the ratio of TfR to GFP band intensity).

    Techniques Used: Immunoprecipitation, Transfection, Affinity Chromatography, Binding Assay, SDS Page

    12) Product Images from "Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer"

    Article Title: Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1029269

    Generation and validation of TCEs targeting CRC cells to encode into oncolytic vaccinia virus as an optimal delivery system. (A) Two novel TCEs were designed by linking a scFv that binds human CEA on the surface of cancer cells to a scFv that recognizes CD3ε on either murine T cells (αCEA:mCD3) or human T cells (αCEA:hCD3) which can be CD4 or CD8 positive. (B) HEK293T cells were transiently transfected with αCEA TCE constructs or pcDNA3.1 empty vector control (EV) and incubated for 48 h. TCE-containing supernatants were collected, spun down to remove cell debris, and concentrated using centrifugal filter units with a 10 kDa molecular weight cutoff. Samples were quantified by BCA assay to load 10 µg of supernatant per lane, separated by SDS-PAGE, and immunoblots were probed with a His antibody to detect His-tagged TCEs. No His tag was detected for the EV control as expected. (C) HT-29 cells were co-cultured with human PBMCs (E:T = 5:1) and αCEA TCE (αCEA:hCD3) or αCEA CTRL (αCEA:mCD3) at indicated concentrations. Resazurin assay was performed at 72 h to determine cancer cell viability after TCE treatment. Results show relative % ± SEM. (D) COLO 205 cells were co-cultured with human PBMCs (E:T = 5:1) and αCEA TCE (αCEA:hCD3) or αCEA CTRL (αCEA:mCD3) at indicated concentrations. Resazurin assay was performed at 72 h to determine cancer cell viability after TCE treatment. Results show relative % ± SEM. (E) In co-cultures with HT-29 cells that express CEA and Jurkat CD69-tdTomato reporter cells (J69; T cells were modified by CRISPR to express tdTomato under the control of the CD69 promoter), the addition of TCEs (1 µg) leads to the visualization of tdTomato-positive Jurkat J69 cells (E:T = 1:1). Scale bar = 400 µm. (F) Three patient-derived CEA-positive colorectal cancer cell lines and other CEA-positive cells (BxPC-3, HT-29, COLO 205) activate J69 cells in co-cultures with αCEA TCE (αCEA:hCD3), but not with αCEA CTRL (αCEA:mCD3) or CEA-negative control cell lines (MIA PaCa-2, HCT 116). Results show MFI ± standard error of the mean (SEM); Two-way ANOVA with Sidak’s correction for multiple comparisons. (G) HT-29, COLO 205, SW620 and MC38 WT spheroids were infected at an MOI 1 of oncolytic vaccinia virus (Copenhagen/Cop strain) VV-CTRL, oncolytic Vesicular Stomatitis virus (VSVΔ51), oncolytic Herpes simplex virus (HSV), oncolytic Measles virus (MeV), or oncolytic Adenovirus (AdV). Spheroids were imaged at 48 hpi to detect transgene expression of enhanced green fluorescent protein (eGFP) or red fluorescent protein (RFP). All colorectal cancer spheroids expressed abundant eGFP levels after VV-CTRL infection, compared to other viruses. (H) Spheroid viability was assessed in triplicate by resazurin assay at 120 hpi, relative to uninfected controls. VV-CTRL decreased cell viability of HT-29, COLO205, SW620 and MC38 WT spheroids. vaccinia virus was able to infect all the colorectal cancer cell lines as spheroids. VV-CTRL decreased cell viability of HT-29, COLO205, SW620 and MC38 WT spheroids. Other viruses also decreased spheroid viability to a lesser extent. Of note, MeV did not significantly change the viability of MC38 WT spheroids, as expected, since this oncolytic virus do not infect murine cancer cell lines but served as a control. Results show relative % ± SEM; Two-way ANOVA.
    Figure Legend Snippet: Generation and validation of TCEs targeting CRC cells to encode into oncolytic vaccinia virus as an optimal delivery system. (A) Two novel TCEs were designed by linking a scFv that binds human CEA on the surface of cancer cells to a scFv that recognizes CD3ε on either murine T cells (αCEA:mCD3) or human T cells (αCEA:hCD3) which can be CD4 or CD8 positive. (B) HEK293T cells were transiently transfected with αCEA TCE constructs or pcDNA3.1 empty vector control (EV) and incubated for 48 h. TCE-containing supernatants were collected, spun down to remove cell debris, and concentrated using centrifugal filter units with a 10 kDa molecular weight cutoff. Samples were quantified by BCA assay to load 10 µg of supernatant per lane, separated by SDS-PAGE, and immunoblots were probed with a His antibody to detect His-tagged TCEs. No His tag was detected for the EV control as expected. (C) HT-29 cells were co-cultured with human PBMCs (E:T = 5:1) and αCEA TCE (αCEA:hCD3) or αCEA CTRL (αCEA:mCD3) at indicated concentrations. Resazurin assay was performed at 72 h to determine cancer cell viability after TCE treatment. Results show relative % ± SEM. (D) COLO 205 cells were co-cultured with human PBMCs (E:T = 5:1) and αCEA TCE (αCEA:hCD3) or αCEA CTRL (αCEA:mCD3) at indicated concentrations. Resazurin assay was performed at 72 h to determine cancer cell viability after TCE treatment. Results show relative % ± SEM. (E) In co-cultures with HT-29 cells that express CEA and Jurkat CD69-tdTomato reporter cells (J69; T cells were modified by CRISPR to express tdTomato under the control of the CD69 promoter), the addition of TCEs (1 µg) leads to the visualization of tdTomato-positive Jurkat J69 cells (E:T = 1:1). Scale bar = 400 µm. (F) Three patient-derived CEA-positive colorectal cancer cell lines and other CEA-positive cells (BxPC-3, HT-29, COLO 205) activate J69 cells in co-cultures with αCEA TCE (αCEA:hCD3), but not with αCEA CTRL (αCEA:mCD3) or CEA-negative control cell lines (MIA PaCa-2, HCT 116). Results show MFI ± standard error of the mean (SEM); Two-way ANOVA with Sidak’s correction for multiple comparisons. (G) HT-29, COLO 205, SW620 and MC38 WT spheroids were infected at an MOI 1 of oncolytic vaccinia virus (Copenhagen/Cop strain) VV-CTRL, oncolytic Vesicular Stomatitis virus (VSVΔ51), oncolytic Herpes simplex virus (HSV), oncolytic Measles virus (MeV), or oncolytic Adenovirus (AdV). Spheroids were imaged at 48 hpi to detect transgene expression of enhanced green fluorescent protein (eGFP) or red fluorescent protein (RFP). All colorectal cancer spheroids expressed abundant eGFP levels after VV-CTRL infection, compared to other viruses. (H) Spheroid viability was assessed in triplicate by resazurin assay at 120 hpi, relative to uninfected controls. VV-CTRL decreased cell viability of HT-29, COLO205, SW620 and MC38 WT spheroids. vaccinia virus was able to infect all the colorectal cancer cell lines as spheroids. VV-CTRL decreased cell viability of HT-29, COLO205, SW620 and MC38 WT spheroids. Other viruses also decreased spheroid viability to a lesser extent. Of note, MeV did not significantly change the viability of MC38 WT spheroids, as expected, since this oncolytic virus do not infect murine cancer cell lines but served as a control. Results show relative % ± SEM; Two-way ANOVA.

    Techniques Used: Transfection, Construct, Plasmid Preparation, Incubation, Molecular Weight, BIA-KA, SDS Page, Western Blot, Cell Culture, Resazurin Assay, Modification, CRISPR, Derivative Assay, Negative Control, Infection, Expressing

    13) Product Images from "Structural insights into the contactin 1 – neurofascin 155 adhesion complex"

    Article Title: Structural insights into the contactin 1 – neurofascin 155 adhesion complex

    Journal: Nature Communications

    doi: 10.1038/s41467-022-34302-9

    Contactin 1–neurofascin 155 expression mediates cell co-clustering. a Representative cell clustering images of K562 cells expressing contactin 1 (mCherry; magenta; CNTN1) and neurofascin 155 (GFP; green; NF155). Wildtype contactin 1–neurofascin 155 co-clusters form when contactin 1 is expressed in the presence of kifunensine (Kif; 10 µM). Neurofascin 155 but not contactin 1-expressing cells exhibit homophilic clustering. Mutation of the competitive interface residues on either protein abolishes co-clustering. Each experiment was repeated three times independently with similar results. Scale bar, 100 µm. Contactin 1 Phe177Asp, Phe180Asp and Phe212Asp is CNTN1 Mut , neurofascin 155 Phe168Asp, Met170Asp, Met174Asp and Ile217Asp is NF155 Mut . b Location of the mutations. Contactin 1–neurofascin 155 interface residues, shown in stick representation, at the “bottom side” of the Ig2 super β-sheet (left panel) are mutated to aspartates and prevent heterophilic trans interactions. The same neurofascin 155 residues are also located in the neurofascin 155 homodimer interface (right panel) and mutating them to aspartates prevents homophilic trans interactions. c Clustering index; the proportion of the total segmented cell area classified as clusters. p values are: 0.9544 for NF155 vs. CNTN1mut + Kif/NF155,
    Figure Legend Snippet: Contactin 1–neurofascin 155 expression mediates cell co-clustering. a Representative cell clustering images of K562 cells expressing contactin 1 (mCherry; magenta; CNTN1) and neurofascin 155 (GFP; green; NF155). Wildtype contactin 1–neurofascin 155 co-clusters form when contactin 1 is expressed in the presence of kifunensine (Kif; 10 µM). Neurofascin 155 but not contactin 1-expressing cells exhibit homophilic clustering. Mutation of the competitive interface residues on either protein abolishes co-clustering. Each experiment was repeated three times independently with similar results. Scale bar, 100 µm. Contactin 1 Phe177Asp, Phe180Asp and Phe212Asp is CNTN1 Mut , neurofascin 155 Phe168Asp, Met170Asp, Met174Asp and Ile217Asp is NF155 Mut . b Location of the mutations. Contactin 1–neurofascin 155 interface residues, shown in stick representation, at the “bottom side” of the Ig2 super β-sheet (left panel) are mutated to aspartates and prevent heterophilic trans interactions. The same neurofascin 155 residues are also located in the neurofascin 155 homodimer interface (right panel) and mutating them to aspartates prevents homophilic trans interactions. c Clustering index; the proportion of the total segmented cell area classified as clusters. p values are: 0.9544 for NF155 vs. CNTN1mut + Kif/NF155,

    Techniques Used: Expressing, Mutagenesis

    14) Product Images from "USP10 strikes down Wnt/β-catenin signaling by dual-wielding deubiquitinase activity and phase transition potential"

    Article Title: USP10 strikes down Wnt/β-catenin signaling by dual-wielding deubiquitinase activity and phase transition potential

    Journal: bioRxiv

    doi: 10.1101/2022.10.31.514466

    USP10 functions in embryonic dorsoventral patterning and axis formation through Wnt/β-catenin signaling (a, b) Embryos were injected with 500pg hUSP10 or hUSP10-CA mRNA at the one-cell stage. Representative embryos of different classes at 24 hpf were shown in (a), lateral views with anterior to the left. The percentage of embryos with indicated phenotype were shown in (b). Scale bar, 200μm. (c-e) The expression analysis of dorsal marker genes (c, boz and d, gsc ) at the sphere stage and ventral marker gene (e, gata2 ) in embryos injected with indicated mRNA at the 75% epi (epiboly) stage. Lateral views. Embryos were injected with 500pg hUSP10 or hUSP10-CA mRNA at one-cell stage, 500pg GFP injection was used as a control. (f-h) Dorsal marker genes (f, boz and g, gsc ) and ventral marker gene (h, gata2 ) were assessed in DMSO and Spautin-1 (5μmoL or 10μmoL) incubated embryos at indicated stage by in situ hybridization. Lateral views. (i, j) Whole-mount in situ hybridization analyzed the transcript of gfp in hUSP10-injected (i) or Spautin-1 (j) treated Tg(TOPdGFP) embryos at shield stage. Animal views with dorsal side to the right. Red arrows point to the dorsal organizer of the embryo. (k, l) Overexpression of ΔN-β-catenin mRNA rescued hUSP10-induced ventralization. Embryos were injected with 500pg of hUSP10 mRNA alone or together with 100pg of ΔN-β-catenin mRNA at the one-cell stage and harvested at the sphere stage for in situ hybridization with the probe of gsc (k) and boz (l). The number of the embryos was indicated within each panel.
    Figure Legend Snippet: USP10 functions in embryonic dorsoventral patterning and axis formation through Wnt/β-catenin signaling (a, b) Embryos were injected with 500pg hUSP10 or hUSP10-CA mRNA at the one-cell stage. Representative embryos of different classes at 24 hpf were shown in (a), lateral views with anterior to the left. The percentage of embryos with indicated phenotype were shown in (b). Scale bar, 200μm. (c-e) The expression analysis of dorsal marker genes (c, boz and d, gsc ) at the sphere stage and ventral marker gene (e, gata2 ) in embryos injected with indicated mRNA at the 75% epi (epiboly) stage. Lateral views. Embryos were injected with 500pg hUSP10 or hUSP10-CA mRNA at one-cell stage, 500pg GFP injection was used as a control. (f-h) Dorsal marker genes (f, boz and g, gsc ) and ventral marker gene (h, gata2 ) were assessed in DMSO and Spautin-1 (5μmoL or 10μmoL) incubated embryos at indicated stage by in situ hybridization. Lateral views. (i, j) Whole-mount in situ hybridization analyzed the transcript of gfp in hUSP10-injected (i) or Spautin-1 (j) treated Tg(TOPdGFP) embryos at shield stage. Animal views with dorsal side to the right. Red arrows point to the dorsal organizer of the embryo. (k, l) Overexpression of ΔN-β-catenin mRNA rescued hUSP10-induced ventralization. Embryos were injected with 500pg of hUSP10 mRNA alone or together with 100pg of ΔN-β-catenin mRNA at the one-cell stage and harvested at the sphere stage for in situ hybridization with the probe of gsc (k) and boz (l). The number of the embryos was indicated within each panel.

    Techniques Used: Injection, Expressing, Marker, Incubation, In Situ Hybridization, Over Expression

    USP10 facilitates the puncta formation of Axin1 via a IDRs-mediated phase separation-like manner independent of DUB activity (a) Representative fluorescent images of Axin1 droplets when co-expressed with shCtrl, shUSP10, EV, USP10 WT, USP10 CA and USP10-ΔDUB. (b-d) Numbers of Axin1 puncta per cell (b), size of Axin1 puncta (c), and immobile fraction (d) of Axin1 puncta. Error bars mean ± SD, two-tailed Student’s t-test. (e) Representative figures of Axin1 puncta by immunostaining of endogenous Axin1 in SW480 cell. Red, Axin1(Alexa 555). Blue, DAPI. All figures are in the same scale in this panel. (f) The statistical analysis of (e). Error bars mean ± SD, by two-tailed Student’s t-test. (g) Representative figures of in vitro phase separation assay by co-incubation of bacterial expressed Axin1-mCherry and USP10-GFP (WT and mutants). Red, Axin1-mCherry. Green, USP10-GFP. All figures are in the same scale in this panel. (h) The statistical analysis of (g). Error bars mean ± SD, by two-tailed Student’s t-test. (i) Immobile fraction of Axin1-DA puncta when co-expressed with USP10 WT and USP10-ΔPBR, as compared to Axin1 WT puncta. (j) Working model of USP10 inhibiting Wnt/β-catenin signaling. The DUB activity contributes to deubiquitination and stabilization of Axin1, and the unstructured region promotes LLPS of the destruction complex through physical interactions. RLU: Relative Luciferase Unit. ns, not significant.
    Figure Legend Snippet: USP10 facilitates the puncta formation of Axin1 via a IDRs-mediated phase separation-like manner independent of DUB activity (a) Representative fluorescent images of Axin1 droplets when co-expressed with shCtrl, shUSP10, EV, USP10 WT, USP10 CA and USP10-ΔDUB. (b-d) Numbers of Axin1 puncta per cell (b), size of Axin1 puncta (c), and immobile fraction (d) of Axin1 puncta. Error bars mean ± SD, two-tailed Student’s t-test. (e) Representative figures of Axin1 puncta by immunostaining of endogenous Axin1 in SW480 cell. Red, Axin1(Alexa 555). Blue, DAPI. All figures are in the same scale in this panel. (f) The statistical analysis of (e). Error bars mean ± SD, by two-tailed Student’s t-test. (g) Representative figures of in vitro phase separation assay by co-incubation of bacterial expressed Axin1-mCherry and USP10-GFP (WT and mutants). Red, Axin1-mCherry. Green, USP10-GFP. All figures are in the same scale in this panel. (h) The statistical analysis of (g). Error bars mean ± SD, by two-tailed Student’s t-test. (i) Immobile fraction of Axin1-DA puncta when co-expressed with USP10 WT and USP10-ΔPBR, as compared to Axin1 WT puncta. (j) Working model of USP10 inhibiting Wnt/β-catenin signaling. The DUB activity contributes to deubiquitination and stabilization of Axin1, and the unstructured region promotes LLPS of the destruction complex through physical interactions. RLU: Relative Luciferase Unit. ns, not significant.

    Techniques Used: Activity Assay, Two Tailed Test, Immunostaining, In Vitro, Incubation, Luciferase

    15) Product Images from "Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol"

    Article Title: Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol

    Journal: bioRxiv

    doi: 10.1101/2022.10.30.514423

    A microscopy-based method to measure MHETase cell surface display efficiency. A . Outline of the microscopy-based method to quantify GFP signal on the cell surface (see text for details). B . Mean fluorescence intensities at each pixel coordinate for the indicated strains. Bars indicate standard deviation. Analysis was performed on at least 40 cells in each replicate. conA-A594: n= 98, Mrh1-GFP and intra-cellular MHETase (intra-M): n = 4, Tif2-GFP and Rrp1A-GFP: n = 2. C . Comparison of mean fluorescence intensities for the -5, -4, and +1 pixel coordinates for the indicated strains (grey shading in B). Bars indicate standard deviation. D, E . Representative fluorescence micrographs for the strains in B and C and for the MHETase surface display chimeras. Scale bar: 5 μm. F . Fraction of MHETase chimeras displayed at the cell surface. Cells were induced for 4 hours, labelled with conA-A594 and imaged. The fraction of displayed chimera is plotted. Horizontal bars indicate the means of the replicates (n = 6). Each replicate included at least 200 cells. G . Abundance of the MHETase chimeras at the cell surface. The fraction of chimera displayed from panel F was used to calculate the cell surface abundance in molecules per cell. Theoretical construct molarity is indicated for a cell density of 10 8 cells/ml. Horizontal bars indicate the means of the replicates (n = 6).
    Figure Legend Snippet: A microscopy-based method to measure MHETase cell surface display efficiency. A . Outline of the microscopy-based method to quantify GFP signal on the cell surface (see text for details). B . Mean fluorescence intensities at each pixel coordinate for the indicated strains. Bars indicate standard deviation. Analysis was performed on at least 40 cells in each replicate. conA-A594: n= 98, Mrh1-GFP and intra-cellular MHETase (intra-M): n = 4, Tif2-GFP and Rrp1A-GFP: n = 2. C . Comparison of mean fluorescence intensities for the -5, -4, and +1 pixel coordinates for the indicated strains (grey shading in B). Bars indicate standard deviation. D, E . Representative fluorescence micrographs for the strains in B and C and for the MHETase surface display chimeras. Scale bar: 5 μm. F . Fraction of MHETase chimeras displayed at the cell surface. Cells were induced for 4 hours, labelled with conA-A594 and imaged. The fraction of displayed chimera is plotted. Horizontal bars indicate the means of the replicates (n = 6). Each replicate included at least 200 cells. G . Abundance of the MHETase chimeras at the cell surface. The fraction of chimera displayed from panel F was used to calculate the cell surface abundance in molecules per cell. Theoretical construct molarity is indicated for a cell density of 10 8 cells/ml. Horizontal bars indicate the means of the replicates (n = 6).

    Techniques Used: Microscopy, Fluorescence, Standard Deviation, Construct

    MHETase display constructs are efficiently expressed at minimal fitness cost. A . GFP calibration standards for measuring abundance of MHETase chimeras in molecules per cell. GFP-fusion strains spanning the range of molecules per cells were selected and GFP fluorescence was measured. The regression analysis line and equation are indicated. Bars indicate standard deviation; n ≥ 7. B . Abundance of the indicated surface display chimeras with (orange) or without (green) MHETase. Abundance was determined using GFP fluorescence after induction with doxycycline for 4 hours and converted to molecules per cell using the equation in A. Theoretical MHETase molarity was inferred from the molecule/cell data for a cell density of 10 8 cells/ml (right y-axis). Horizontal bars indicate the means of the replicates. Asterisks indicate p-values ≤ 0.05 (unpaired Student’s t-test; n = 7). Intracellular MHETase (intra-M) and secreted MHETase (secreted-M) are indicated. C . Fitness of cells expressing the surface display chimeras. Cells expressing the indicated chimeras were grown in presence of doxycycline in YPD medium for 24h. Fitness is expressed as a ratio of the growth rate of each strain to that of the wild-type. Horizontal bars indicate the means of the replicates. Asterisks indicate p-values ≤ 0.05 (unpaired Student’s t-test; n = 4).
    Figure Legend Snippet: MHETase display constructs are efficiently expressed at minimal fitness cost. A . GFP calibration standards for measuring abundance of MHETase chimeras in molecules per cell. GFP-fusion strains spanning the range of molecules per cells were selected and GFP fluorescence was measured. The regression analysis line and equation are indicated. Bars indicate standard deviation; n ≥ 7. B . Abundance of the indicated surface display chimeras with (orange) or without (green) MHETase. Abundance was determined using GFP fluorescence after induction with doxycycline for 4 hours and converted to molecules per cell using the equation in A. Theoretical MHETase molarity was inferred from the molecule/cell data for a cell density of 10 8 cells/ml (right y-axis). Horizontal bars indicate the means of the replicates. Asterisks indicate p-values ≤ 0.05 (unpaired Student’s t-test; n = 7). Intracellular MHETase (intra-M) and secreted MHETase (secreted-M) are indicated. C . Fitness of cells expressing the surface display chimeras. Cells expressing the indicated chimeras were grown in presence of doxycycline in YPD medium for 24h. Fitness is expressed as a ratio of the growth rate of each strain to that of the wild-type. Horizontal bars indicate the means of the replicates. Asterisks indicate p-values ≤ 0.05 (unpaired Student’s t-test; n = 4).

    Techniques Used: Construct, Fluorescence, Standard Deviation, Expressing

    16) Product Images from "Evasion of cGAS and TRIM5 defines pandemic HIV"

    Article Title: Evasion of cGAS and TRIM5 defines pandemic HIV

    Journal: Nature Microbiology

    doi: 10.1038/s41564-022-01247-0

    a. Replication of HIV-1(M) NL4.3 (Bal Env) bearing HIV-2 ROD10 or HIV-1(O) MVP5180 Capsid in permissive GHOST cells (flow cytometry for induced GFP expression) representative of 2 independent experiments. b . Binding of fluorescently labeled nucleotides to HIV-1(M) or HIV-1(O) recombinant CA hexamers in the presence or absence of DTT to reduce monomer cross links. c . Titres of HIV-1(M), HIV-2 or HIV-1(O) -GFP made by mixing WT and CA R18G bearing packing constructs (left axis, white circles) and DNA synthesis measured at 6 hours post infection (right axis, black circles). d . Amino acids in BHP hinge region influencing BHP position with overlay of HIV-1(O) (PDB ID:7T12) and open HIV-1(M) (PDB ID:5HGL) hexamers. e . Capsid survival curves for CA mutant Q50Y generated from pooled data from two (no IP6) or three (100 μM IP6) independent experiments showing IP-mediated capsid stabilisation. f . Single round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) (R9) CA Q50Y –GFP measured 48 h post-infection by flow. Mean + /− SD, n = 3 independent experiments and 3 donors for MDM. Source data
    Figure Legend Snippet: a. Replication of HIV-1(M) NL4.3 (Bal Env) bearing HIV-2 ROD10 or HIV-1(O) MVP5180 Capsid in permissive GHOST cells (flow cytometry for induced GFP expression) representative of 2 independent experiments. b . Binding of fluorescently labeled nucleotides to HIV-1(M) or HIV-1(O) recombinant CA hexamers in the presence or absence of DTT to reduce monomer cross links. c . Titres of HIV-1(M), HIV-2 or HIV-1(O) -GFP made by mixing WT and CA R18G bearing packing constructs (left axis, white circles) and DNA synthesis measured at 6 hours post infection (right axis, black circles). d . Amino acids in BHP hinge region influencing BHP position with overlay of HIV-1(O) (PDB ID:7T12) and open HIV-1(M) (PDB ID:5HGL) hexamers. e . Capsid survival curves for CA mutant Q50Y generated from pooled data from two (no IP6) or three (100 μM IP6) independent experiments showing IP-mediated capsid stabilisation. f . Single round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) (R9) CA Q50Y –GFP measured 48 h post-infection by flow. Mean + /− SD, n = 3 independent experiments and 3 donors for MDM. Source data

    Techniques Used: Flow Cytometry, Expressing, Binding Assay, Labeling, Recombinant, Construct, DNA Synthesis, Infection, Mutagenesis, Generated

    Pandemic-associated adaptation of HIV capsid at position 120. a , Maximum-likelihood phylogenetic tree of primate lentiviral capsid genes coloured to illustrate the residues equivalent to HIV-1(O) CA 120. Grey and (_) branch labels denote a gap in the alignment. b , Structures showing salt bridges in HIV-1(O) (PDB ID:7T12) (E98-R120), HIV-2 (PDB ID:2 × 82) (E96-R118), SIVmac (PDB ID:7T14) (E95-R117) and HIV-1(M) + R120 (PDB ID:7QDF) (E98-R120). The salt bridge is absent in WT HIV-1(M) CA (PDB ID:5HGN) and SIVcpz (PDB ID:7T15) because R120 is absent. Helix bearing R120 in HIV is coloured blue. Salt bridges are shown as dashed lines. CypA binding loop is coloured wheat. c , Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) or HIV-1(M) CA Q50Y 120R GFP measured at 48 h post infection by flow cytometry. Mean ± s.e.m. n = 3 donors. Source data
    Figure Legend Snippet: Pandemic-associated adaptation of HIV capsid at position 120. a , Maximum-likelihood phylogenetic tree of primate lentiviral capsid genes coloured to illustrate the residues equivalent to HIV-1(O) CA 120. Grey and (_) branch labels denote a gap in the alignment. b , Structures showing salt bridges in HIV-1(O) (PDB ID:7T12) (E98-R120), HIV-2 (PDB ID:2 × 82) (E96-R118), SIVmac (PDB ID:7T14) (E95-R117) and HIV-1(M) + R120 (PDB ID:7QDF) (E98-R120). The salt bridge is absent in WT HIV-1(M) CA (PDB ID:5HGN) and SIVcpz (PDB ID:7T15) because R120 is absent. Helix bearing R120 in HIV is coloured blue. Salt bridges are shown as dashed lines. CypA binding loop is coloured wheat. c , Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M) or HIV-1(M) CA Q50Y 120R GFP measured at 48 h post infection by flow cytometry. Mean ± s.e.m. n = 3 donors. Source data

    Techniques Used: Binding Assay, Infection, Flow Cytometry

    a. NF-kB - secreted alkaline phosphatase reporter 24 or 72 hours after treatment with increasing doses of viral-like particles (VLP, made with packaging plasmid and VSV-G but no genome). N = 2 independent experiments. b . Quantification of IL-1β in supernatants from MDM mock infected or infected with HIV-1(M), HIV-2 or HIV-1(O). N = 4 donors. c . IRF-reporter activation after interferon β (IFNβ) or IL-1β treatment of THP-1 cells. N = 4 independent experiments. d . % of infection in THP-1 cells after addition of IFNβ or IL-1β at different time points. Dotted line indicates the % of infection of untreated cells. N = 3 independent experiments e . Infection levels in MDM depleted of TRIM5 with equal genome copies of VSV-G pseudotyped HIV-1(M), HIV-2 or HIV-1(O) –GFP measured 48 h post-infection. N = 2 donors. Data shows mean + SD. Source data
    Figure Legend Snippet: a. NF-kB - secreted alkaline phosphatase reporter 24 or 72 hours after treatment with increasing doses of viral-like particles (VLP, made with packaging plasmid and VSV-G but no genome). N = 2 independent experiments. b . Quantification of IL-1β in supernatants from MDM mock infected or infected with HIV-1(M), HIV-2 or HIV-1(O). N = 4 donors. c . IRF-reporter activation after interferon β (IFNβ) or IL-1β treatment of THP-1 cells. N = 4 independent experiments. d . % of infection in THP-1 cells after addition of IFNβ or IL-1β at different time points. Dotted line indicates the % of infection of untreated cells. N = 3 independent experiments e . Infection levels in MDM depleted of TRIM5 with equal genome copies of VSV-G pseudotyped HIV-1(M), HIV-2 or HIV-1(O) –GFP measured 48 h post-infection. N = 2 donors. Data shows mean + SD. Source data

    Techniques Used: Plasmid Preparation, Infection, Activation Assay

    a. Infection of MDM with HIV, measured at 48 h by counting Gag positive cells, in the presence of anti-interferon α/β receptor (IFNα/β-R), or control, antibody (cAb). b . Replication of HIV-1(M), HIV-2 or HIV-1(O) isolates in permissive GHOST cells measuring induced GFP expression by flow. Representative experiment of 2 independent replicates. c, d . Activation of (c) IRF-luciferase reporter or (d) NF-kB secreted alkaline phosphatase reporter 48 h after infection by equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 or HIV-1(O) -GFP. e, f . Measurement of VSV-G pseudotyped HIV-1(M), HIV-1(O) and HIV-2 DNA synthesis (GFP primers) during a 20 h time course in THP-1 cells. g . Infection measured at 48 hours in wells parallel to (e) by flow. h . Viral DNA (GFP) copy number at 20 h post-infection per infected cell using data from (e-f). Mean + /− SD, N = 3 donors (MDM) or independent experiments (THP-1 c,d). N = 4 independent experiments ThP1 e-h. Source data
    Figure Legend Snippet: a. Infection of MDM with HIV, measured at 48 h by counting Gag positive cells, in the presence of anti-interferon α/β receptor (IFNα/β-R), or control, antibody (cAb). b . Replication of HIV-1(M), HIV-2 or HIV-1(O) isolates in permissive GHOST cells measuring induced GFP expression by flow. Representative experiment of 2 independent replicates. c, d . Activation of (c) IRF-luciferase reporter or (d) NF-kB secreted alkaline phosphatase reporter 48 h after infection by equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 or HIV-1(O) -GFP. e, f . Measurement of VSV-G pseudotyped HIV-1(M), HIV-1(O) and HIV-2 DNA synthesis (GFP primers) during a 20 h time course in THP-1 cells. g . Infection measured at 48 hours in wells parallel to (e) by flow. h . Viral DNA (GFP) copy number at 20 h post-infection per infected cell using data from (e-f). Mean + /− SD, N = 3 donors (MDM) or independent experiments (THP-1 c,d). N = 4 independent experiments ThP1 e-h. Source data

    Techniques Used: Infection, Expressing, Activation Assay, Luciferase, DNA Synthesis

    HIV activation of innate immune responses in macrophages. a – c , Replication of HIV-1(M) ( a ), HIV-2 ( b ) or HIV-1(O) ( c ) isolates in human MDM in the presence of interferon α/β receptor (IFNα/β-R) or control antibody (CAb). Two-way ANOVA vs CAb, ROD10 P = 0.0001, pSTbx P = 0.0001, ps7312s P = 0,0001, RBF206 P = 0.033. d , Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. e , Secreted IL-8 and CXCL10 from infections in d measured by ELISA 48 h post infection. f , GAPDH-normalized mRNA levels in infections from d expressed as fold induction over untreated MDM 24 h post infection or after HT-DNA transfection (1 ug ml −1 ) or LPS stimulation (100 ng ml −1 ). g , Infection of THP-1 cells with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. h , GAPDH-normalized mRNA levels from infections in g expressed as fold induction over untreated THP-1 cells 24 h post infection. Mean ± s.d., n = 3 donors ( a – e ) or independent experiments ( f – h ). Two-tailed unpaired t -test vs untreated MDM ( d – f ), paired t -test vs untreated THP-1 cells ( g , h ). * P
    Figure Legend Snippet: HIV activation of innate immune responses in macrophages. a – c , Replication of HIV-1(M) ( a ), HIV-2 ( b ) or HIV-1(O) ( c ) isolates in human MDM in the presence of interferon α/β receptor (IFNα/β-R) or control antibody (CAb). Two-way ANOVA vs CAb, ROD10 P = 0.0001, pSTbx P = 0.0001, ps7312s P = 0,0001, RBF206 P = 0.033. d , Single-round infection of MDM with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. e , Secreted IL-8 and CXCL10 from infections in d measured by ELISA 48 h post infection. f , GAPDH-normalized mRNA levels in infections from d expressed as fold induction over untreated MDM 24 h post infection or after HT-DNA transfection (1 ug ml −1 ) or LPS stimulation (100 ng ml −1 ). g , Infection of THP-1 cells with equal genome copies of VSV-G-pseudotyped HIV-1(M), HIV-2 and HIV-1(O)-GFP measured 48 h post infection. h , GAPDH-normalized mRNA levels from infections in g expressed as fold induction over untreated THP-1 cells 24 h post infection. Mean ± s.d., n = 3 donors ( a – e ) or independent experiments ( f – h ). Two-tailed unpaired t -test vs untreated MDM ( d – f ), paired t -test vs untreated THP-1 cells ( g , h ). * P

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

    17) Product Images from "Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin"

    Article Title: Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-022-03705-z

    RHM treatment counteracts LIG3 activity and induces DNA damage in MM cells. KMS26 and AMO1 cells were treated with increasing dose of RHM or vehicle. A: Immunoblot analysis of DDR markers was performed 48 h after treatment. B: γ-H2AX foci evaluation by immunofluorescence 48 h after treatment. Representative images of unrepaired DSBs are shown. DAPI (blue) was used for nuclear staining. C . Immunoblot analysis of nuclear soluble and chromatin bound fractions prepared from AMO1 cells treated with or without RHM (5 μM). D. KMS26 and AMO1 cells were treated with RHM (5 μM) or vehicle. Left: Mitochondrial DNA copy number, as measured by qRT-PCR 48 h after treatment. Right: Mitochondria were stained with Mitotracker Red and visualized by fluorescence microscopy 48 h after treatment. E. Alt-NHEJ repair was evaluated by EJ2- GFP assay on AMO1 cells 72 h after treatment with RHM (5 μM) or vehicle. F. Affymetrix CytoScan HD Array analysis, using genomic DNA from AMO1 treated with RHM (2,5 μM) or vehicle. Representative images of deletions or gains acquisition on chromosome 16 (16p11.2) and 1(1p36.3), respectively. Red lines represent deletions, while blue lines represent gains Results are representative of three independent experiments ± SD. *, P
    Figure Legend Snippet: RHM treatment counteracts LIG3 activity and induces DNA damage in MM cells. KMS26 and AMO1 cells were treated with increasing dose of RHM or vehicle. A: Immunoblot analysis of DDR markers was performed 48 h after treatment. B: γ-H2AX foci evaluation by immunofluorescence 48 h after treatment. Representative images of unrepaired DSBs are shown. DAPI (blue) was used for nuclear staining. C . Immunoblot analysis of nuclear soluble and chromatin bound fractions prepared from AMO1 cells treated with or without RHM (5 μM). D. KMS26 and AMO1 cells were treated with RHM (5 μM) or vehicle. Left: Mitochondrial DNA copy number, as measured by qRT-PCR 48 h after treatment. Right: Mitochondria were stained with Mitotracker Red and visualized by fluorescence microscopy 48 h after treatment. E. Alt-NHEJ repair was evaluated by EJ2- GFP assay on AMO1 cells 72 h after treatment with RHM (5 μM) or vehicle. F. Affymetrix CytoScan HD Array analysis, using genomic DNA from AMO1 treated with RHM (2,5 μM) or vehicle. Representative images of deletions or gains acquisition on chromosome 16 (16p11.2) and 1(1p36.3), respectively. Red lines represent deletions, while blue lines represent gains Results are representative of three independent experiments ± SD. *, P

    Techniques Used: Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Fluorescence, Microscopy, Non-Homologous End Joining

    18) Product Images from "Single Cell lineage Tracing Identifies Cancer Testis Antigens as Mediators of Chemoresistance in Small Cell Lung Cancer"

    Article Title: Single Cell lineage Tracing Identifies Cancer Testis Antigens as Mediators of Chemoresistance in Small Cell Lung Cancer

    Journal: bioRxiv

    doi: 10.1101/2022.10.20.513051

    Generation and validation of barcoded xenografts and PCR error rate correction. A, The xenograft barcoding system. The barcode is inserted retrovirally, and contains a CAG promoter, GFP, the lineage barcode (LBC), and a polyA tail. B, Overview of the lineage tracing process. Cells are barcoded in culture and a portion taken for scRNA-seq. The remainder of the cells are injected as a xenograft. Half of this xenograft undergoes scRNA-seq, and the other half is injected as a serial xenograft into a new mouse, which is treated with chemotherapy and then undergoes scRNA-seq. C, Doubling time of the two SCLC cell lines used for making xenografts, NCI-H209 and NCI-H82 D, Process of sequencing LBCs for diversity validation. E, PCR purification of the LBC and subsequent sequencing. Diversity estimates from each read direction of the PCR products are shown. F, Radial plot showing the unbiased clustering of LBC similarity for a single sample. A single A > G transition is observed which is interpreted as a PCR error as its relative occurrence in the population is less than the calculated PCR error rate determined by variants in the LBC constant regions. G, Representation of the degree of overlap between two subdivided samples split at various cellular doublings measuring in ( C ). H , Degree of overlap between a single sample split in half to represent the labeled starting cell line and the cells to be divided into four subpopulations for xenograft injection.
    Figure Legend Snippet: Generation and validation of barcoded xenografts and PCR error rate correction. A, The xenograft barcoding system. The barcode is inserted retrovirally, and contains a CAG promoter, GFP, the lineage barcode (LBC), and a polyA tail. B, Overview of the lineage tracing process. Cells are barcoded in culture and a portion taken for scRNA-seq. The remainder of the cells are injected as a xenograft. Half of this xenograft undergoes scRNA-seq, and the other half is injected as a serial xenograft into a new mouse, which is treated with chemotherapy and then undergoes scRNA-seq. C, Doubling time of the two SCLC cell lines used for making xenografts, NCI-H209 and NCI-H82 D, Process of sequencing LBCs for diversity validation. E, PCR purification of the LBC and subsequent sequencing. Diversity estimates from each read direction of the PCR products are shown. F, Radial plot showing the unbiased clustering of LBC similarity for a single sample. A single A > G transition is observed which is interpreted as a PCR error as its relative occurrence in the population is less than the calculated PCR error rate determined by variants in the LBC constant regions. G, Representation of the degree of overlap between two subdivided samples split at various cellular doublings measuring in ( C ). H , Degree of overlap between a single sample split in half to represent the labeled starting cell line and the cells to be divided into four subpopulations for xenograft injection.

    Techniques Used: Polymerase Chain Reaction, Injection, Sequencing, Purification, Labeling

    Validation of the LBC labeling and xenograft growth. Confocal imagery of the NCI-H209 ( A ) and NCI-H82 ( B ) cell lines expressing GFP after transduction with the Retro-LBC virus. Scale bars = 20 μm. Growth of the NCI-H209 xenografts in the primary xenograft without treatment ( C ) and the secondary xenograft during chemotherapy treatment ( D ). Growth of the NCI-H82 xenografts in the primary xenograft without treatment ( E ) and the secondary xenograft during chemotherapy treatment ( F ). Dashed lines represent the measured size of the xenografts and the solid line is the best-fit model of xenograft growth.
    Figure Legend Snippet: Validation of the LBC labeling and xenograft growth. Confocal imagery of the NCI-H209 ( A ) and NCI-H82 ( B ) cell lines expressing GFP after transduction with the Retro-LBC virus. Scale bars = 20 μm. Growth of the NCI-H209 xenografts in the primary xenograft without treatment ( C ) and the secondary xenograft during chemotherapy treatment ( D ). Growth of the NCI-H82 xenografts in the primary xenograft without treatment ( E ) and the secondary xenograft during chemotherapy treatment ( F ). Dashed lines represent the measured size of the xenografts and the solid line is the best-fit model of xenograft growth.

    Techniques Used: Labeling, Expressing, Transduction

    Single-Cell RNA sequencing reveals two distinct subpopulations in SCLC. A, Overview of the in situ barcoding model. Tumors in the RPR2-Cas9 model were initiated, and barcoded at one-month intervals, up to 5 months post-initiation. Tumors were harvested at one-month intervals following barcoding, up to 6 months, and GFP + cells were isolated and scRNA-seq was performed. B, Multiple cellular populations were detected in the scRNA-seq due to the presence of microdissected tumors in this analysis, including a large population of SCLC cells. C, Proportion of cells in each stage of the cell cycle at varying months of tumor development. D, UMAP plot of the SCLC cells labeled by months of tumor development. Black outlines describe the clusters identified in ( E ). E, Unbiased clustering of the SCLC cells into two clusters. Due to the distribution of timepoints in ( D ), clusters are labeled “early” or “late”. F, Proportion of cells in the early and late populations at each timepoint after tumor initiation. G, Gene modules that differentiate early and late populations. Significance determined by an unpaired student’s t-test where (*) P
    Figure Legend Snippet: Single-Cell RNA sequencing reveals two distinct subpopulations in SCLC. A, Overview of the in situ barcoding model. Tumors in the RPR2-Cas9 model were initiated, and barcoded at one-month intervals, up to 5 months post-initiation. Tumors were harvested at one-month intervals following barcoding, up to 6 months, and GFP + cells were isolated and scRNA-seq was performed. B, Multiple cellular populations were detected in the scRNA-seq due to the presence of microdissected tumors in this analysis, including a large population of SCLC cells. C, Proportion of cells in each stage of the cell cycle at varying months of tumor development. D, UMAP plot of the SCLC cells labeled by months of tumor development. Black outlines describe the clusters identified in ( E ). E, Unbiased clustering of the SCLC cells into two clusters. Due to the distribution of timepoints in ( D ), clusters are labeled “early” or “late”. F, Proportion of cells in the early and late populations at each timepoint after tumor initiation. G, Gene modules that differentiate early and late populations. Significance determined by an unpaired student’s t-test where (*) P

    Techniques Used: RNA Sequencing Assay, In Situ, Isolation, Labeling

    Validation of in situ barcoding model. A, Separate lung sections displaying positivity for both CAS9 and GFP. B, Co-staining of representative sections showing colocalization of both CAS9 and GFP. C, Flow-sorting strategy for isolating GFP+ cells from harvested lungs transduced with Adeno-Cre and the AAV-LBC viruses. D, UMAP plot displaying expression of GFP in the in situ scRNA-seq data.
    Figure Legend Snippet: Validation of in situ barcoding model. A, Separate lung sections displaying positivity for both CAS9 and GFP. B, Co-staining of representative sections showing colocalization of both CAS9 and GFP. C, Flow-sorting strategy for isolating GFP+ cells from harvested lungs transduced with Adeno-Cre and the AAV-LBC viruses. D, UMAP plot displaying expression of GFP in the in situ scRNA-seq data.

    Techniques Used: In Situ, Staining, Transduction, Expressing

    19) Product Images from "In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death"

    Article Title: In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death

    Journal: Regenerative Therapy

    doi: 10.1016/j.reth.2022.10.003

    Immunofluorescence staining of transplanted murine quadriceps. Mice transplanted by the various cell methods were sacrificed at 4-weeks old and subjected to immunofluorescent staining. Quadriceps, tibialis anterior, and diaphragm muscles were stained, and the quadriceps muscle is presented in this figure. No rats were GFP-positive other than GFP rats stained as controls. Bars = 100 μm.
    Figure Legend Snippet: Immunofluorescence staining of transplanted murine quadriceps. Mice transplanted by the various cell methods were sacrificed at 4-weeks old and subjected to immunofluorescent staining. Quadriceps, tibialis anterior, and diaphragm muscles were stained, and the quadriceps muscle is presented in this figure. No rats were GFP-positive other than GFP rats stained as controls. Bars = 100 μm.

    Techniques Used: Immunofluorescence, Staining, Mouse Assay

    20) Product Images from "A synthetic transcription platform for programmable gene expression in mammalian cells"

    Article Title: A synthetic transcription platform for programmable gene expression in mammalian cells

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33287-9

    Precision control of human monoclonal antibody (mAb) production. a Schematic illustrations of the sequential, site-specific genomic integration of two payload gene circuits into the CHO cells engineered with a double landing pad (dLP) for human mAb production. First, a synthetic gene circuit encoding one copy of dCas9-VPR gene and 2 flanking mammalian selection marker genes, hygromycin (5′-end) and G418 (3′-end), was integrated into dLP1-1 site with BxB1 integrase. Selected cells with single dLP1-1 occupancy were clonally sorted based on EYFP−/EBFP+ signals. A single clone with the most consistent outputs of dCas9-VPR and EBFP was chosen for the second BxB1-mediated integration targeting the free dLP1-2 site. The second integration gene circuit contained independent TUs encoding either mKate and TagBFP reporter genes (control circuit) or the light chain and heavy chain genes of a human mAb JUG444 (mAb circuit) as well as gRNA10, one copy of dCas9-VPR gene, and two additional flanking selection marker genes: puromycin (5′-end) and blasticidin (3′-end). Integrated cells selected with four antibiotics were then pool-sorted based on EYFP−/EBFP− signals. b The mKate and TagBFP expression of the integrated payload control circuits in dLP-CHO cells with two distinct configurations (8× BS without SI and 16× BS with SI). c The mAb production of the integrated payload circuits to express the light chain and heavy chain of JUG444 with four gRNA10 operator configurations: JUGAb1 (8× BS), JUGAb2 (8× BS with SI), JUGAb3 (16× BS), and JUGAb4 (16× BS with SI). Octet mAb titer quantitation over five weeks showed stable, differential JUG444 production by all four integration circuits. d Pearson correlation analysis to determine the relationship between JUG444 mAb titers and crisprTF promoter strengths over the course of five weeks. The Pearson correlation coefficients ( r ) at Week 1 through Week 5 were: r = 0.98 ( R 2 = 0.96, p = 0.0194), r = 0.97 ( R 2 = 0.94, p = 0.0317), r = 0.99 ( R 2 = 0.97, p = 0.0132), r = 0.99 ( R 2 = 0.97, p = 0.015), and r = 0.99 ( R 2 = 0.99, p = 0.0069), respectively. e The doubling time of mAb-producing cell lines (JUGAb3 and JUGAb4 vs. dLP-CHO: p = 0.0049 and p
    Figure Legend Snippet: Precision control of human monoclonal antibody (mAb) production. a Schematic illustrations of the sequential, site-specific genomic integration of two payload gene circuits into the CHO cells engineered with a double landing pad (dLP) for human mAb production. First, a synthetic gene circuit encoding one copy of dCas9-VPR gene and 2 flanking mammalian selection marker genes, hygromycin (5′-end) and G418 (3′-end), was integrated into dLP1-1 site with BxB1 integrase. Selected cells with single dLP1-1 occupancy were clonally sorted based on EYFP−/EBFP+ signals. A single clone with the most consistent outputs of dCas9-VPR and EBFP was chosen for the second BxB1-mediated integration targeting the free dLP1-2 site. The second integration gene circuit contained independent TUs encoding either mKate and TagBFP reporter genes (control circuit) or the light chain and heavy chain genes of a human mAb JUG444 (mAb circuit) as well as gRNA10, one copy of dCas9-VPR gene, and two additional flanking selection marker genes: puromycin (5′-end) and blasticidin (3′-end). Integrated cells selected with four antibiotics were then pool-sorted based on EYFP−/EBFP− signals. b The mKate and TagBFP expression of the integrated payload control circuits in dLP-CHO cells with two distinct configurations (8× BS without SI and 16× BS with SI). c The mAb production of the integrated payload circuits to express the light chain and heavy chain of JUG444 with four gRNA10 operator configurations: JUGAb1 (8× BS), JUGAb2 (8× BS with SI), JUGAb3 (16× BS), and JUGAb4 (16× BS with SI). Octet mAb titer quantitation over five weeks showed stable, differential JUG444 production by all four integration circuits. d Pearson correlation analysis to determine the relationship between JUG444 mAb titers and crisprTF promoter strengths over the course of five weeks. The Pearson correlation coefficients ( r ) at Week 1 through Week 5 were: r = 0.98 ( R 2 = 0.96, p = 0.0194), r = 0.97 ( R 2 = 0.94, p = 0.0317), r = 0.99 ( R 2 = 0.97, p = 0.0132), r = 0.99 ( R 2 = 0.97, p = 0.015), and r = 0.99 ( R 2 = 0.99, p = 0.0069), respectively. e The doubling time of mAb-producing cell lines (JUGAb3 and JUGAb4 vs. dLP-CHO: p = 0.0049 and p

    Techniques Used: Selection, Marker, Expressing, Quantitation Assay

    21) Product Images from "Thymic mesenchymal niche cells drive T cell immune regeneration"

    Article Title: Thymic mesenchymal niche cells drive T cell immune regeneration

    Journal: bioRxiv

    doi: 10.1101/2022.10.12.511184

    Postn+ ThyMCs maintain and recruit T cell progenitors and regenerate T cell immunity (E) Schematic illustration of experimental design for adoptive transfer of mesenchymal cells in the context of HSCT. (E) FACS quantification of early thymic progenitors after Sham (PBS), GFP+ (Penk+) ThyMC or tdTomato+ (Postn+) ThyMC treatment displayed as bar graphs. (Dose of Penk+ and Postn+ ThyMCs: 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10) Three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Bar graphs displaying the flow cytometric quantification of early thymic progenitors 6 days following an intrathymic sham injection or adoptive transfer of CD8+ T cells and CD248- ThyMCs in HSCT recipients. (Dose of CD8+ T cells and CD248- ThyMCs: 2000-4000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (Sham n= 4, CD8+ T cells n=6, CD248- ThyMC n=8). Two independent experiments. (E) Bar graphs showing the results of FACS quantification of early thymic progenitors 6 days after an intrathymic sham injection or adoptive transfer of Cas9-GFP CD248- ThyMCs following knockout of either GFP control (KO Ctrl) or Ccl19 (KO) in mice undergoing HSCT. (Dose of GFP KO Ctrl and Ccl19 KO cells: 4000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (n= 15 Sham, n=11 GFP KO Ctrl, n=10 Ccl19 KO). Three independent experiments. (E) Bar graphs showing the results of FACS analysis of early thymic progenitors and endothelial cells, 6 days following an intrathymic sham injection or adoptive transfer of Cas9-GFP bone marrow stromal cells following infection with of either mCherry control (Ctrl) or Ccl19 mCherry overexpression (Ccl19 OE) vectors in HSCT recipients. (Dose of mCherry Ctrl and Ccl19 OE cells: 50 000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (n= 6 Sham, n=9 mCherry Ctrl, n=9 Ccl19 OE). Two independent experiments. (E) Flow cytometric analysis of cytotoxic T lymphocytes (T CTL cells) and T helper cells (T H cells) in peripheral blood following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs 2-16 weeks post-bone marrow transplantation. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=7, CD8+ T cells=9, CD248- ThyMCs=9) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. # denotes statistical significance for CD248- ThyMC vs CD8+ T cell comparison and * denotes statistical significance for CD248- ThyMC vs Sham comparison. Gray shaded area denotes the peripheral blood parameters of untreated control mice (n=12) housed in the animal facility at the same time as the transplant recipients. (E) Quantification of de novo generated T cells following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs by way of signal-joint T cell receptor excision circles (sjTRECs) 4 weeks following bone marrow transplantation. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=8, CD8+ T cells=12, CD248- ThyMCs= 14) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Diversity in T cell receptors (TCRs) as analyzed using the sequencing of the CDR3 β chain 4 weeks following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs and HSCT. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) Each colored square represents a single clone. Samples were pooled from sorted CD3+ splenocytes from four mice for each group and the combined data is represented. (E) Overview of ovalbumin vaccination response assessment after adoptive transfer of CD248- ThyMCs in HSCT recipients. (E) Bar graphs showing the flow cytometric quantification of absolute numbers of ovalbumin specific T CTL cells in recipients of CD248- ThyMCs and HSCT. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=4-9, CD8+ T cells=6-11, CD248- ThyMCs= 4-8) Statistical significance was determined by one-way ANOVA followed by Tukey’s post- hoc analysis. Two independent experiments. (E) Bar graph representation of IFNg production by ovalbumin specific T CTL cells as assessed by ELISpot. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=4-9, CD8+ T cells=6-11, CD248- ThyMCs= 4-8) Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Schematic depiction of ovalbumin vaccination experimental design for aged recipients of Ccl19 overexpressing bone marrow stroma. (E) Bar graphs showing the results of flow cytometric analysis of ETPs in Young controls (Ctrl), Aged mCherry controls (Ctrl) and Aged Ccl19 mCherry overexpression (Ccl19 OE) recipients. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged Ccl19 OE n=7). Two independent experiments. (E) Bar graphs representing the flow cytometric analysis of TECs in Young Ctrl, Aged mCherry Ctrl and Aged Ccl19 OE treated mice. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged Ccl19 OE n=7). Two independent experiments. (E) Bar graphs showing the number of animals that failed or succeeded in mounting an ovalbumin specific CD8+ T CTL cell response as assessed by flow cytometric analysis of SINFEKL labeling, after an intrathymic injection of bone marrow stromal cells overexpressing either mCherry Ctrl or Ccl19 OE. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged mCherry Ccl19 OE n=7). Two independent experiments.
    Figure Legend Snippet: Postn+ ThyMCs maintain and recruit T cell progenitors and regenerate T cell immunity (E) Schematic illustration of experimental design for adoptive transfer of mesenchymal cells in the context of HSCT. (E) FACS quantification of early thymic progenitors after Sham (PBS), GFP+ (Penk+) ThyMC or tdTomato+ (Postn+) ThyMC treatment displayed as bar graphs. (Dose of Penk+ and Postn+ ThyMCs: 4000-8000) (Sham=9, Penk+ ThyMC=14, Postn+ ThyMCs=10) Three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Bar graphs displaying the flow cytometric quantification of early thymic progenitors 6 days following an intrathymic sham injection or adoptive transfer of CD8+ T cells and CD248- ThyMCs in HSCT recipients. (Dose of CD8+ T cells and CD248- ThyMCs: 2000-4000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (Sham n= 4, CD8+ T cells n=6, CD248- ThyMC n=8). Two independent experiments. (E) Bar graphs showing the results of FACS quantification of early thymic progenitors 6 days after an intrathymic sham injection or adoptive transfer of Cas9-GFP CD248- ThyMCs following knockout of either GFP control (KO Ctrl) or Ccl19 (KO) in mice undergoing HSCT. (Dose of GFP KO Ctrl and Ccl19 KO cells: 4000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (n= 15 Sham, n=11 GFP KO Ctrl, n=10 Ccl19 KO). Three independent experiments. (E) Bar graphs showing the results of FACS analysis of early thymic progenitors and endothelial cells, 6 days following an intrathymic sham injection or adoptive transfer of Cas9-GFP bone marrow stromal cells following infection with of either mCherry control (Ctrl) or Ccl19 mCherry overexpression (Ccl19 OE) vectors in HSCT recipients. (Dose of mCherry Ctrl and Ccl19 OE cells: 50 000) Values are presented as percent of Sham treated animals and statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis (n= 6 Sham, n=9 mCherry Ctrl, n=9 Ccl19 OE). Two independent experiments. (E) Flow cytometric analysis of cytotoxic T lymphocytes (T CTL cells) and T helper cells (T H cells) in peripheral blood following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs 2-16 weeks post-bone marrow transplantation. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=7, CD8+ T cells=9, CD248- ThyMCs=9) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. # denotes statistical significance for CD248- ThyMC vs CD8+ T cell comparison and * denotes statistical significance for CD248- ThyMC vs Sham comparison. Gray shaded area denotes the peripheral blood parameters of untreated control mice (n=12) housed in the animal facility at the same time as the transplant recipients. (E) Quantification of de novo generated T cells following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs by way of signal-joint T cell receptor excision circles (sjTRECs) 4 weeks following bone marrow transplantation. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=8, CD8+ T cells=12, CD248- ThyMCs= 14) Two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Diversity in T cell receptors (TCRs) as analyzed using the sequencing of the CDR3 β chain 4 weeks following adoptive transfer of Sham, CD8+ T cells or CD248- ThyMCs and HSCT. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) Each colored square represents a single clone. Samples were pooled from sorted CD3+ splenocytes from four mice for each group and the combined data is represented. (E) Overview of ovalbumin vaccination response assessment after adoptive transfer of CD248- ThyMCs in HSCT recipients. (E) Bar graphs showing the flow cytometric quantification of absolute numbers of ovalbumin specific T CTL cells in recipients of CD248- ThyMCs and HSCT. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=4-9, CD8+ T cells=6-11, CD248- ThyMCs= 4-8) Statistical significance was determined by one-way ANOVA followed by Tukey’s post- hoc analysis. Two independent experiments. (E) Bar graph representation of IFNg production by ovalbumin specific T CTL cells as assessed by ELISpot. (Dose of CD8+ T cells and CD248- ThyMCs: 10 000) (Sham=4-9, CD8+ T cells=6-11, CD248- ThyMCs= 4-8) Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis. (E) Schematic depiction of ovalbumin vaccination experimental design for aged recipients of Ccl19 overexpressing bone marrow stroma. (E) Bar graphs showing the results of flow cytometric analysis of ETPs in Young controls (Ctrl), Aged mCherry controls (Ctrl) and Aged Ccl19 mCherry overexpression (Ccl19 OE) recipients. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged Ccl19 OE n=7). Two independent experiments. (E) Bar graphs representing the flow cytometric analysis of TECs in Young Ctrl, Aged mCherry Ctrl and Aged Ccl19 OE treated mice. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged Ccl19 OE n=7). Two independent experiments. (E) Bar graphs showing the number of animals that failed or succeeded in mounting an ovalbumin specific CD8+ T CTL cell response as assessed by flow cytometric analysis of SINFEKL labeling, after an intrathymic injection of bone marrow stromal cells overexpressing either mCherry Ctrl or Ccl19 OE. (Dose of mCherry Ctrl and Ccl19 OE cells: 150 000) Statistical significance was determined using Student’s t-test comparing Aged mCherry Ctrl with Aged Ccl19 OE. (Young n= 9, Aged mCherry Ctrl n=9, Aged mCherry Ccl19 OE n=7). Two independent experiments.

    Techniques Used: Adoptive Transfer Assay, FACS, Injection, Knock-Out, Mouse Assay, Infection, Over Expression, Transplantation Assay, Generated, Sequencing, Enzyme-linked Immunospot, Labeling

    Postn+ ThyMCs are lost during transplant conditioning and aging (A) Schematic overview of sample collection from Transplantation, IL7RKO and Aging stress conditions. (B) UMAP embeddings showing detailed annotation of different stromal cell populations ( Cd3e-, Cd4-, Cd8a-, Ptprc-) in Control (n=4, total number of stromal cells=5451), Transplant (n=3, total number of stromal cells=8057), IL7RKO (n=4, total number of stromal cells=4551) and Aging (n=4, total number of stromal cells=16178) samples. (C) The difference between Control and Transplantation, and Aging samples visualized via condition-specific densities in a joint ThyMC specific UMAP embedding (left). Detailed annotation of ThyMC subsets shown in ThyMC specific UMAP embedding (right). Control (n=4), Transplantation (n=3), and Aging (n=4), total number of ThyMC = 19739. (D) Bar graphs comparing proportional shifts of individual ThyMC subsets between Control, Transplant, and Aging samples as determined by scRNAseq. (Control n= 4, Transplant n= 3, Aging n=4) Statistical significance is based on beta regression with Benjamini- Hochberg FDR control of these comparisons. (E) Bar graphs showing flow cytometric quantification of absolute numbers of different ThyMC subsets in Control, Transplant (day 3) or Aged (2 years old) Penk-Cre-tdTomato mice. Statistical significance was calculated using one-way ANOVA followed by Dunnet’s post-hoc analysis. (F) 3D confocal microscopy images from Control and Day 3 post-transplant Postn-CreER- tdTomato thymi stained with DAPI (white; cell nuclei), and tdTomato (red; Postn+ ThyMCs). (G) 3D confocal microscopy images of thymic tissue from an irradiated Postn-CreER- tdTomato mouse transplanted with 40 000 GFP+ lymphoid progenitors cells 2 days post- transplantation. Tissue was stained with DAPI (white; cell nuclei), tdTomato (red; Postn+ ThyMCs) and GFP (green; lymphoid progenitor donor cells). (H) Schematic illustration of experimental design for depletion of Postn+ cells in Postn- CreER-tdT/iDTA mice. (I) Flow cytometric quantification of early thymic progenitors and DN1 thymocytes in Control (iDTA Ctrl n=4) and Postn-CreER-tdT/iDTA (Postn iDTA n=6) mice 6 days post-bone marrow transplantation. Statistical significance was calculated using unpaired two-tailed student’s t-test. Two independent experiments. (J) Schematic illustration of experimental design for depletion of Penk+ cells in Penk-Cre- tdT/iDTR mice. (K) FACS analysis of early thymic progenitors and DN1 thymocytes in Control (iDTR Ctrl n=7) and Penk-Cre-tdT/iDTA (Penk iDTA n=7) mice 6 days post-bone marrow transplantation. Statistical significance was calculated using unpaired two-tailed student’s t-test. Two independent experiments.
    Figure Legend Snippet: Postn+ ThyMCs are lost during transplant conditioning and aging (A) Schematic overview of sample collection from Transplantation, IL7RKO and Aging stress conditions. (B) UMAP embeddings showing detailed annotation of different stromal cell populations ( Cd3e-, Cd4-, Cd8a-, Ptprc-) in Control (n=4, total number of stromal cells=5451), Transplant (n=3, total number of stromal cells=8057), IL7RKO (n=4, total number of stromal cells=4551) and Aging (n=4, total number of stromal cells=16178) samples. (C) The difference between Control and Transplantation, and Aging samples visualized via condition-specific densities in a joint ThyMC specific UMAP embedding (left). Detailed annotation of ThyMC subsets shown in ThyMC specific UMAP embedding (right). Control (n=4), Transplantation (n=3), and Aging (n=4), total number of ThyMC = 19739. (D) Bar graphs comparing proportional shifts of individual ThyMC subsets between Control, Transplant, and Aging samples as determined by scRNAseq. (Control n= 4, Transplant n= 3, Aging n=4) Statistical significance is based on beta regression with Benjamini- Hochberg FDR control of these comparisons. (E) Bar graphs showing flow cytometric quantification of absolute numbers of different ThyMC subsets in Control, Transplant (day 3) or Aged (2 years old) Penk-Cre-tdTomato mice. Statistical significance was calculated using one-way ANOVA followed by Dunnet’s post-hoc analysis. (F) 3D confocal microscopy images from Control and Day 3 post-transplant Postn-CreER- tdTomato thymi stained with DAPI (white; cell nuclei), and tdTomato (red; Postn+ ThyMCs). (G) 3D confocal microscopy images of thymic tissue from an irradiated Postn-CreER- tdTomato mouse transplanted with 40 000 GFP+ lymphoid progenitors cells 2 days post- transplantation. Tissue was stained with DAPI (white; cell nuclei), tdTomato (red; Postn+ ThyMCs) and GFP (green; lymphoid progenitor donor cells). (H) Schematic illustration of experimental design for depletion of Postn+ cells in Postn- CreER-tdT/iDTA mice. (I) Flow cytometric quantification of early thymic progenitors and DN1 thymocytes in Control (iDTA Ctrl n=4) and Postn-CreER-tdT/iDTA (Postn iDTA n=6) mice 6 days post-bone marrow transplantation. Statistical significance was calculated using unpaired two-tailed student’s t-test. Two independent experiments. (J) Schematic illustration of experimental design for depletion of Penk+ cells in Penk-Cre- tdT/iDTR mice. (K) FACS analysis of early thymic progenitors and DN1 thymocytes in Control (iDTR Ctrl n=7) and Penk-Cre-tdT/iDTA (Penk iDTA n=7) mice 6 days post-bone marrow transplantation. Statistical significance was calculated using unpaired two-tailed student’s t-test. Two independent experiments.

    Techniques Used: Transplantation Assay, Mouse Assay, Confocal Microscopy, Staining, Irradiation, Two Tailed Test, FACS

    22) Product Images from "Depletion of Scleraxis-lineage cells during tendon healing transiently impairs multi-scale restoration of tendon structure during early healing"

    Article Title: Depletion of Scleraxis-lineage cells during tendon healing transiently impairs multi-scale restoration of tendon structure during early healing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0274227

    αSMA myofibroblasts are transiently present between D14 and D35 post-surgery and retain Scx expression. A. Hind paws from the Scx-Cre ERT2 , Ai9 ; Scx GFP mice were probed with Green Fluorescence Protein (GFP) to visualize Scx GFP + cells, stained with αSMA to visualize myofibroblasts, and were counterstained with the nuclear dye DAPI. White dashed lines represent the tendon stub. Yellow dashed lines represent the bridging tissue between the tendon stubs. White arrows represent blood vessels. B. Cell density of a SMA + cells overtime. Significance was set to p
    Figure Legend Snippet: αSMA myofibroblasts are transiently present between D14 and D35 post-surgery and retain Scx expression. A. Hind paws from the Scx-Cre ERT2 , Ai9 ; Scx GFP mice were probed with Green Fluorescence Protein (GFP) to visualize Scx GFP + cells, stained with αSMA to visualize myofibroblasts, and were counterstained with the nuclear dye DAPI. White dashed lines represent the tendon stub. Yellow dashed lines represent the bridging tissue between the tendon stubs. White arrows represent blood vessels. B. Cell density of a SMA + cells overtime. Significance was set to p

    Techniques Used: Expressing, Mouse Assay, Fluorescence, Staining

    Scx Lin cells continuously expand with additonal cells expressing Scx during tendon healing. A. Schematic of the mouse model used and timeline for tamoxifen injections, tendon surgeries, and tissue harvesting. B. Hind paws from Scx-Cre ERT2 , Ai9 ; Scx-GFP were probed Red Fluorescence Protein (RFP), Green Fluorescence Protein (GFP), and were counterstained with the nuclear dye DAPI. C. Cell density of Scx LinAdult + , Scx GFP + , Scx LinAdult +;Scx GFP + and only DAPI+ . White dashed lines represent the tendon stub. Yellow dashed lines represent the bridging tissue between the tendon stubs. per timepoint. N = 3–5 per timepoint. Significance was set to p
    Figure Legend Snippet: Scx Lin cells continuously expand with additonal cells expressing Scx during tendon healing. A. Schematic of the mouse model used and timeline for tamoxifen injections, tendon surgeries, and tissue harvesting. B. Hind paws from Scx-Cre ERT2 , Ai9 ; Scx-GFP were probed Red Fluorescence Protein (RFP), Green Fluorescence Protein (GFP), and were counterstained with the nuclear dye DAPI. C. Cell density of Scx LinAdult + , Scx GFP + , Scx LinAdult +;Scx GFP + and only DAPI+ . White dashed lines represent the tendon stub. Yellow dashed lines represent the bridging tissue between the tendon stubs. per timepoint. N = 3–5 per timepoint. Significance was set to p

    Techniques Used: Expressing, Fluorescence

    23) Product Images from "The Hippo pathway links adipocyte plasticity to adipose tissue fibrosis"

    Article Title: The Hippo pathway links adipocyte plasticity to adipose tissue fibrosis

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33800-0

    Lats1/2 deficiency promotes cell fate conversion from adipocytes to myofibroblasts. a Immunoblot analysis of protein expression of GFP in SVF and adipocyte from 8-week-old Adipoq Cre Rosa26 mTmG scWAT. Independent experiments were performed twice with similar results. b Flow cytometry analysis of percentages of live CD45 − CD31 − GFP + DPP4 + cells in scWAT SVF of P21 L1L2-FF LSL-CAS9-EGFP or L1L2-AKO LSL-CAS9-EGFP reporter mice ( n = 5). c Representative sections of scWAT of P14 or P28 L1L2-AKO LSL-CAS9-EGFP mice stained for αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). Arrowheads indicate examples of GFP + DPP4 + cells. Arrows point to GFP + αSMA + cells. Independent experiments were performed three times with similar results. d Sorted cell subsets from P21 L1L2-AKO mTmG scWAT SVF were plated and stained for αSMA (AF647, red) and nucleus (DAPI, blue). e Quantification of percentages of GFP+DPP4+ ( n = 6), GFP+DPP4- ( n = 7), GFP-DPP4+ ( n = 7), GFP-DPP4- ( n = 7) cells. Dots represent cell percentages of random visual fields from P21 L1L2-AKOmTmG mice. Independent experiments were performed twice with similar results. f Sorted GFP + DPP4 + cells from P21 L1L2-AKO LSL-CAS9-EGFP scWAT SVF were treated with TGF-β1 (10 ng/ml) for 0 h or 24 h. Cells were stained with αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). An arrowhead points to the GFP + αSMA − DPP4 + cell. Independent experiments were performed twice with similar results. g Sorted GFP + DPP4 + cells from L1L2-AKO LSL-CAS9-EGFP reporter scWAT SVF were analyzed for DPP4 expression in GFP + cells before or after transplantation into scWAT of 8-day-old L1L2-AKO recipient mice. One representative transplant from n = 3 biological replicates. Data are means ± SEM. One-way ANOVA with Bonferroni’s multiple-comparisons test in e ; two-tailed unpaired Student’s t test in b . * P
    Figure Legend Snippet: Lats1/2 deficiency promotes cell fate conversion from adipocytes to myofibroblasts. a Immunoblot analysis of protein expression of GFP in SVF and adipocyte from 8-week-old Adipoq Cre Rosa26 mTmG scWAT. Independent experiments were performed twice with similar results. b Flow cytometry analysis of percentages of live CD45 − CD31 − GFP + DPP4 + cells in scWAT SVF of P21 L1L2-FF LSL-CAS9-EGFP or L1L2-AKO LSL-CAS9-EGFP reporter mice ( n = 5). c Representative sections of scWAT of P14 or P28 L1L2-AKO LSL-CAS9-EGFP mice stained for αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). Arrowheads indicate examples of GFP + DPP4 + cells. Arrows point to GFP + αSMA + cells. Independent experiments were performed three times with similar results. d Sorted cell subsets from P21 L1L2-AKO mTmG scWAT SVF were plated and stained for αSMA (AF647, red) and nucleus (DAPI, blue). e Quantification of percentages of GFP+DPP4+ ( n = 6), GFP+DPP4- ( n = 7), GFP-DPP4+ ( n = 7), GFP-DPP4- ( n = 7) cells. Dots represent cell percentages of random visual fields from P21 L1L2-AKOmTmG mice. Independent experiments were performed twice with similar results. f Sorted GFP + DPP4 + cells from P21 L1L2-AKO LSL-CAS9-EGFP scWAT SVF were treated with TGF-β1 (10 ng/ml) for 0 h or 24 h. Cells were stained with αSMA (red), DPP4 (purple), nucleus (DAPI, blue) and GFP (green). An arrowhead points to the GFP + αSMA − DPP4 + cell. Independent experiments were performed twice with similar results. g Sorted GFP + DPP4 + cells from L1L2-AKO LSL-CAS9-EGFP reporter scWAT SVF were analyzed for DPP4 expression in GFP + cells before or after transplantation into scWAT of 8-day-old L1L2-AKO recipient mice. One representative transplant from n = 3 biological replicates. Data are means ± SEM. One-way ANOVA with Bonferroni’s multiple-comparisons test in e ; two-tailed unpaired Student’s t test in b . * P

    Techniques Used: Expressing, Flow Cytometry, Mouse Assay, Staining, Transplantation Assay, Two Tailed Test

    TGFβ stimulation is coupled with Hippo pathway inactivation to promote AT fibrosis. a Quantification of phosphorylation levels of SMAD2 in scWAT during growth ( n = 4 mice). b Eight-week-old male L1L2-FF or Lats1 f/f Lats2 f/f Adipoq CreERT2 (L1L2-iAKO) mice were locally injected with AAV-CAG-GFP and AAV-CAG-TGFβR1 (T204D) respectively in scWAT. After 3 days, all mice were intraperitoneally (i.p.) administered 3 doses of tamoxifen every other day and then analyzed 4 weeks later. c Immunostaining of αSMA + cells in the indicated mice. Independent experiments were performed twice with similar results. d mRNA expression of fibrotic and adipocyte markers in L1L2-FF or L1L2-iAKO scWAT transduced with AAV-CAG-GFP ( n = 5 mice) or AAV-CAG-TGFβR1 (T204D) ( n = 6 mice) followed by tamoxifen administration. e , f L1L2-FF or L1L2-iAKO mice were locally injected with AAV-CAG-TGFβ1 (2CS) in scWAT. e mRNA expression of fibrotic and adipocyte markers ( n = 5 mice). f Immunostaining of αSMA + cells. g AAV vectors for inducible expression of TGFβR1 (T204D) by Double-Floxed Inverted Open reading frame system. h , i , mRNA expression of fibrotic and adipocyte markers ( h ) ( n = 4 mice) or immunostaining of αSMA + cells ( i ) in L1L2-FF or L1L2-iAKO scWAT transduced with AAV-CAG-GFP or AAV-EF1A-DIO-TGFβR1. j Eight-week-old male L1L2-FF and L1L2-iAKO mice were subcutaneously co-injected with AAV-ADP-FLPo and AAV-EF1A-FIO-TGFβR1 (T204D) for 3 weeks, followed by i.p. injection of tamoxifen. k , l mRNA expression of fibrosis markers ( k ) ( n = 6 mice) or immunostaining of αSMA + cells ( l ) of mice in j . m mRNA expression of Acta2 of Cas9 Tg/+ SVF transduced with vector ( n = 4 biologically independent cell cultures) or YT-gRNA ( n = 3 biologically independent cell cultures) in the presence or absence of TGFβ1 for 36 h. Data are means ± SEM. Two-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparisons test in d , e , h , k , m ; * P
    Figure Legend Snippet: TGFβ stimulation is coupled with Hippo pathway inactivation to promote AT fibrosis. a Quantification of phosphorylation levels of SMAD2 in scWAT during growth ( n = 4 mice). b Eight-week-old male L1L2-FF or Lats1 f/f Lats2 f/f Adipoq CreERT2 (L1L2-iAKO) mice were locally injected with AAV-CAG-GFP and AAV-CAG-TGFβR1 (T204D) respectively in scWAT. After 3 days, all mice were intraperitoneally (i.p.) administered 3 doses of tamoxifen every other day and then analyzed 4 weeks later. c Immunostaining of αSMA + cells in the indicated mice. Independent experiments were performed twice with similar results. d mRNA expression of fibrotic and adipocyte markers in L1L2-FF or L1L2-iAKO scWAT transduced with AAV-CAG-GFP ( n = 5 mice) or AAV-CAG-TGFβR1 (T204D) ( n = 6 mice) followed by tamoxifen administration. e , f L1L2-FF or L1L2-iAKO mice were locally injected with AAV-CAG-TGFβ1 (2CS) in scWAT. e mRNA expression of fibrotic and adipocyte markers ( n = 5 mice). f Immunostaining of αSMA + cells. g AAV vectors for inducible expression of TGFβR1 (T204D) by Double-Floxed Inverted Open reading frame system. h , i , mRNA expression of fibrotic and adipocyte markers ( h ) ( n = 4 mice) or immunostaining of αSMA + cells ( i ) in L1L2-FF or L1L2-iAKO scWAT transduced with AAV-CAG-GFP or AAV-EF1A-DIO-TGFβR1. j Eight-week-old male L1L2-FF and L1L2-iAKO mice were subcutaneously co-injected with AAV-ADP-FLPo and AAV-EF1A-FIO-TGFβR1 (T204D) for 3 weeks, followed by i.p. injection of tamoxifen. k , l mRNA expression of fibrosis markers ( k ) ( n = 6 mice) or immunostaining of αSMA + cells ( l ) of mice in j . m mRNA expression of Acta2 of Cas9 Tg/+ SVF transduced with vector ( n = 4 biologically independent cell cultures) or YT-gRNA ( n = 3 biologically independent cell cultures) in the presence or absence of TGFβ1 for 36 h. Data are means ± SEM. Two-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparisons test in d , e , h , k , m ; * P

    Techniques Used: Mouse Assay, Injection, Immunostaining, Expressing, Transduction, Plasmid Preparation

    Lats1/2 deficiency in adipocytes elicits a CCL2/CCL7-macrophage feedforward loop that increases TGFβ expression in macrophages. a Expression of F4/80 , Col1a1 , and Acta2 of L1L2-FF ( n = 6) and L1L2-AKO ( n = 5 at P1, n = 6 at P7, P14 and P21) mice during growth. b Representative scWAT sections stained for F4/80 of 3-week-old male L1L2-FF or L1L2-AKO mice. Independent experiments were performed three times with similar results. c Quantification of F4/80 + cell percentages in scWAT SVF of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). d , e Quantification of CD206 + CD11c − ( d ) and CD206 − CD11c + ( e ) cell percentages in scWAT SVF of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). f mRNA expression of inflammatory markers in scWAT of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). g mRNA expression of inflammatory genes in male L1L2-FF and L1L2-iAKO mice that were locally injected with AAV-CAG-GFP ( n = 5) or AAV-CAG-TGFβR1 (T204D) ( n = 6) in scWAT. h mRNA expression of inflammatory genes in male L1L2-FF or L1L2-iAKO mice that were locally injected with AAV-CAG-TGFβ1 (2CS) in scWAT ( n = 5). i mRNA expression of inflammatory genes in male L1L2-FF or L1L2-iAKO mice that were locally injected with AAV-EF1A-DIO-TGFβR1 (T204D) in scWAT ( n = 4). j Eight-week-old male L1L2-iAKO mice were subcutaneously injected with AAV-CAG-GFP ( n = 7) or AAV-CAG-CCL2/CCL7 ( n = 5) and analyzed for mRNA expression of inflammatory genes and fibrosis markers 4 weeks later. k F4/80 − and F4/80 + cells were magnetically sorted from pooled WT scWAT SVF and analyzed for mRNA expression of Tgfb1/2/3 ( n = 3). scWAT from three male mice were pooled as one sample. l BMDMs were treated with Veh, and IL4 plus IL13 for 24 h and analyzed for Mrc1 , Ccl2 and Ccl7 mRNA expression ( n = 4 biologically independent cell cultures). Data are means ± SEM. Two-tailed unpaired Student’s t test in a , c – f , h – l ; two-way ANOVA with Bonferroni’s multiple-comparisons test in g ; * P
    Figure Legend Snippet: Lats1/2 deficiency in adipocytes elicits a CCL2/CCL7-macrophage feedforward loop that increases TGFβ expression in macrophages. a Expression of F4/80 , Col1a1 , and Acta2 of L1L2-FF ( n = 6) and L1L2-AKO ( n = 5 at P1, n = 6 at P7, P14 and P21) mice during growth. b Representative scWAT sections stained for F4/80 of 3-week-old male L1L2-FF or L1L2-AKO mice. Independent experiments were performed three times with similar results. c Quantification of F4/80 + cell percentages in scWAT SVF of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). d , e Quantification of CD206 + CD11c − ( d ) and CD206 − CD11c + ( e ) cell percentages in scWAT SVF of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). f mRNA expression of inflammatory markers in scWAT of 3-week-old male L1L2-FF or L1L2-AKO mice ( n = 6). g mRNA expression of inflammatory genes in male L1L2-FF and L1L2-iAKO mice that were locally injected with AAV-CAG-GFP ( n = 5) or AAV-CAG-TGFβR1 (T204D) ( n = 6) in scWAT. h mRNA expression of inflammatory genes in male L1L2-FF or L1L2-iAKO mice that were locally injected with AAV-CAG-TGFβ1 (2CS) in scWAT ( n = 5). i mRNA expression of inflammatory genes in male L1L2-FF or L1L2-iAKO mice that were locally injected with AAV-EF1A-DIO-TGFβR1 (T204D) in scWAT ( n = 4). j Eight-week-old male L1L2-iAKO mice were subcutaneously injected with AAV-CAG-GFP ( n = 7) or AAV-CAG-CCL2/CCL7 ( n = 5) and analyzed for mRNA expression of inflammatory genes and fibrosis markers 4 weeks later. k F4/80 − and F4/80 + cells were magnetically sorted from pooled WT scWAT SVF and analyzed for mRNA expression of Tgfb1/2/3 ( n = 3). scWAT from three male mice were pooled as one sample. l BMDMs were treated with Veh, and IL4 plus IL13 for 24 h and analyzed for Mrc1 , Ccl2 and Ccl7 mRNA expression ( n = 4 biologically independent cell cultures). Data are means ± SEM. Two-tailed unpaired Student’s t test in a , c – f , h – l ; two-way ANOVA with Bonferroni’s multiple-comparisons test in g ; * P

    Techniques Used: Expressing, Mouse Assay, Staining, Injection, Two Tailed Test

    Lats1/2 deficiency induces AT fibrosis in obese mice. a mRNA expression of Tgfb1/2/3 in scWAT of male mice fed a ND or HFD for 18 weeks from 6 weeks old ( n = 5 mice). b Immunoblot analysis of phosphorylation of SMAD2/3 (p-SMAD2/3) of mice fed a ND or HFD for 18 weeks. Right, quantification of protein and relative phosphorylation levels of SMAD2/3 ( n = 3 mice). c mRNA expression of Tgfb1/2/3 in scWAT of 12-week-old male WT or ob/ob mice ( n = 5 mice). d Immunoblot analysis of protein expression of p-SMAD2/3 of mice in 12-week-old male WT or ob/ob mice. Right, quantification of protein and relative phosphorylation levels of SMAD2/3 ( n = 4 mice). e , f Eight-week-old male Lats1 f/f Lats2 f/f mice were injected with AAV-ADP-GFP or AAV-ADP-Cre in scWAT for 4 weeks. e Immunoblot analysis of LATS1 or LATS2 knockout efficiency in scWAT. f mRNA expression of fibrotic and adipocyte markers in scWAT ( n = 6 mice). g Experimental outline: male L1L2-FF mice were fed a HFD for 16 weeks from 6 weeks old, followed by a local injection with AAV-ADP-GFP or AAV-ADP-Cre in scWAT, and all were analyzed after 4 weeks. h RT-qPCR analysis of fibrosis markers in scWAT of mice in g ( n = 5 mice). i RT-qPCR analysis of fibrosis markers in scWAT of 12-week-old male Lats1 f/f Lats2 f/f ob/ob mice injected with AAV-ADP-GFP or AAV-ADP-Cre ( n = 5 mice). j , k Representative scWAT sections with Masson’s trichrome staining ( j ) or Picrosirius red staining ( k ) of mice in i . Data are means ± SEM. Two-tailed unpaired Student’s t test; * P
    Figure Legend Snippet: Lats1/2 deficiency induces AT fibrosis in obese mice. a mRNA expression of Tgfb1/2/3 in scWAT of male mice fed a ND or HFD for 18 weeks from 6 weeks old ( n = 5 mice). b Immunoblot analysis of phosphorylation of SMAD2/3 (p-SMAD2/3) of mice fed a ND or HFD for 18 weeks. Right, quantification of protein and relative phosphorylation levels of SMAD2/3 ( n = 3 mice). c mRNA expression of Tgfb1/2/3 in scWAT of 12-week-old male WT or ob/ob mice ( n = 5 mice). d Immunoblot analysis of protein expression of p-SMAD2/3 of mice in 12-week-old male WT or ob/ob mice. Right, quantification of protein and relative phosphorylation levels of SMAD2/3 ( n = 4 mice). e , f Eight-week-old male Lats1 f/f Lats2 f/f mice were injected with AAV-ADP-GFP or AAV-ADP-Cre in scWAT for 4 weeks. e Immunoblot analysis of LATS1 or LATS2 knockout efficiency in scWAT. f mRNA expression of fibrotic and adipocyte markers in scWAT ( n = 6 mice). g Experimental outline: male L1L2-FF mice were fed a HFD for 16 weeks from 6 weeks old, followed by a local injection with AAV-ADP-GFP or AAV-ADP-Cre in scWAT, and all were analyzed after 4 weeks. h RT-qPCR analysis of fibrosis markers in scWAT of mice in g ( n = 5 mice). i RT-qPCR analysis of fibrosis markers in scWAT of 12-week-old male Lats1 f/f Lats2 f/f ob/ob mice injected with AAV-ADP-GFP or AAV-ADP-Cre ( n = 5 mice). j , k Representative scWAT sections with Masson’s trichrome staining ( j ) or Picrosirius red staining ( k ) of mice in i . Data are means ± SEM. Two-tailed unpaired Student’s t test; * P

    Techniques Used: Mouse Assay, Expressing, Injection, Knock-Out, Quantitative RT-PCR, Staining, Two Tailed Test

    24) Product Images from "Mesenchymal stem cell transplantation improves biomechanical properties of vaginal tissue following full-thickness incision in aged rats"

    Article Title: Mesenchymal stem cell transplantation improves biomechanical properties of vaginal tissue following full-thickness incision in aged rats

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2022.09.005

    Homing and survival of transplanted MSCs following full-thickness vaginal incision (A and B) Low power images of H E staining show the location of the injury site, just opposite to the urethra in sham (A) and MSC-transplanted (B) old rats at day 3 post-transplantation. (C) Bar graphs displaying the maximal distance between epithelial edges of the wound (μm) in old and young rats at 3 days post-transplantation. (D and E) A higher magnification of the incision sites of sham (D) and MSC-transplanted (E) rats are shown. (F) Immunofluorescence image of a section of a sham-operated rat at day 3 post-injury, stained with anti-cytokeratin and counterstained with DAPI. (G) A dual-color immunofluorescence image showing cytokeratin staining and PKH26 + cells in an old MSC-transplanted rat at day 3 post-transplantation. (H and I) DAPI-stained vaginal section from an old MSC-transplanted rat at day 30 post-injury showing a venule/arteriole (H), which is composed of GFP-labeled cells (I). (J) A merged image of (H) and (I). (K and L) Transplanted PKH-26-labeled MSCs (K) in the venule/arteriole wall expressed CD31 (L). (M) Nuclei are counterstained with DAPI. (N) A merged image showing the co-localization of PKH-26 and CD31. Scale bars: (A and B) 500, (D and E) 200, (E and F) 100, and (H–N) 50 μm. Mean ± SD are presented (C). Two-way ANOVA performed in (C). Post hoc analysis between specific groups was performed using Bonferroni correction. ∗∗ p
    Figure Legend Snippet: Homing and survival of transplanted MSCs following full-thickness vaginal incision (A and B) Low power images of H E staining show the location of the injury site, just opposite to the urethra in sham (A) and MSC-transplanted (B) old rats at day 3 post-transplantation. (C) Bar graphs displaying the maximal distance between epithelial edges of the wound (μm) in old and young rats at 3 days post-transplantation. (D and E) A higher magnification of the incision sites of sham (D) and MSC-transplanted (E) rats are shown. (F) Immunofluorescence image of a section of a sham-operated rat at day 3 post-injury, stained with anti-cytokeratin and counterstained with DAPI. (G) A dual-color immunofluorescence image showing cytokeratin staining and PKH26 + cells in an old MSC-transplanted rat at day 3 post-transplantation. (H and I) DAPI-stained vaginal section from an old MSC-transplanted rat at day 30 post-injury showing a venule/arteriole (H), which is composed of GFP-labeled cells (I). (J) A merged image of (H) and (I). (K and L) Transplanted PKH-26-labeled MSCs (K) in the venule/arteriole wall expressed CD31 (L). (M) Nuclei are counterstained with DAPI. (N) A merged image showing the co-localization of PKH-26 and CD31. Scale bars: (A and B) 500, (D and E) 200, (E and F) 100, and (H–N) 50 μm. Mean ± SD are presented (C). Two-way ANOVA performed in (C). Post hoc analysis between specific groups was performed using Bonferroni correction. ∗∗ p

    Techniques Used: Staining, Transplantation Assay, Immunofluorescence, Labeling

    25) Product Images from "LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study"

    Article Title: LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2022.10.009

    Knockdown of apo(a) in LPA +/0 Ldlr −/− mice by AAV-CRISPR (A) Male and female LPA +/0 Ldlr −/− mice were injected at 8 weeks of age with 1 × 10 12 genome copies of AAV expressing either GFP as a control (AAV-GFP) or Cas9 and an sgRNA targeting LPA exon 2 in the KIV 1 domain of the LPA transgene (AAV-CRISPR). Plasma and body weights were collected weekly until 12 weeks of age, at which point plasma and liver tissue were harvested. (B) Weekly body weights in male and female mice. (C) Liver to body weight ratios and (D) fasted plasma cholesterol levels in male and female mice given either AAV-GFP or AAV-CRISPR. (E) Western blot for GFP in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (F) Quantification for GFP western blot. All groups normalized to male mice given AAV-GFP vector. (G) Western blot for SaCas9 in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (H) Quantification for SaCas9 western blot. All groups normalized to male mice given AAV-GFP vector. (I) Western blot for apo(a) in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (J) Time course for apo(a) expression in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR measured by ELISA (n ≥ 6). Statistical analysis was done using Mann-Whitney test on non-normally distributed data with ∗p
    Figure Legend Snippet: Knockdown of apo(a) in LPA +/0 Ldlr −/− mice by AAV-CRISPR (A) Male and female LPA +/0 Ldlr −/− mice were injected at 8 weeks of age with 1 × 10 12 genome copies of AAV expressing either GFP as a control (AAV-GFP) or Cas9 and an sgRNA targeting LPA exon 2 in the KIV 1 domain of the LPA transgene (AAV-CRISPR). Plasma and body weights were collected weekly until 12 weeks of age, at which point plasma and liver tissue were harvested. (B) Weekly body weights in male and female mice. (C) Liver to body weight ratios and (D) fasted plasma cholesterol levels in male and female mice given either AAV-GFP or AAV-CRISPR. (E) Western blot for GFP in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (F) Quantification for GFP western blot. All groups normalized to male mice given AAV-GFP vector. (G) Western blot for SaCas9 in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (H) Quantification for SaCas9 western blot. All groups normalized to male mice given AAV-GFP vector. (I) Western blot for apo(a) in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR. (J) Time course for apo(a) expression in male and female LPA +/0 Ldlr −/− mice given AAV-GFP or AAV-CRISPR measured by ELISA (n ≥ 6). Statistical analysis was done using Mann-Whitney test on non-normally distributed data with ∗p

    Techniques Used: Mouse Assay, CRISPR, Injection, Expressing, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    26) Product Images from "Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms"

    Article Title: Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms

    Journal: bioRxiv

    doi: 10.1101/2022.10.06.511083

    SCPs are sensed by RE02 and RLP30. A, Ethylene accumulation in Arabidopsis wild-type plants (Col-0) or indicated mutants 4 h after treatment with water (mock), 1 μM nlp20, 1 μM SCP Ss , or homologs from B. cinerea ( Bc ) and Phytophthora infestans ( Pi ). b, N. benthamiana plants were silenced for RE02 , and transiently transformed with RLP23-GFP, RLP30-GFP , or RE02-GFP , respectively. The TRV2:GUS construct was used as a control. Ethylene production was measured 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM SCP Ss . c, Ethylene accumulation in Arabidopsis rlp30-2 mutants or lines stably expressing RLP30-YFP or RE02-GFP (line 4 and 5) 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM SCP Ss . Data points are indicated as dots ( n = 6) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from control treatments are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.
    Figure Legend Snippet: SCPs are sensed by RE02 and RLP30. A, Ethylene accumulation in Arabidopsis wild-type plants (Col-0) or indicated mutants 4 h after treatment with water (mock), 1 μM nlp20, 1 μM SCP Ss , or homologs from B. cinerea ( Bc ) and Phytophthora infestans ( Pi ). b, N. benthamiana plants were silenced for RE02 , and transiently transformed with RLP23-GFP, RLP30-GFP , or RE02-GFP , respectively. The TRV2:GUS construct was used as a control. Ethylene production was measured 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM SCP Ss . c, Ethylene accumulation in Arabidopsis rlp30-2 mutants or lines stably expressing RLP30-YFP or RE02-GFP (line 4 and 5) 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM SCP Ss . Data points are indicated as dots ( n = 6) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from control treatments are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.

    Techniques Used: Transformation Assay, Construct, Stable Transfection, Expressing

    RLP30 expression in N. tabacum confers increased resistance to bacterial, fungal, and oomycete pathogens. a, Ethylene accumulation in N. tabacum wild-type plants (Wt) or two transgenic lines ( #49 and #55 ) stably expressing RLP30-RFP and SOBIR1-GFP after 4 h treatment with water (mock), 1 μM SCP from indicated sources, or 1.5 μg/ml SCP-like Psp . b, Bacterial growth of P. syringae pv. tabaci in N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (#49 and #55). Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. c, B. cinerea infected area on leaves of N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (left, shown are representative leaves) and determination of lesion diameter on day 2 after drop inoculation (right). d, Growth of Phytophthora capsici on leaves of N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines by determination of lesion size (right) of lesions observed under UV light (left, shown are representative leaves) on day 2 after drop inoculation. Data points are indicated as dots ( n = 6 for a; n = 20 for b, n = 10 for c, d) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from wild-type (Wt) plants are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.
    Figure Legend Snippet: RLP30 expression in N. tabacum confers increased resistance to bacterial, fungal, and oomycete pathogens. a, Ethylene accumulation in N. tabacum wild-type plants (Wt) or two transgenic lines ( #49 and #55 ) stably expressing RLP30-RFP and SOBIR1-GFP after 4 h treatment with water (mock), 1 μM SCP from indicated sources, or 1.5 μg/ml SCP-like Psp . b, Bacterial growth of P. syringae pv. tabaci in N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (#49 and #55). Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. c, B. cinerea infected area on leaves of N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines (left, shown are representative leaves) and determination of lesion diameter on day 2 after drop inoculation (right). d, Growth of Phytophthora capsici on leaves of N. tabacum wild-type plants (Wt) or transgenic RLP30/SOBIR1 lines by determination of lesion size (right) of lesions observed under UV light (left, shown are representative leaves) on day 2 after drop inoculation. Data points are indicated as dots ( n = 6 for a; n = 20 for b, n = 10 for c, d) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from wild-type (Wt) plants are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.

    Techniques Used: Expressing, Transgenic Assay, Stable Transfection, Infection

    Pseudomonads produce a RLP30-dependent elicitor activity. A, Ethylene accumulation in Col-0 wild-type plants, rlp30-2 mutants or an rlp30-2 line complemented with a p35S::RL30-YFP construct 4 h after treatment with water (mock), 1 μM SCP Ss or 1.5 μg/ml SCP-like from Pseudomonas syringae pv. tomato (Pst), P. syringae pv. phaseolicola ( Psp ), P. fluorescens ( Pflu ), P. protegens ( Ppr ), and P. stutzeri ( Pstu ). b, Bacterial growth in Col-0 wild-type plants, rlp30-2 mutants, or the rlp30-2/RLP30-YFP complementation line treated with water (mock), 1 μM nlp20, or 1.5 μg/ml SCP-like Psp 24 h before infiltration of Pst DC3000. Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. c, Ethylene accumulation in N. benthamiana ( Nb ) plants transiently expressing RLP23-GFP or RLP30-GFP and treated for 4 h with water (mock), 1 μM nlp20, or 1.5 μg/ml SCP-like Psp . Data points are indicated as dots ( n = 6 for a, c; n = 20 for b) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from Col-0 plants ( a ) or mock treatments ( b,c ) are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.
    Figure Legend Snippet: Pseudomonads produce a RLP30-dependent elicitor activity. A, Ethylene accumulation in Col-0 wild-type plants, rlp30-2 mutants or an rlp30-2 line complemented with a p35S::RL30-YFP construct 4 h after treatment with water (mock), 1 μM SCP Ss or 1.5 μg/ml SCP-like from Pseudomonas syringae pv. tomato (Pst), P. syringae pv. phaseolicola ( Psp ), P. fluorescens ( Pflu ), P. protegens ( Ppr ), and P. stutzeri ( Pstu ). b, Bacterial growth in Col-0 wild-type plants, rlp30-2 mutants, or the rlp30-2/RLP30-YFP complementation line treated with water (mock), 1 μM nlp20, or 1.5 μg/ml SCP-like Psp 24 h before infiltration of Pst DC3000. Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. c, Ethylene accumulation in N. benthamiana ( Nb ) plants transiently expressing RLP23-GFP or RLP30-GFP and treated for 4 h with water (mock), 1 μM nlp20, or 1.5 μg/ml SCP-like Psp . Data points are indicated as dots ( n = 6 for a, c; n = 20 for b) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from Col-0 plants ( a ) or mock treatments ( b,c ) are indicated (two-sided Student’s t-test, *** P ≤ 0.001). Each experiment was repeated three times with similar results.

    Techniques Used: Activity Assay, Construct, Expressing

    SCP Ss is immunogenic and binds to RLP30. a, Ethylene accumulation in Arabidopsis Col-0 wildtype plants, rlp30-2 mutants, or an rlp30-2 line complemented with a p35S::RL30-YFP construct 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM P. pastoris -expressed SCP Ss . b, B. cinerea infected area as determined by lesion diameter on day 2 after 24 h-treatment of Col-0 wild-type plants, rlp30-2 mutants or the rlp30-2/RLP30-YFP complementation line with water (mock), 1 μM nlp20, or 1 μM SCP Ss . c, Bacterial growth in plants pre-treated with water (mock), 1 μM nlp20, or 1 μM SCP Ss 24 h before infiltration of Pst DC3000. Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. d, Ethylene accumulation in plants of the Brassicaceae and Solanaceae family 4 h after treatment with water (mock), 1 μM flg22, or 1 μM SCP Ss . e, Ligand-binding assay in N. benthamiana transiently co-expressing SCP Ss -myc and either RLP30-GFP or RLP23-GFP. Leaf protein extracts (Input) were used for co-immunoprecipitation with GFP-trap beads (IP:GFP) and immunoblotting with tag-specific antibodies. For a-d, data points are indicated as dots ( n = 6 for a, b, d; n = 20 for c) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from mock treatments in the respective plants are indicated (two-sided Student’s t-test, ** P ≤ 0.01, *** P ≤ 0.001). All assays were performed at least three times with similar results.
    Figure Legend Snippet: SCP Ss is immunogenic and binds to RLP30. a, Ethylene accumulation in Arabidopsis Col-0 wildtype plants, rlp30-2 mutants, or an rlp30-2 line complemented with a p35S::RL30-YFP construct 4 h after treatment with water (mock), 1 μM nlp20, or 1 μM P. pastoris -expressed SCP Ss . b, B. cinerea infected area as determined by lesion diameter on day 2 after 24 h-treatment of Col-0 wild-type plants, rlp30-2 mutants or the rlp30-2/RLP30-YFP complementation line with water (mock), 1 μM nlp20, or 1 μM SCP Ss . c, Bacterial growth in plants pre-treated with water (mock), 1 μM nlp20, or 1 μM SCP Ss 24 h before infiltration of Pst DC3000. Bacteria (colony forming units, CFU) were quantified in extracts of leaves 3 days after inoculation. d, Ethylene accumulation in plants of the Brassicaceae and Solanaceae family 4 h after treatment with water (mock), 1 μM flg22, or 1 μM SCP Ss . e, Ligand-binding assay in N. benthamiana transiently co-expressing SCP Ss -myc and either RLP30-GFP or RLP23-GFP. Leaf protein extracts (Input) were used for co-immunoprecipitation with GFP-trap beads (IP:GFP) and immunoblotting with tag-specific antibodies. For a-d, data points are indicated as dots ( n = 6 for a, b, d; n = 20 for c) and plotted as box plots (centre line, median; bounds of box, the first and third quartiles; whiskers, 1.5 times the interquartile range; error bar, minima and maxima). Statistically significant differences from mock treatments in the respective plants are indicated (two-sided Student’s t-test, ** P ≤ 0.01, *** P ≤ 0.001). All assays were performed at least three times with similar results.

    Techniques Used: Construct, Infection, Ligand Binding Assay, Expressing, Immunoprecipitation

    27) Product Images from "ZIP1+ fibroblasts protect lung cancer against chemotherapy via connexin-43 mediated intercellular Zn2+ transfer"

    Article Title: ZIP1+ fibroblasts protect lung cancer against chemotherapy via connexin-43 mediated intercellular Zn2+ transfer

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33521-4

    Labile Zn 2+ induces ABCB1-mediated drug extrusion. a Zn 2+ uptake by LLC cells treated with paclitaxel (PTX). ZnCl 2 (10 μM) and PTX were added simultaneously. Ctl, no ZnCl 2 . n = 3. b DOX accumulation (determined by DOX fluorescence in individual cells) and DNA damage (indicated by γ-H2AX expression in individual cells) in LLC-GFP-luc cells co-cultured with MEFs in DMEM + 10% FBS + TPEN (5 μM) for 4 h. TPEN: 50 μM, pre-treating MEFs for 5 min. HEPT: 2 mM. DOX: 3 μM. Median ± interquartile range. RFU, relative fluorescence unit. c ABCB1 expression in LLC-GFP-luc cells co-cultured with mCAFs in DMEM + 10% FBS for 24 h. A representative result from three independent experiments is shown. d LLC-GFP-luc cells viability co-cultured with mCAFs under indicated conditions in DMEM + 10% FBS + 5 μM TPEN for 24 h, determined by luciferase activity. DOX: 3 μM. Mean ± SD, n = 6. Two-tailed t -test. e Inhibition rate of PTX on LLC cells with or without TPEN (0.3 μM) in DMEM determined by CCK8. Mean ± SD, n = 3 for PTX 30 nM group. n = 4 for other groups. One-way ANOVA with multiple comparisons. f Inhibition rate of DOX on LLC-GFP-luc cells with different concentrations of ZnCl 2 determined by CCK8. DOX: 3 μM. Cells were treated for 24 h in DMEM + 10% FBS + TPEN (5 μM). Mean ± SEM, n = 3. Two-tailed t -test. g , h ABCB1 expression in LLC-GFP-luc cells treated with indicated concentrations of ZnCl 2 for 4 h ( g ), or with 10 µM ZnCl 2 for the indicated time ( h ). Representative results from three ( g ) or four ( h ) independent experiments are shown. i ABCB1 expression on LLC-GFP-luc tumour cells treated with indicated concentrations of ZnCl 2 for 4 h. Mean ± SEM, n = 3. Two-tailed t -test. j DOX accumulation in LLC-GFP-luc cells treated with 3 μM DOX combined with indicated concentrations of ZnCl 2 for 4 h. Blank: no DOX treatment. Mean ± SEM, n = 4. Two-tailed t -test. k ABCB1 expression in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) and LY294002 (50 μM) for 3 h. A representative result from three independent experiments is shown. Two-way ANOVA test for curve comparison. Source data are provided as a Source Data file ( a–k ).
    Figure Legend Snippet: Labile Zn 2+ induces ABCB1-mediated drug extrusion. a Zn 2+ uptake by LLC cells treated with paclitaxel (PTX). ZnCl 2 (10 μM) and PTX were added simultaneously. Ctl, no ZnCl 2 . n = 3. b DOX accumulation (determined by DOX fluorescence in individual cells) and DNA damage (indicated by γ-H2AX expression in individual cells) in LLC-GFP-luc cells co-cultured with MEFs in DMEM + 10% FBS + TPEN (5 μM) for 4 h. TPEN: 50 μM, pre-treating MEFs for 5 min. HEPT: 2 mM. DOX: 3 μM. Median ± interquartile range. RFU, relative fluorescence unit. c ABCB1 expression in LLC-GFP-luc cells co-cultured with mCAFs in DMEM + 10% FBS for 24 h. A representative result from three independent experiments is shown. d LLC-GFP-luc cells viability co-cultured with mCAFs under indicated conditions in DMEM + 10% FBS + 5 μM TPEN for 24 h, determined by luciferase activity. DOX: 3 μM. Mean ± SD, n = 6. Two-tailed t -test. e Inhibition rate of PTX on LLC cells with or without TPEN (0.3 μM) in DMEM determined by CCK8. Mean ± SD, n = 3 for PTX 30 nM group. n = 4 for other groups. One-way ANOVA with multiple comparisons. f Inhibition rate of DOX on LLC-GFP-luc cells with different concentrations of ZnCl 2 determined by CCK8. DOX: 3 μM. Cells were treated for 24 h in DMEM + 10% FBS + TPEN (5 μM). Mean ± SEM, n = 3. Two-tailed t -test. g , h ABCB1 expression in LLC-GFP-luc cells treated with indicated concentrations of ZnCl 2 for 4 h ( g ), or with 10 µM ZnCl 2 for the indicated time ( h ). Representative results from three ( g ) or four ( h ) independent experiments are shown. i ABCB1 expression on LLC-GFP-luc tumour cells treated with indicated concentrations of ZnCl 2 for 4 h. Mean ± SEM, n = 3. Two-tailed t -test. j DOX accumulation in LLC-GFP-luc cells treated with 3 μM DOX combined with indicated concentrations of ZnCl 2 for 4 h. Blank: no DOX treatment. Mean ± SEM, n = 4. Two-tailed t -test. k ABCB1 expression in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) and LY294002 (50 μM) for 3 h. A representative result from three independent experiments is shown. Two-way ANOVA test for curve comparison. Source data are provided as a Source Data file ( a–k ).

    Techniques Used: Fluorescence, Expressing, Cell Culture, Luciferase, Activity Assay, Two Tailed Test, Inhibition

    ZIP1 + fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo. a Experimental design (left) and results of DNA damage (γ-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs for 24 h. Mean ± SEM. Ctl, Zip1 +/+ : n = 3; Zip1 −/− : n = 6. Two-tailed t -test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1 +/+ , Zip1 +/− and Zip1 −/− mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1 +/+ : n = 4; Zip1 +/− , Zip1 −/− : n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1 +/+ and Zip1 −/− mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t -test. f Tumour growth curves for Zip1 +/+ and Zip1 −/− mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1 +/+ PBS ( n = 11), Zip1 −/− PBS ( n = 12), Zip1 +/+ DOX ( n = 8), Zip1 −/− MEFs DOX ( n = 10). g Tumour growth curves for co-injection of Zip1 +/+ or Zip1 −/− MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1 +/+ , Zip1 −/− MEFs PBS, Zip1 −/− MEFs DOX: n = 9; Zip1 +/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET- Zip1 ). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET- Zip1 PBS: n = 6; TET- Zip1 PTX, TET- Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file ( a–h ).
    Figure Legend Snippet: ZIP1 + fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo. a Experimental design (left) and results of DNA damage (γ-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs for 24 h. Mean ± SEM. Ctl, Zip1 +/+ : n = 3; Zip1 −/− : n = 6. Two-tailed t -test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1 +/+ , Zip1 +/− and Zip1 −/− mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1 +/+ : n = 4; Zip1 +/− , Zip1 −/− : n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1 +/+ and Zip1 −/− mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t -test. f Tumour growth curves for Zip1 +/+ and Zip1 −/− mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1 +/+ PBS ( n = 11), Zip1 −/− PBS ( n = 12), Zip1 +/+ DOX ( n = 8), Zip1 −/− MEFs DOX ( n = 10). g Tumour growth curves for co-injection of Zip1 +/+ or Zip1 −/− MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1 +/+ , Zip1 −/− MEFs PBS, Zip1 −/− MEFs DOX: n = 9; Zip1 +/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET- Zip1 ). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET- Zip1 PBS: n = 6; TET- Zip1 PTX, TET- Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file ( a–h ).

    Techniques Used: In Vitro, In Vivo, Expressing, Cell Culture, Two Tailed Test, Injection, Mouse Assay, Transplantation Assay, One-tailed Test

    S100A4 increases ZIP1 expression in fibroblasts. a , b ZIP1 expression in mCAF treated with DOX at the indicated doses ( a ) or for the indicated times ( b ). Representative results from three ( a , b ) independent experiments are shown. c S100A4 levels in the same volume of LLC-GFP-luc cell culture mediums (CMs) with or without DOX treatment determined by western blotting. The same number of tumour cells were seeded and pre-treated with DOX (1 μM) for 6 h. Twenty-four hours after changing the fresh medium, CMs were collected. The same volume of CM from different treatment groups was analysed by Western blotting. A representative result from three independe nt experiments is shown. d ZIP1 expression in mCAF treated with indicated CM. For S100A4 neutralisation, CM was pre-incubated with α-S100A4 antibody 3B11 (6 μg/mL) for 1 h. A representative result from four independent experiments is shown. e , f ZIP1 expression in mCAF treated with S100A4 at the indicated doses ( e ) or for the indicated times ( f ). β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( e , f ) independent experiments are shown. g , h Expression of phosphorylated p65 (pp65), p65, phosphorylated ERK (pERK) ( g ), phosphorylated AKT (pAKT), AKT, phosphorylated p38 (pp38), and p38 ( h ) in mCAF treated with S100A4 for the indicated time periods. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( g , h ) independent experiments are shown. i Expression of ZIP1 in mCAF treated with S100A4, SB202190 (p38 inhibitor, 50 μM), U0126 (ERK inhibitor, 10 μM), SC75741 (p65 inhibitor, 8 μM), and LY294002 (AKT inhibitor, 25 μM) as indicated. A representative result from three independent experiments is shown. j Expression of ZIP1 in mCAF treated with S100A4, TLR4-IN-C34 (TLR4 inhibitor, 10 μM), and FPS-ZM1 (RAGE inhibitor, 1 μM), as indicated. A representative result from three independent experiments is shown. k Diagram showing the signalling pathways for the regulation of ZIP1 by S100A4. l Extracellular S100A4 in LLC-GFP-luc tumours, with or without DOX treatment. n = 6 for the PBS group and n = 7 for the DOX group. Data are presented as mean ± SEM, n = 6 for PBS, n = 7 for DOX. Two-tailed t -test. Source data are provided as a Source Data file ( a–j , l ).
    Figure Legend Snippet: S100A4 increases ZIP1 expression in fibroblasts. a , b ZIP1 expression in mCAF treated with DOX at the indicated doses ( a ) or for the indicated times ( b ). Representative results from three ( a , b ) independent experiments are shown. c S100A4 levels in the same volume of LLC-GFP-luc cell culture mediums (CMs) with or without DOX treatment determined by western blotting. The same number of tumour cells were seeded and pre-treated with DOX (1 μM) for 6 h. Twenty-four hours after changing the fresh medium, CMs were collected. The same volume of CM from different treatment groups was analysed by Western blotting. A representative result from three independe nt experiments is shown. d ZIP1 expression in mCAF treated with indicated CM. For S100A4 neutralisation, CM was pre-incubated with α-S100A4 antibody 3B11 (6 μg/mL) for 1 h. A representative result from four independent experiments is shown. e , f ZIP1 expression in mCAF treated with S100A4 at the indicated doses ( e ) or for the indicated times ( f ). β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( e , f ) independent experiments are shown. g , h Expression of phosphorylated p65 (pp65), p65, phosphorylated ERK (pERK) ( g ), phosphorylated AKT (pAKT), AKT, phosphorylated p38 (pp38), and p38 ( h ) in mCAF treated with S100A4 for the indicated time periods. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( g , h ) independent experiments are shown. i Expression of ZIP1 in mCAF treated with S100A4, SB202190 (p38 inhibitor, 50 μM), U0126 (ERK inhibitor, 10 μM), SC75741 (p65 inhibitor, 8 μM), and LY294002 (AKT inhibitor, 25 μM) as indicated. A representative result from three independent experiments is shown. j Expression of ZIP1 in mCAF treated with S100A4, TLR4-IN-C34 (TLR4 inhibitor, 10 μM), and FPS-ZM1 (RAGE inhibitor, 1 μM), as indicated. A representative result from three independent experiments is shown. k Diagram showing the signalling pathways for the regulation of ZIP1 by S100A4. l Extracellular S100A4 in LLC-GFP-luc tumours, with or without DOX treatment. n = 6 for the PBS group and n = 7 for the DOX group. Data are presented as mean ± SEM, n = 6 for PBS, n = 7 for DOX. Two-tailed t -test. Source data are provided as a Source Data file ( a–j , l ).

    Techniques Used: Expressing, Cell Culture, Western Blot, Incubation, Derivative Assay, Two Tailed Test

    ZIP1 + fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. a UMAP plot showing 12 cell subsets in PBS group, DOX group and their combined. b Bar plots showing the percentages of cells in each subset with PBS and DOX treatment. c Gene-set variation analysis of KEGG signalling pathway terms within the 8 fibroblast subsets (CA0–7). d , e ZIP1 and CX43 expression in d human lung adenocarcinoma-associated fibroblast (hCAF) and e prostate-cancer cancer-associated fibroblast (PCCAF) transfected with control (mock) or ZIP1-overexpression vector ( ZIP1 ). Representative results from four ( d ), three ( e , ZIP1) or five ( e , CX43) independent experiments are shown. f CX43 expression in mouse embryonic fibroblasts (MEFs) transfected with control (siCtl) or Zip1 -silencing (si Zip1 ) siRNA. A representative result from five independent experiments is shown. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel ( e-f ). g CX43 expression on the cell surface of Zip1 +/+ , Zip1 +/− and Zip1 −/− CAFs. Single-cell suspensions of LLC-GFP-luc tumours from indicated mice were analysed by FACS on day 22. CD45 − CD31 − GFP − CAFs were gated. Relative mean fluorescence intensity (MFI) was calculated as MFI[sample]/MFI[negative control]−1. Data are presented as mean ± SEM, n = 3 for Zip1 +/+ , n = 5 for Zip1 +/− and Zip1 −/− . Two-tailed t test. h Direct CX43-CX43 interaction between A549 and PCCAF. A549-CX43-RFP and PCCAF-CX43-GFP were constructed and cocultured. Co-immunoprecipitation (IP) of CX43-RFP and CX43-GFP was examined by western blotting. A representative result from two independent experiments is shown. ( i ) Calcein transfer from hCAFs to A549 tumour cells. A549 cells (pre-labelled with CM-Dil) and hCAFs (loaded with 0.5 μM calcein-AM) were co-cultured for 2 h in DMEM + 10% FBS. Heptanol (HEPT, 2 mM) was used to block gap junctions. Calcein fluorescence in tumour cells was determined by FACS. Arrow, the position of hCAFs. Mean ± SEM, n = 3 for each group. Two-tailed t test. j Calcein transfer from hCAF-mock/hCAF- ZIP1 to A549 tumour cells at the indicated time. A549 alone without fibroblasts were used as a control. Calcein relative MFI in A549 cells of different groups was compared. Mean ± SEM, n = 4 for hCAF-mock 20, 40 min and hCAF- ZIP1 20 min, n = 3 for hCAF-mock 60 min and hCAF- ZIP1 40, 60 min. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ).
    Figure Legend Snippet: ZIP1 + fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. a UMAP plot showing 12 cell subsets in PBS group, DOX group and their combined. b Bar plots showing the percentages of cells in each subset with PBS and DOX treatment. c Gene-set variation analysis of KEGG signalling pathway terms within the 8 fibroblast subsets (CA0–7). d , e ZIP1 and CX43 expression in d human lung adenocarcinoma-associated fibroblast (hCAF) and e prostate-cancer cancer-associated fibroblast (PCCAF) transfected with control (mock) or ZIP1-overexpression vector ( ZIP1 ). Representative results from four ( d ), three ( e , ZIP1) or five ( e , CX43) independent experiments are shown. f CX43 expression in mouse embryonic fibroblasts (MEFs) transfected with control (siCtl) or Zip1 -silencing (si Zip1 ) siRNA. A representative result from five independent experiments is shown. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel ( e-f ). g CX43 expression on the cell surface of Zip1 +/+ , Zip1 +/− and Zip1 −/− CAFs. Single-cell suspensions of LLC-GFP-luc tumours from indicated mice were analysed by FACS on day 22. CD45 − CD31 − GFP − CAFs were gated. Relative mean fluorescence intensity (MFI) was calculated as MFI[sample]/MFI[negative control]−1. Data are presented as mean ± SEM, n = 3 for Zip1 +/+ , n = 5 for Zip1 +/− and Zip1 −/− . Two-tailed t test. h Direct CX43-CX43 interaction between A549 and PCCAF. A549-CX43-RFP and PCCAF-CX43-GFP were constructed and cocultured. Co-immunoprecipitation (IP) of CX43-RFP and CX43-GFP was examined by western blotting. A representative result from two independent experiments is shown. ( i ) Calcein transfer from hCAFs to A549 tumour cells. A549 cells (pre-labelled with CM-Dil) and hCAFs (loaded with 0.5 μM calcein-AM) were co-cultured for 2 h in DMEM + 10% FBS. Heptanol (HEPT, 2 mM) was used to block gap junctions. Calcein fluorescence in tumour cells was determined by FACS. Arrow, the position of hCAFs. Mean ± SEM, n = 3 for each group. Two-tailed t test. j Calcein transfer from hCAF-mock/hCAF- ZIP1 to A549 tumour cells at the indicated time. A549 alone without fibroblasts were used as a control. Calcein relative MFI in A549 cells of different groups was compared. Mean ± SEM, n = 4 for hCAF-mock 20, 40 min and hCAF- ZIP1 20 min, n = 3 for hCAF-mock 60 min and hCAF- ZIP1 40, 60 min. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ).

    Techniques Used: Expressing, Transfection, Over Expression, Plasmid Preparation, Derivative Assay, Mouse Assay, FACS, Fluorescence, Negative Control, Two Tailed Test, Construct, Immunoprecipitation, Western Blot, Cell Culture, Blocking Assay

    Labile Zn 2+ upregulates CX43 by modulation of the PTEN/AKT pathway. a Expression of CX43, pAKT and AKT in mCAFs stimulated with different concentrations of ZnCl 2 for 20 min in DMEM + 10%FBS + 5 μM TPEN. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. b Expression of PTEN in mCAFs stimulated with different concentrations of ZnCl 2 for 5 min in DMEM + 10%FBS + 5 μM TPEN. A representative result from three independent experiments is shown. c Expression of CX43 in mCAFs stimulated with ZnCl 2 ± AKT inhibitor (50 μM LY294002) for 20 min. A representative result from three independent experiments is shown. d Expression of pAKT and PTEN in Zip1 +/+ and Zip1 −/− mouse embryonic fibroblasts (MEFs). β-actin was used as a control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. e Expression of CX43 in LLC-GFP-luc cells co-cultured with mCAFs. Cells were cultured in DMEM + 10%FBS for 24 h. mCAFs were pre-treated with TPEN (50 μM). HEPT; 2 mM. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from four independent experiments is shown. f Expression of CX43 in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) for different times under indicated conditions. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. g Expression of CX43 on the cell surface of LLC-GFP-luc tumour cells treated with different concentrations of ZnCl 2 for 4 h in DMEM + 10%FBS + 5 μM TPEN. Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. h Expression of CX43 on the cell surface of Zip1 +/+ and Zip1 −/− MEFs and LLC-GFP-luc cells co-cultured with MEFs (for 1 h in DMEM + 10%FBS + 5 μM TPEN), determined by FACS. LLC-GFP-luc without MEFs were used as a control (Ctl). Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ). Source data are provided as a Source Data file ( a–h ).
    Figure Legend Snippet: Labile Zn 2+ upregulates CX43 by modulation of the PTEN/AKT pathway. a Expression of CX43, pAKT and AKT in mCAFs stimulated with different concentrations of ZnCl 2 for 20 min in DMEM + 10%FBS + 5 μM TPEN. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. b Expression of PTEN in mCAFs stimulated with different concentrations of ZnCl 2 for 5 min in DMEM + 10%FBS + 5 μM TPEN. A representative result from three independent experiments is shown. c Expression of CX43 in mCAFs stimulated with ZnCl 2 ± AKT inhibitor (50 μM LY294002) for 20 min. A representative result from three independent experiments is shown. d Expression of pAKT and PTEN in Zip1 +/+ and Zip1 −/− mouse embryonic fibroblasts (MEFs). β-actin was used as a control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. e Expression of CX43 in LLC-GFP-luc cells co-cultured with mCAFs. Cells were cultured in DMEM + 10%FBS for 24 h. mCAFs were pre-treated with TPEN (50 μM). HEPT; 2 mM. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from four independent experiments is shown. f Expression of CX43 in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) for different times under indicated conditions. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. g Expression of CX43 on the cell surface of LLC-GFP-luc tumour cells treated with different concentrations of ZnCl 2 for 4 h in DMEM + 10%FBS + 5 μM TPEN. Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. h Expression of CX43 on the cell surface of Zip1 +/+ and Zip1 −/− MEFs and LLC-GFP-luc cells co-cultured with MEFs (for 1 h in DMEM + 10%FBS + 5 μM TPEN), determined by FACS. LLC-GFP-luc without MEFs were used as a control (Ctl). Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ). Source data are provided as a Source Data file ( a–h ).

    Techniques Used: Expressing, Derivative Assay, Cell Culture, Two Tailed Test, FACS

    28) Product Images from "ZIP1+ fibroblasts protect lung cancer against chemotherapy via connexin-43 mediated intercellular Zn2+ transfer"

    Article Title: ZIP1+ fibroblasts protect lung cancer against chemotherapy via connexin-43 mediated intercellular Zn2+ transfer

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33521-4

    Labile Zn 2+ induces ABCB1-mediated drug extrusion. a Zn 2+ uptake by LLC cells treated with paclitaxel (PTX). ZnCl 2 (10 μM) and PTX were added simultaneously. Ctl, no ZnCl 2 . n = 3. b DOX accumulation (determined by DOX fluorescence in individual cells) and DNA damage (indicated by γ-H2AX expression in individual cells) in LLC-GFP-luc cells co-cultured with MEFs in DMEM + 10% FBS + TPEN (5 μM) for 4 h. TPEN: 50 μM, pre-treating MEFs for 5 min. HEPT: 2 mM. DOX: 3 μM. Median ± interquartile range. RFU, relative fluorescence unit. c ABCB1 expression in LLC-GFP-luc cells co-cultured with mCAFs in DMEM + 10% FBS for 24 h. A representative result from three independent experiments is shown. d LLC-GFP-luc cells viability co-cultured with mCAFs under indicated conditions in DMEM + 10% FBS + 5 μM TPEN for 24 h, determined by luciferase activity. DOX: 3 μM. Mean ± SD, n = 6. Two-tailed t -test. e Inhibition rate of PTX on LLC cells with or without TPEN (0.3 μM) in DMEM determined by CCK8. Mean ± SD, n = 3 for PTX 30 nM group. n = 4 for other groups. One-way ANOVA with multiple comparisons. f Inhibition rate of DOX on LLC-GFP-luc cells with different concentrations of ZnCl 2 determined by CCK8. DOX: 3 μM. Cells were treated for 24 h in DMEM + 10% FBS + TPEN (5 μM). Mean ± SEM, n = 3. Two-tailed t -test. g , h ABCB1 expression in LLC-GFP-luc cells treated with indicated concentrations of ZnCl 2 for 4 h ( g ), or with 10 µM ZnCl 2 for the indicated time ( h ). Representative results from three ( g ) or four ( h ) independent experiments are shown. i ABCB1 expression on LLC-GFP-luc tumour cells treated with indicated concentrations of ZnCl 2 for 4 h. Mean ± SEM, n = 3. Two-tailed t -test. j DOX accumulation in LLC-GFP-luc cells treated with 3 μM DOX combined with indicated concentrations of ZnCl 2 for 4 h. Blank: no DOX treatment. Mean ± SEM, n = 4. Two-tailed t -test. k ABCB1 expression in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) and LY294002 (50 μM) for 3 h. A representative result from three independent experiments is shown. Two-way ANOVA test for curve comparison. Source data are provided as a Source Data file ( a–k ).
    Figure Legend Snippet: Labile Zn 2+ induces ABCB1-mediated drug extrusion. a Zn 2+ uptake by LLC cells treated with paclitaxel (PTX). ZnCl 2 (10 μM) and PTX were added simultaneously. Ctl, no ZnCl 2 . n = 3. b DOX accumulation (determined by DOX fluorescence in individual cells) and DNA damage (indicated by γ-H2AX expression in individual cells) in LLC-GFP-luc cells co-cultured with MEFs in DMEM + 10% FBS + TPEN (5 μM) for 4 h. TPEN: 50 μM, pre-treating MEFs for 5 min. HEPT: 2 mM. DOX: 3 μM. Median ± interquartile range. RFU, relative fluorescence unit. c ABCB1 expression in LLC-GFP-luc cells co-cultured with mCAFs in DMEM + 10% FBS for 24 h. A representative result from three independent experiments is shown. d LLC-GFP-luc cells viability co-cultured with mCAFs under indicated conditions in DMEM + 10% FBS + 5 μM TPEN for 24 h, determined by luciferase activity. DOX: 3 μM. Mean ± SD, n = 6. Two-tailed t -test. e Inhibition rate of PTX on LLC cells with or without TPEN (0.3 μM) in DMEM determined by CCK8. Mean ± SD, n = 3 for PTX 30 nM group. n = 4 for other groups. One-way ANOVA with multiple comparisons. f Inhibition rate of DOX on LLC-GFP-luc cells with different concentrations of ZnCl 2 determined by CCK8. DOX: 3 μM. Cells were treated for 24 h in DMEM + 10% FBS + TPEN (5 μM). Mean ± SEM, n = 3. Two-tailed t -test. g , h ABCB1 expression in LLC-GFP-luc cells treated with indicated concentrations of ZnCl 2 for 4 h ( g ), or with 10 µM ZnCl 2 for the indicated time ( h ). Representative results from three ( g ) or four ( h ) independent experiments are shown. i ABCB1 expression on LLC-GFP-luc tumour cells treated with indicated concentrations of ZnCl 2 for 4 h. Mean ± SEM, n = 3. Two-tailed t -test. j DOX accumulation in LLC-GFP-luc cells treated with 3 μM DOX combined with indicated concentrations of ZnCl 2 for 4 h. Blank: no DOX treatment. Mean ± SEM, n = 4. Two-tailed t -test. k ABCB1 expression in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) and LY294002 (50 μM) for 3 h. A representative result from three independent experiments is shown. Two-way ANOVA test for curve comparison. Source data are provided as a Source Data file ( a–k ).

    Techniques Used: Fluorescence, Expressing, Cell Culture, Luciferase, Activity Assay, Two Tailed Test, Inhibition

    ZIP1 + fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo. a Experimental design (left) and results of DNA damage (γ-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs for 24 h. Mean ± SEM. Ctl, Zip1 +/+ : n = 3; Zip1 −/− : n = 6. Two-tailed t -test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1 +/+ , Zip1 +/− and Zip1 −/− mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1 +/+ : n = 4; Zip1 +/− , Zip1 −/− : n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1 +/+ and Zip1 −/− mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t -test. f Tumour growth curves for Zip1 +/+ and Zip1 −/− mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1 +/+ PBS ( n = 11), Zip1 −/− PBS ( n = 12), Zip1 +/+ DOX ( n = 8), Zip1 −/− MEFs DOX ( n = 10). g Tumour growth curves for co-injection of Zip1 +/+ or Zip1 −/− MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1 +/+ , Zip1 −/− MEFs PBS, Zip1 −/− MEFs DOX: n = 9; Zip1 +/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET- Zip1 ). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET- Zip1 PBS: n = 6; TET- Zip1 PTX, TET- Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file ( a–h ).
    Figure Legend Snippet: ZIP1 + fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo. a Experimental design (left) and results of DNA damage (γ-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs with DOX (3 μM) treatment. Mean ± SEM, n = 4. Two-tailed t -test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1 +/+ or Zip1 −/− MEFs for 24 h. Mean ± SEM. Ctl, Zip1 +/+ : n = 3; Zip1 −/− : n = 6. Two-tailed t -test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1 +/+ , Zip1 +/− and Zip1 −/− mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1 +/+ : n = 4; Zip1 +/− , Zip1 −/− : n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1 +/+ and Zip1 −/− mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t -test. f Tumour growth curves for Zip1 +/+ and Zip1 −/− mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1 +/+ PBS ( n = 11), Zip1 −/− PBS ( n = 12), Zip1 +/+ DOX ( n = 8), Zip1 −/− MEFs DOX ( n = 10). g Tumour growth curves for co-injection of Zip1 +/+ or Zip1 −/− MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1 +/+ , Zip1 −/− MEFs PBS, Zip1 −/− MEFs DOX: n = 9; Zip1 +/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET- Zip1 ). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET- Zip1 PBS: n = 6; TET- Zip1 PTX, TET- Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file ( a–h ).

    Techniques Used: In Vitro, In Vivo, Expressing, Cell Culture, Two Tailed Test, Injection, Mouse Assay, Transplantation Assay, One-tailed Test

    S100A4 increases ZIP1 expression in fibroblasts. a , b ZIP1 expression in mCAF treated with DOX at the indicated doses ( a ) or for the indicated times ( b ). Representative results from three ( a , b ) independent experiments are shown. c S100A4 levels in the same volume of LLC-GFP-luc cell culture mediums (CMs) with or without DOX treatment determined by western blotting. The same number of tumour cells were seeded and pre-treated with DOX (1 μM) for 6 h. Twenty-four hours after changing the fresh medium, CMs were collected. The same volume of CM from different treatment groups was analysed by Western blotting. A representative result from three independe nt experiments is shown. d ZIP1 expression in mCAF treated with indicated CM. For S100A4 neutralisation, CM was pre-incubated with α-S100A4 antibody 3B11 (6 μg/mL) for 1 h. A representative result from four independent experiments is shown. e , f ZIP1 expression in mCAF treated with S100A4 at the indicated doses ( e ) or for the indicated times ( f ). β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( e , f ) independent experiments are shown. g , h Expression of phosphorylated p65 (pp65), p65, phosphorylated ERK (pERK) ( g ), phosphorylated AKT (pAKT), AKT, phosphorylated p38 (pp38), and p38 ( h ) in mCAF treated with S100A4 for the indicated time periods. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( g , h ) independent experiments are shown. i Expression of ZIP1 in mCAF treated with S100A4, SB202190 (p38 inhibitor, 50 μM), U0126 (ERK inhibitor, 10 μM), SC75741 (p65 inhibitor, 8 μM), and LY294002 (AKT inhibitor, 25 μM) as indicated. A representative result from three independent experiments is shown. j Expression of ZIP1 in mCAF treated with S100A4, TLR4-IN-C34 (TLR4 inhibitor, 10 μM), and FPS-ZM1 (RAGE inhibitor, 1 μM), as indicated. A representative result from three independent experiments is shown. k Diagram showing the signalling pathways for the regulation of ZIP1 by S100A4. l Extracellular S100A4 in LLC-GFP-luc tumours, with or without DOX treatment. n = 6 for the PBS group and n = 7 for the DOX group. Data are presented as mean ± SEM, n = 6 for PBS, n = 7 for DOX. Two-tailed t -test. Source data are provided as a Source Data file ( a–j , l ).
    Figure Legend Snippet: S100A4 increases ZIP1 expression in fibroblasts. a , b ZIP1 expression in mCAF treated with DOX at the indicated doses ( a ) or for the indicated times ( b ). Representative results from three ( a , b ) independent experiments are shown. c S100A4 levels in the same volume of LLC-GFP-luc cell culture mediums (CMs) with or without DOX treatment determined by western blotting. The same number of tumour cells were seeded and pre-treated with DOX (1 μM) for 6 h. Twenty-four hours after changing the fresh medium, CMs were collected. The same volume of CM from different treatment groups was analysed by Western blotting. A representative result from three independe nt experiments is shown. d ZIP1 expression in mCAF treated with indicated CM. For S100A4 neutralisation, CM was pre-incubated with α-S100A4 antibody 3B11 (6 μg/mL) for 1 h. A representative result from four independent experiments is shown. e , f ZIP1 expression in mCAF treated with S100A4 at the indicated doses ( e ) or for the indicated times ( f ). β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( e , f ) independent experiments are shown. g , h Expression of phosphorylated p65 (pp65), p65, phosphorylated ERK (pERK) ( g ), phosphorylated AKT (pAKT), AKT, phosphorylated p38 (pp38), and p38 ( h ) in mCAF treated with S100A4 for the indicated time periods. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. Representative results from three ( g , h ) independent experiments are shown. i Expression of ZIP1 in mCAF treated with S100A4, SB202190 (p38 inhibitor, 50 μM), U0126 (ERK inhibitor, 10 μM), SC75741 (p65 inhibitor, 8 μM), and LY294002 (AKT inhibitor, 25 μM) as indicated. A representative result from three independent experiments is shown. j Expression of ZIP1 in mCAF treated with S100A4, TLR4-IN-C34 (TLR4 inhibitor, 10 μM), and FPS-ZM1 (RAGE inhibitor, 1 μM), as indicated. A representative result from three independent experiments is shown. k Diagram showing the signalling pathways for the regulation of ZIP1 by S100A4. l Extracellular S100A4 in LLC-GFP-luc tumours, with or without DOX treatment. n = 6 for the PBS group and n = 7 for the DOX group. Data are presented as mean ± SEM, n = 6 for PBS, n = 7 for DOX. Two-tailed t -test. Source data are provided as a Source Data file ( a–j , l ).

    Techniques Used: Expressing, Cell Culture, Western Blot, Incubation, Derivative Assay, Two Tailed Test

    ZIP1 + fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. a UMAP plot showing 12 cell subsets in PBS group, DOX group and their combined. b Bar plots showing the percentages of cells in each subset with PBS and DOX treatment. c Gene-set variation analysis of KEGG signalling pathway terms within the 8 fibroblast subsets (CA0–7). d , e ZIP1 and CX43 expression in d human lung adenocarcinoma-associated fibroblast (hCAF) and e prostate-cancer cancer-associated fibroblast (PCCAF) transfected with control (mock) or ZIP1-overexpression vector ( ZIP1 ). Representative results from four ( d ), three ( e , ZIP1) or five ( e , CX43) independent experiments are shown. f CX43 expression in mouse embryonic fibroblasts (MEFs) transfected with control (siCtl) or Zip1 -silencing (si Zip1 ) siRNA. A representative result from five independent experiments is shown. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel ( e-f ). g CX43 expression on the cell surface of Zip1 +/+ , Zip1 +/− and Zip1 −/− CAFs. Single-cell suspensions of LLC-GFP-luc tumours from indicated mice were analysed by FACS on day 22. CD45 − CD31 − GFP − CAFs were gated. Relative mean fluorescence intensity (MFI) was calculated as MFI[sample]/MFI[negative control]−1. Data are presented as mean ± SEM, n = 3 for Zip1 +/+ , n = 5 for Zip1 +/− and Zip1 −/− . Two-tailed t test. h Direct CX43-CX43 interaction between A549 and PCCAF. A549-CX43-RFP and PCCAF-CX43-GFP were constructed and cocultured. Co-immunoprecipitation (IP) of CX43-RFP and CX43-GFP was examined by western blotting. A representative result from two independent experiments is shown. ( i ) Calcein transfer from hCAFs to A549 tumour cells. A549 cells (pre-labelled with CM-Dil) and hCAFs (loaded with 0.5 μM calcein-AM) were co-cultured for 2 h in DMEM + 10% FBS. Heptanol (HEPT, 2 mM) was used to block gap junctions. Calcein fluorescence in tumour cells was determined by FACS. Arrow, the position of hCAFs. Mean ± SEM, n = 3 for each group. Two-tailed t test. j Calcein transfer from hCAF-mock/hCAF- ZIP1 to A549 tumour cells at the indicated time. A549 alone without fibroblasts were used as a control. Calcein relative MFI in A549 cells of different groups was compared. Mean ± SEM, n = 4 for hCAF-mock 20, 40 min and hCAF- ZIP1 20 min, n = 3 for hCAF-mock 60 min and hCAF- ZIP1 40, 60 min. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ).
    Figure Legend Snippet: ZIP1 + fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. a UMAP plot showing 12 cell subsets in PBS group, DOX group and their combined. b Bar plots showing the percentages of cells in each subset with PBS and DOX treatment. c Gene-set variation analysis of KEGG signalling pathway terms within the 8 fibroblast subsets (CA0–7). d , e ZIP1 and CX43 expression in d human lung adenocarcinoma-associated fibroblast (hCAF) and e prostate-cancer cancer-associated fibroblast (PCCAF) transfected with control (mock) or ZIP1-overexpression vector ( ZIP1 ). Representative results from four ( d ), three ( e , ZIP1) or five ( e , CX43) independent experiments are shown. f CX43 expression in mouse embryonic fibroblasts (MEFs) transfected with control (siCtl) or Zip1 -silencing (si Zip1 ) siRNA. A representative result from five independent experiments is shown. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel ( e-f ). g CX43 expression on the cell surface of Zip1 +/+ , Zip1 +/− and Zip1 −/− CAFs. Single-cell suspensions of LLC-GFP-luc tumours from indicated mice were analysed by FACS on day 22. CD45 − CD31 − GFP − CAFs were gated. Relative mean fluorescence intensity (MFI) was calculated as MFI[sample]/MFI[negative control]−1. Data are presented as mean ± SEM, n = 3 for Zip1 +/+ , n = 5 for Zip1 +/− and Zip1 −/− . Two-tailed t test. h Direct CX43-CX43 interaction between A549 and PCCAF. A549-CX43-RFP and PCCAF-CX43-GFP were constructed and cocultured. Co-immunoprecipitation (IP) of CX43-RFP and CX43-GFP was examined by western blotting. A representative result from two independent experiments is shown. ( i ) Calcein transfer from hCAFs to A549 tumour cells. A549 cells (pre-labelled with CM-Dil) and hCAFs (loaded with 0.5 μM calcein-AM) were co-cultured for 2 h in DMEM + 10% FBS. Heptanol (HEPT, 2 mM) was used to block gap junctions. Calcein fluorescence in tumour cells was determined by FACS. Arrow, the position of hCAFs. Mean ± SEM, n = 3 for each group. Two-tailed t test. j Calcein transfer from hCAF-mock/hCAF- ZIP1 to A549 tumour cells at the indicated time. A549 alone without fibroblasts were used as a control. Calcein relative MFI in A549 cells of different groups was compared. Mean ± SEM, n = 4 for hCAF-mock 20, 40 min and hCAF- ZIP1 20 min, n = 3 for hCAF-mock 60 min and hCAF- ZIP1 40, 60 min. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ).

    Techniques Used: Expressing, Transfection, Over Expression, Plasmid Preparation, Derivative Assay, Mouse Assay, FACS, Fluorescence, Negative Control, Two Tailed Test, Construct, Immunoprecipitation, Western Blot, Cell Culture, Blocking Assay

    Labile Zn 2+ upregulates CX43 by modulation of the PTEN/AKT pathway. a Expression of CX43, pAKT and AKT in mCAFs stimulated with different concentrations of ZnCl 2 for 20 min in DMEM + 10%FBS + 5 μM TPEN. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. b Expression of PTEN in mCAFs stimulated with different concentrations of ZnCl 2 for 5 min in DMEM + 10%FBS + 5 μM TPEN. A representative result from three independent experiments is shown. c Expression of CX43 in mCAFs stimulated with ZnCl 2 ± AKT inhibitor (50 μM LY294002) for 20 min. A representative result from three independent experiments is shown. d Expression of pAKT and PTEN in Zip1 +/+ and Zip1 −/− mouse embryonic fibroblasts (MEFs). β-actin was used as a control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. e Expression of CX43 in LLC-GFP-luc cells co-cultured with mCAFs. Cells were cultured in DMEM + 10%FBS for 24 h. mCAFs were pre-treated with TPEN (50 μM). HEPT; 2 mM. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from four independent experiments is shown. f Expression of CX43 in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) for different times under indicated conditions. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. g Expression of CX43 on the cell surface of LLC-GFP-luc tumour cells treated with different concentrations of ZnCl 2 for 4 h in DMEM + 10%FBS + 5 μM TPEN. Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. h Expression of CX43 on the cell surface of Zip1 +/+ and Zip1 −/− MEFs and LLC-GFP-luc cells co-cultured with MEFs (for 1 h in DMEM + 10%FBS + 5 μM TPEN), determined by FACS. LLC-GFP-luc without MEFs were used as a control (Ctl). Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ). Source data are provided as a Source Data file ( a–h ).
    Figure Legend Snippet: Labile Zn 2+ upregulates CX43 by modulation of the PTEN/AKT pathway. a Expression of CX43, pAKT and AKT in mCAFs stimulated with different concentrations of ZnCl 2 for 20 min in DMEM + 10%FBS + 5 μM TPEN. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. b Expression of PTEN in mCAFs stimulated with different concentrations of ZnCl 2 for 5 min in DMEM + 10%FBS + 5 μM TPEN. A representative result from three independent experiments is shown. c Expression of CX43 in mCAFs stimulated with ZnCl 2 ± AKT inhibitor (50 μM LY294002) for 20 min. A representative result from three independent experiments is shown. d Expression of pAKT and PTEN in Zip1 +/+ and Zip1 −/− mouse embryonic fibroblasts (MEFs). β-actin was used as a control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. e Expression of CX43 in LLC-GFP-luc cells co-cultured with mCAFs. Cells were cultured in DMEM + 10%FBS for 24 h. mCAFs were pre-treated with TPEN (50 μM). HEPT; 2 mM. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from four independent experiments is shown. f Expression of CX43 in LLC-GFP-luc cells stimulated with ZnCl 2 (30 μM) for different times under indicated conditions. β-actin was used as control. The samples derived from the same experiment and blots were processed in parallel. A representative result from three independent experiments is shown. g Expression of CX43 on the cell surface of LLC-GFP-luc tumour cells treated with different concentrations of ZnCl 2 for 4 h in DMEM + 10%FBS + 5 μM TPEN. Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. h Expression of CX43 on the cell surface of Zip1 +/+ and Zip1 −/− MEFs and LLC-GFP-luc cells co-cultured with MEFs (for 1 h in DMEM + 10%FBS + 5 μM TPEN), determined by FACS. LLC-GFP-luc without MEFs were used as a control (Ctl). Data are presented as mean ± SEM, n = 3 for each group. Two-tailed t -test. Source data are provided as a Source Data file ( b , d–j ). Source data are provided as a Source Data file ( a–h ).

    Techniques Used: Expressing, Derivative Assay, Cell Culture, Two Tailed Test, FACS

    29) Product Images from "Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function"

    Article Title: Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function

    Journal: bioRxiv

    doi: 10.1101/2022.10.01.510473

    AMPKγ2 transduced Tcells enhance oxidative metabolism. (A) Human T cells were transduced with AMPKγ2 or Empty lentiviral vectors and expanded in IL-2. On day 9, a portion of cells were stimulated overnight with anti-CD3/CD28 Dynabeads and the next day both resting (left) and activated (right) cells were assessed for oxidative capacity utilizing the Seahorse Metabolic Analyzer. Bar graphs represent data from 3 individual human donors. (B) AMPKγ2- and Empty-transduced T cells were assessed for mitochondrial density utilizing MitoTracker Red (left). Fluorescence was compared to GFP neg controls within each sample to standardize staining between groups. Differences in median fluorescence intensity (MFI) between GFP+ and GFP neg cells was normalized to differences seen in the Empty control from 5 human donors (right). (C-E) GFP+ AMPK-versus Empty-transduced T cells were sorted into CD4+ and CD8+ subsets on Day 9 post-stimulation and protein expression of the transcriptional co-activator PGClα (C) or mitochondrial fusion proteins OPA1 (D) and MFN1 (E) assessed by immunoblot. Bar graphs represent data from 3-4 human donors. *p
    Figure Legend Snippet: AMPKγ2 transduced Tcells enhance oxidative metabolism. (A) Human T cells were transduced with AMPKγ2 or Empty lentiviral vectors and expanded in IL-2. On day 9, a portion of cells were stimulated overnight with anti-CD3/CD28 Dynabeads and the next day both resting (left) and activated (right) cells were assessed for oxidative capacity utilizing the Seahorse Metabolic Analyzer. Bar graphs represent data from 3 individual human donors. (B) AMPKγ2- and Empty-transduced T cells were assessed for mitochondrial density utilizing MitoTracker Red (left). Fluorescence was compared to GFP neg controls within each sample to standardize staining between groups. Differences in median fluorescence intensity (MFI) between GFP+ and GFP neg cells was normalized to differences seen in the Empty control from 5 human donors (right). (C-E) GFP+ AMPK-versus Empty-transduced T cells were sorted into CD4+ and CD8+ subsets on Day 9 post-stimulation and protein expression of the transcriptional co-activator PGClα (C) or mitochondrial fusion proteins OPA1 (D) and MFN1 (E) assessed by immunoblot. Bar graphs represent data from 3-4 human donors. *p

    Techniques Used: Transduction, Fluorescence, Staining, Expressing

    AMPK activation increases T cell expansion, cell cycling, and mTOR activity. (A) AMPK- and Empty-transduced T cells were expanded in vitro for 7-9 days, incubated with BrdU for the final 2 hours, and co-stained with 7AAD. Plots are divided into resting (G0/G1) and cycling (S, G2/M) phases. Graphs represent data from 3 human donors. (B) AMPKγ2-transduced human T cells (GFP+) were manually counted between Day 5 and 7 to calculate the doubling rate per 24 hours, which was then normalized to the Empty-transduced control for each donor. Graph represents data from 6 independent human donors. (C) Transduced cells were harvested on Day 9 of culture and stained for CD25. MFI values were compared between multiple donors. (D) Transduced human T cells were assessed on day 9 for baseline extracellular acidification rates (ECAR) using the Seahorse Metabolic Analyzer. (E-F) Empty- and AMPKγ2-transduced T cells were expanded until day 9, followed by assessment of mTOR activity using antibodies against phosphorylated S6 (E) and 4EBP1 (F). Cells were assessed either at rest (day 9) or following 24 hours of CD3/CD28 stimulation (24hr stim). The MFI for *P-S6 or *P-4EBP1 from each donor was normalized to expression in Empty-transduced controls and this value was then compared between donors. Bar graphs represent data from 4 human donors.*p
    Figure Legend Snippet: AMPK activation increases T cell expansion, cell cycling, and mTOR activity. (A) AMPK- and Empty-transduced T cells were expanded in vitro for 7-9 days, incubated with BrdU for the final 2 hours, and co-stained with 7AAD. Plots are divided into resting (G0/G1) and cycling (S, G2/M) phases. Graphs represent data from 3 human donors. (B) AMPKγ2-transduced human T cells (GFP+) were manually counted between Day 5 and 7 to calculate the doubling rate per 24 hours, which was then normalized to the Empty-transduced control for each donor. Graph represents data from 6 independent human donors. (C) Transduced cells were harvested on Day 9 of culture and stained for CD25. MFI values were compared between multiple donors. (D) Transduced human T cells were assessed on day 9 for baseline extracellular acidification rates (ECAR) using the Seahorse Metabolic Analyzer. (E-F) Empty- and AMPKγ2-transduced T cells were expanded until day 9, followed by assessment of mTOR activity using antibodies against phosphorylated S6 (E) and 4EBP1 (F). Cells were assessed either at rest (day 9) or following 24 hours of CD3/CD28 stimulation (24hr stim). The MFI for *P-S6 or *P-4EBP1 from each donor was normalized to expression in Empty-transduced controls and this value was then compared between donors. Bar graphs represent data from 4 human donors.*p

    Techniques Used: Activation Assay, Activity Assay, In Vitro, Incubation, Staining, Expressing

    AMPKγ2 overexpression increases AMPK activity in human T cells. (A) Schematic of AMPKγ2 and GFP-only “Empty” control vectors with an EF1α promoter and either GFP or RQR8 expression tag. (B-D) Primary human T cells were mock transduced or transduced with AMPKγ2 or Empty plasmids. Expression was verified by flow cytometry for GFP or the CD34 motif of the RQR8 construct (B), fold-change in AMPKγ2 mRNA expression using qRT-PCR (C), and immunoblot to detect protein expression of AMPKγ2 (D). To note, studies utilized a shorter isoform of AMPKγ2 (isoform C), with lower expression levels at baseline, allowing for easy identification of a distinct, lower molecular weight band. (E) Human T cells were transduced with AMPKγ2-versus Empty constructs, cells lysates collected on days 9-12, and phosphorylation of AMPKα Thr172 (to detect AMPK activation), ACC Ser79, and ULK-1 Ser555 measured by immunoblot. (F) Densitometry was measured on immunoblots from multiple donors using ImageJ software. Values for AMPKγ2-transduced cells in each sample were normalized to Empty controls. All data represent 3 or more independent human donor samples. *p
    Figure Legend Snippet: AMPKγ2 overexpression increases AMPK activity in human T cells. (A) Schematic of AMPKγ2 and GFP-only “Empty” control vectors with an EF1α promoter and either GFP or RQR8 expression tag. (B-D) Primary human T cells were mock transduced or transduced with AMPKγ2 or Empty plasmids. Expression was verified by flow cytometry for GFP or the CD34 motif of the RQR8 construct (B), fold-change in AMPKγ2 mRNA expression using qRT-PCR (C), and immunoblot to detect protein expression of AMPKγ2 (D). To note, studies utilized a shorter isoform of AMPKγ2 (isoform C), with lower expression levels at baseline, allowing for easy identification of a distinct, lower molecular weight band. (E) Human T cells were transduced with AMPKγ2-versus Empty constructs, cells lysates collected on days 9-12, and phosphorylation of AMPKα Thr172 (to detect AMPK activation), ACC Ser79, and ULK-1 Ser555 measured by immunoblot. (F) Densitometry was measured on immunoblots from multiple donors using ImageJ software. Values for AMPKγ2-transduced cells in each sample were normalized to Empty controls. All data represent 3 or more independent human donor samples. *p

    Techniques Used: Over Expression, Activity Assay, Expressing, Transduction, Flow Cytometry, Construct, Quantitative RT-PCR, Molecular Weight, Activation Assay, Western Blot, Software

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    LSSL has strong bacterial agglutination effect. A ELISA showing the interaction between LSSL and microbial components and glycans. Plates were coated with 20 μg components, washed, and incubated overnight with LSSL at 4 °C, followed by detection using an anti-LSSL antibody. One representative experiment of three is shown ( n = 3 technical replicates, data are representative of more than three independent experiments). Background absorbance without protein was subtracted. B The determination of bacterial activities of serum, LSSL, and LSSL-depleted serum on E. coli and S. aureus after 12 or 24 h. Data are presented as the mean percentage ± SD of three independent experiments. C Scanning electron microscopy (SEM) analysis of E. coli and S. aureus after treatment with LSSL. E. coli and S. aureus were incubated with PBS and used as controls. The concentration of EDTA used was 50 μg/mL. Scale bar, 5 µm. D Agglutination <t>of</t> <t>GFP-</t> E. coli by LSSL. Different components were incubated with FITC-labeled E. coli (10 5 cells per well) in PBS for 1 h at room temperature and were examined using a high-content screening system ( n = 3, **** P
    Gfp E, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gfp tag antibody abfinity rabbit monoclonal
    LSSL has strong bacterial agglutination effect. A ELISA showing the interaction between LSSL and microbial components and glycans. Plates were coated with 20 μg components, washed, and incubated overnight with LSSL at 4 °C, followed by detection using an anti-LSSL antibody. One representative experiment of three is shown ( n = 3 technical replicates, data are representative of more than three independent experiments). Background absorbance without protein was subtracted. B The determination of bacterial activities of serum, LSSL, and LSSL-depleted serum on E. coli and S. aureus after 12 or 24 h. Data are presented as the mean percentage ± SD of three independent experiments. C Scanning electron microscopy (SEM) analysis of E. coli and S. aureus after treatment with LSSL. E. coli and S. aureus were incubated with PBS and used as controls. The concentration of EDTA used was 50 μg/mL. Scale bar, 5 µm. D Agglutination <t>of</t> <t>GFP-</t> E. coli by LSSL. Different components were incubated with FITC-labeled E. coli (10 5 cells per well) in PBS for 1 h at room temperature and were examined using a high-content screening system ( n = 3, **** P
    Gfp Tag Antibody Abfinity Rabbit Monoclonal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp tag antibody abfinity rabbit monoclonal/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp tag antibody abfinity rabbit monoclonal - by Bioz Stars, 2022-11
    86/100 stars
      Buy from Supplier

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    LSSL has strong bacterial agglutination effect. A ELISA showing the interaction between LSSL and microbial components and glycans. Plates were coated with 20 μg components, washed, and incubated overnight with LSSL at 4 °C, followed by detection using an anti-LSSL antibody. One representative experiment of three is shown ( n = 3 technical replicates, data are representative of more than three independent experiments). Background absorbance without protein was subtracted. B The determination of bacterial activities of serum, LSSL, and LSSL-depleted serum on E. coli and S. aureus after 12 or 24 h. Data are presented as the mean percentage ± SD of three independent experiments. C Scanning electron microscopy (SEM) analysis of E. coli and S. aureus after treatment with LSSL. E. coli and S. aureus were incubated with PBS and used as controls. The concentration of EDTA used was 50 μg/mL. Scale bar, 5 µm. D Agglutination of GFP- E. coli by LSSL. Different components were incubated with FITC-labeled E. coli (10 5 cells per well) in PBS for 1 h at room temperature and were examined using a high-content screening system ( n = 3, **** P

    Journal: Cellular & Molecular Biology Letters

    Article Title: A novel serum spherical lectin from lamprey reveals a more efficient mechanism of immune initiation and regulation in jawless vertebrates

    doi: 10.1186/s11658-022-00401-0

    Figure Lengend Snippet: LSSL has strong bacterial agglutination effect. A ELISA showing the interaction between LSSL and microbial components and glycans. Plates were coated with 20 μg components, washed, and incubated overnight with LSSL at 4 °C, followed by detection using an anti-LSSL antibody. One representative experiment of three is shown ( n = 3 technical replicates, data are representative of more than three independent experiments). Background absorbance without protein was subtracted. B The determination of bacterial activities of serum, LSSL, and LSSL-depleted serum on E. coli and S. aureus after 12 or 24 h. Data are presented as the mean percentage ± SD of three independent experiments. C Scanning electron microscopy (SEM) analysis of E. coli and S. aureus after treatment with LSSL. E. coli and S. aureus were incubated with PBS and used as controls. The concentration of EDTA used was 50 μg/mL. Scale bar, 5 µm. D Agglutination of GFP- E. coli by LSSL. Different components were incubated with FITC-labeled E. coli (10 5 cells per well) in PBS for 1 h at room temperature and were examined using a high-content screening system ( n = 3, **** P

    Article Snippet: Fixed GFP-E. coli (Thermo Fisher Scientific, Waltham, MA, USA) suspension (OD600 0.4–0.6) was treated with equal volumes of PBS, 30% concentration of lamprey serum, or LSSL (50 μg) for 12 h at 4 °C [ ].

    Techniques: Agglutination, Enzyme-linked Immunosorbent Assay, Incubation, Electron Microscopy, Concentration Assay, Labeling, High Content Screening