Journal: Journal of Clinical Investigation

Article Title: Myeloid cell genome-wide screen identifies variants associated with Mycobacterium tuberculosis-induced cytokine transcriptional responses

doi: 10.1172/jci179822

Figure Lengend Snippet: Figure 3. Multiple SNPs associated with Mtb-induced cytokine expression in the Uganda cohort show association with cytokine expression in a Seattle population cohort. Lead SNPs that passed the suggestive threshold for a particular cytokine within the Uganda population (n = 100) were tested for association with the corresponding cytokine in the Seattle population (n = 40). Four loci were associated with cytokine expression in the 2 populations (P < 0.05). To account for differences between populations, all SNPs within the 2 loci were assessed for linkage disequilibrium (LD) and association with corresponding cytokine expression. (A) P values of SNPs of the chr 9p24.2 locus, within the gene SLC1A1, with coloration of individual SNP points indicating LD with rs10974620 within each population. (B) Differences in Mtb-induced IL1B expression are plotted according to genotype for the chr 9p24.2 locus lead SNP of the Uganda population, rs10974620. (C) P values of SNPs of the chr 5q34 locus, within the gene SLIT3, with coloration of individual SNP points indi- cating LD with rs79380271 within each population. (D) Differences in Mtb-induced IL1B expression are plotted according to genotype for the chr 5q34 locus lead SNP of the Uganda population, rs79380271. Threshold lines indicate nominal P value threshold (blue, P < 0.05 unadjusted) and multiple corrected threshold (red, 0.05/number of SNPs tested for each cytokine). For both loci, multiple SNPs are significantly associated with decreased Mtb-induced IL1B expression in both populations. (E and F) Global minor allele frequency of the SLC1A1 locus Uganda sentinel SNP, rs10974620 (E), and SLIT3 locus Uganda sentinel SNP, rs79380271 (F), plotted using Geography of Genetic Variants browser.

Article Snippet: Assessment of supernatant and lysate SLIT3 protein abundance was performed using a human SLIT3 protein ELISA kit (CUSABIO).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: Myeloid cell genome-wide screen identifies variants associated with Mycobacterium tuberculosis-induced cytokine transcriptional responses

doi: 10.1172/jci179822

Figure Lengend Snippet: Figure 4. In vitro investigation of SNP-associated genes identifies SLC1A1 and SLIT3 as major regulators of the Mtb-induced cytokine response. (A and B) siRNA knockdown of SLC1A1 and SLIT3 expression was used to determine effect on IL1B, IL6, and TNF secretion measured by ELISA after 24 hours of Mtb infection (MOI = 1) (A) and IL1B, IL6, and TNF mRNA expression after 6-hour mock and Mtb infection (B). (C) Purified hSLIT3 N- and C-terminal fragments were tested for effect on IL1B, IL6, and TNF secretion measured by ELISA after 24-hour mock and Mtb infection. Results are from 3 biological replicates in 3 human donors per siRNA or treatment. ND, not detected. Significance was assessed using linear mixed model adjusting for random effect of donor followed by ANOVA and pairwise comparisons. Individual data points were plotted according to model calculated values adjusted for donor inter- cept. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Individual donor values are presented in Supplemental Figure 6, B–D.

Article Snippet: Assessment of supernatant and lysate SLIT3 protein abundance was performed using a human SLIT3 protein ELISA kit (CUSABIO).

Techniques: In Vitro, Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Infection, Purification

Journal: Journal of Clinical Investigation

Article Title: Myeloid cell genome-wide screen identifies variants associated with Mycobacterium tuberculosis-induced cytokine transcriptional responses

doi: 10.1172/jci179822

Figure Lengend Snippet: Figure 5. Evaluation of SNP-associated gene effect on other Mtb-response phenotypes reveals SLIT3 effect on intracellular Mtb. Intracellular Mtb replication in human macrophages assessed after hSLIT3 N- and C-terminal cotreatment using fluorescence microscopy to measure Mtb-mCherry surface area within each well over 72 hours. Four-hour time point quantifies differences in initial phagocytic uptake and shows no significant differ- ences between groups. Results shown are from 6 biological replicates in 3 human donors. Significant differences were evaluated at each time point and show a significant increase in intracellular Mtb replication with cotreatment of N- and C-terminal SLIT3 at 48 and 72 hours. Significance was assessed using a linear mixed model adjusting for random effect of donor followed by ANOVA and pairwise comparisons for each time point. Indi- vidual data points were plotted according to model calculated values adjusted for donor intercept. *P < 0.05. Individual donor values are presented in Supplemental Figure 6E.

Article Snippet: Assessment of supernatant and lysate SLIT3 protein abundance was performed using a human SLIT3 protein ELISA kit (CUSABIO).

Techniques: Fluorescence, Microscopy

Journal: International immunopharmacology

Article Title: Angiogenesis-related immune response may be the prelude to the syndesmophyte formation in Ankylosing spondylitis.

doi: 10.1016/j.intimp.2024.112040

Figure Lengend Snippet: Fig. 5. TNF-α and SLIT3 were present in high levels at the angiogenesis-coupled osteogenic lesions in AS cynomolgus monkeys. (A) Immunohistochemical staining of TNF-α. TNF-α was found in the annulus fibrosus and ligament in the intervertebral discs of the diseased animals. (a)(b)The endothelial cells of the vessels were positive for TNF-α. Red arrows indicate TNF-α-positive blood vessels. Black scale bar = 100 μm. Red scale bar = 20 μm. n = 3 in the normal group, n = 16 in the AS early stage, and n = 12 in the AS late stage. The staining was repeated 3 times per sample. (B) Immunohistochemical staining of SLIT3. SLIT3 was mainly expressed in the blood vessels at the annulus fibrosus and ligament in the AS lumbar spine. (a)(b) High magnification of SLIT3-positive blood vessels. Red arrows indicate positive- stained blood vessels. Black scale bar = 100 μm. Yellow/Red scale bar = 20 μm. n = 3 in the normal group, n = 15 in the AS early stage, and n = 14 in the AS late stage. The staining was repeated 3 times per sample. (C) Colocalization of SLIT3 and CD31 in the vertebra. Red arrows indicate SLIT3/CD31 double-positive blood vessels. CD31: green; SLIT3: red; Merge: yellow. Red scale bar = 50 μm. White scale bar = 20 μm. n = 3 in the normal group, n = 4 in the AS group. The staining was repeated 3 times per sample. (D) Immunohistochemical staining of MMP-3. MMP-3 was mainly expressed in the cartilage plate, annulus fibrosus in the AS lumbar spine. (a)(c)MMP-3-positive vascular invasion in the annulus fibrosus. (b) MMP-3-positive heterotopic hypertrophic chondrocytes (inside the green dotted area). The blue dotted line showed the positive area in the cartilage endplate. Red arrows indicate positive blood vessels. Black scale bar = 100 μm. Blue scale bar = 50 μm. Red scale bar = 20 μm. AF: annulus fibrosus, LM: ligament. n = 3 in the normal group, n = 15 in the AS early stage, and n = 14 in the AS late stage. The staining was repeated 3 times per sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After blocking with hydrogen peroxide and serum, the tissue sections were subjected to antigen repair by enzyme repair method [38]; after 5 % BSA blocking, the tissue sections were incubated with the following antibodies: ICOS (1:200, 14–9949-82, Abcam), CD4(1:150, 48274, Abcam), CXCR5 (1:200, bsm54319R, Bioss), TNF-α (1:50, sc-52746, Santa cruz), CD31 (1:200, NBP2-44342, Novus), Endomucin (EMCN) (1:100, sc-65495, Novus), SLIT3 (1:100, TA351721, Origene), and MMP-3 (1:100, 66338–1-lg, Proteintech) overnight at 4 ◦C.

Techniques: Immunohistochemical staining, Staining

Journal: International immunopharmacology

Article Title: Angiogenesis-related immune response may be the prelude to the syndesmophyte formation in Ankylosing spondylitis.

doi: 10.1016/j.intimp.2024.112040

Figure Lengend Snippet: Fig. 6. Schematic illustration of the immune-angiogenesis-osteogenesis coupling mechanism. Tfh cells would mingle in close proximity to sprouting lesions, forming localized ELSs with high ICOS activities. This facilitated recruitment of monocytes and DC to relay these molecular signals to promote nascent vasculogenesis, and increased local TNF-α level in bone lesions. TNF-α could promote the secretion of Slit3, which further promoted Type H vessel (CD31+EMCN+) hyperplasia and led to syndesmophyte formation.

Article Snippet: After blocking with hydrogen peroxide and serum, the tissue sections were subjected to antigen repair by enzyme repair method [38]; after 5 % BSA blocking, the tissue sections were incubated with the following antibodies: ICOS (1:200, 14–9949-82, Abcam), CD4(1:150, 48274, Abcam), CXCR5 (1:200, bsm54319R, Bioss), TNF-α (1:50, sc-52746, Santa cruz), CD31 (1:200, NBP2-44342, Novus), Endomucin (EMCN) (1:100, sc-65495, Novus), SLIT3 (1:100, TA351721, Origene), and MMP-3 (1:100, 66338–1-lg, Proteintech) overnight at 4 ◦C.

Techniques:

Journal: FEBS letters

Article Title: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

doi: 10.1016/j.febslet.2007.02.001

Figure Lengend Snippet: Fig. 1. LPS stimulation induces slit3 mRNA expression in primary mouse peritoneal macrophages. (A) The macrophages were cultured for the indicated periods of time with or without 100 ng/ml LPS. (B) The cells were treated with 10, 100, or 1000 ng/ml LPS for 2 h and analyzed by RT-PCR.

Article Snippet: The supernatant was subjected to immunoblot analysis conducted using the anti-slit3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti- b-tubulin (Sigma) antibodies.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: FEBS letters

Article Title: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

doi: 10.1016/j.febslet.2007.02.001

Figure Lengend Snippet: Fig. 2. Slit3 expression in RAW264.7 cells stimulated with LPS. Cells were cultured in the presence or absence of LPS (100 ng/ml) for the indicated periods of time. (A) RT-PCR analysis of slit1, slit2, and slit3 expression. (B) The relative expression levels of slit3 were assessed by real time-PCR analysis. Quantitative results of data from individual processes in the presence of LPS (n = 3) or absence of LPS (n = 3). (C) Immonoblot analysis for slit3 protein in the cells. Immunoblotting with the anti-tubulin antibody indicates that an equal amount of protein was loaded in each lane.

Article Snippet: The supernatant was subjected to immunoblot analysis conducted using the anti-slit3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti- b-tubulin (Sigma) antibodies.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: FEBS letters

Article Title: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

doi: 10.1016/j.febslet.2007.02.001

Figure Lengend Snippet: Fig. 3. Localization of slit3 protein in RAW264.7 cells. (A) Immuno- blot analysis for slit3 secretion. The cells were treated with or without LPS. The cultured supernatant and the high-salt wash were mixed (SW) and the cells were lysed (CL). Slit3 and slit1-tagged with FLAG were probed by anti-slit3 antibody and anti-FLAG antibody, respec- tively. (B) Differential interference microscope image. Cells were stimulated with LPS (a–c) or incubated without LPS (d–f). Immuno- fluorescence with slit3 antibody (b,e) and merged images with Mitotracker labeling for mitochondria (c,f). Merged images demon- strate colocalization of slit3 with the mitochondria (arrow). (C) The cells were stimulated with (a–c) or without (d–f) LPS. Immunofluo- rescence with slit3 antibody (b,e) and merged images with rhodamine- phalloidin labeling of F-actin (c,f). Merged image demonstrates colocalization of slit3 with F-actin (arrow).

Article Snippet: The supernatant was subjected to immunoblot analysis conducted using the anti-slit3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti- b-tubulin (Sigma) antibodies.

Techniques: Cell Culture, Microscopy, Incubation, Labeling

Journal: FEBS letters

Article Title: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

doi: 10.1016/j.febslet.2007.02.001

Figure Lengend Snippet: Fig. 4. Effect of slit3-RNAi on the spreading of RAW264.7 cells. (A) RT-PCR and immunoblot analysis for slit3 expression in cells transfected with or without the slit3-RNAi vector. Cells were stimulated with or without LPS. (B) RT-PCR analysis of slit3, TNFa, and IL-1b expression. (C) The cells transfected with the control vector (a) or the slit3-RNAi construct (c) were stimulated with LPS. Cells transfected with the vectors containing GFP gene were confirmed by GFP observation (b, d). (D) The cells transfected with or without the RNAi vector were fixed, and the percentage of cell spreading was determined. Results are from one of three independent experiments (means ± S.E.M.).

Article Snippet: The supernatant was subjected to immunoblot analysis conducted using the anti-slit3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti- b-tubulin (Sigma) antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Construct

Journal: FEBS letters

Article Title: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages.

doi: 10.1016/j.febslet.2007.02.001

Figure Lengend Snippet: Fig. 5. Effect of slit3-RNAi on Rac and Cdc42 activation. (A and B) The cells transfected with the control vector or the slit3-RNAi construct were cultured in the presence or absence of LPS. (C) The cells were incubated for 24 h. The images were photographed under phase contrast (a, c) and fluorescent light (b, d). Cells transfected with the RNAi vectors including the GFP gene, were confirmed by GFP observation. Photographs of results are from one of three independent experiments.

Article Snippet: The supernatant was subjected to immunoblot analysis conducted using the anti-slit3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti- b-tubulin (Sigma) antibodies.

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Construct, Cell Culture, Incubation

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Transcriptional mechanism underpinning Slit3 secretion. a mRNA expression levels of Ets1, E47, FoxJ2 , and FoxA2 genes in MC3T3 cells cultured in osteoblast differentiation-inducing medium and treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, t -test). b , c Protein expression level of E47, FoxA2 in MC3T3 cells cultured in stimulated medium and treated with PTH or Veh (PBS) for 3 days ( n = 3, t -test). Representative images depicting E47 IF staining (green) in lumbar vertebral body and endplate sections ( d ), quantitative analysis of the number of E47 + cells in the vertebral body ( e ) and endplate ( f ) of aged mice treated with Veh or PTH ( n = 5, t -tset). Scale bar: 100 µm. Representative images showing FoxA2 IF staining (red) in lumbar vertebral body and endplate sections ( g ), quantitative analysis of the number of FoxA2 + cells in the vertebral body ( h ) and endplate ( i ) of aged mice treated with PTH or vehicle for 2 months ( n = 5, t -tset). Scale bar: 100 µm. j Relative fold enrichment of the E47 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). k Relative fold enrichment of the FoxA2 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Binding Assay

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Efficacy of PTH treatment is diminished in osteoblastic Slit3 knockout mice with spinal degeneration. Vertebral endplate bone structure analyses by micro-CT: bone volume per tissue volume (BV/TV) percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in Slit3 OCN −/− LSI mice with PTH (40 µg/kg/d) or Veh treatment for 2 months ( n = 7, t -test). Behavior evaluations included pressure tolerance of the lumbar spine assessed via the force threshold ( d ), total distance covered in spontaneous activity in 2 days ( e ), and latency of paw withdrawal post-thermal stimulation ( f ) for Slit3 OCN −/− LSI mice with PTH or Veh treatment for 2 months ( n = 7, t -test). g Western blot analysis of protein expression levels of β3 tubulin, CGRP, PGP9.5 relative to GAPDH in endplate tissue (L3-L5) in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months. ( n = 5). Representative images of PGP9.5 (red) and CGRP (green)-positive fibers ( h ) and quantitative analysis of fiber length in the lumbar vertebral body of Slit3 OCN −/− LSI mice with PTH or Veh treatment ( i , j ) ( n = 5, t -tset). Scale bar: 100 µm. k Western blot analysis of protein expression levels of CGRP in DRG tissue (L1–L2), normalized to GAPDH expression, in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5). Representative IF images showing CGRP-positive (green) neurons in DRG sections ( l ), followed by quantitative analysis of the mean IF intensity for CGRP ( m ) in the DRG of Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5, t -tset). Scale bar: 100 µm

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Knock-Out, Micro-CT, Activity Assay, Western Blot, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rac1 inhibition via Srgap2 restrains inflammatory osteoclastogenesis and limits the clastokine, SLIT3

doi: 10.1002/jbmr.3945

Figure Lengend Snippet: Conditioned medium from Srgap2 deficient osteoclastic cells promotes osteoblastic differentiation in vitro. Recombinant SLIT3 (rSLIT3), conditioned medium of osteoclast cultures from Srgap2-cKO or control mice were added to osteoblasts cultured with β-glycerophosphate and ascorbic acid. (A) Representative ALP and ARS staining of osteoblastic cultures. (B) Alkaline phosphatase (ALP) activity assay and Alizarin red S (ARS) eluted from stained wells was measured at 415nm absorbance. (C) Osteoblastic gene expression levels, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (Ocn) relative to Gapdh were analyzed by quantitative real-time PCR. (D) ALP activity and ARS measurement after treating the cells with control or Srgap2-cKO osteoclast conditioned medium in the presence or absence of SLIT3 neutralizing antibody, to block SLIT3 activity. *, p<0.05, **, p<0.01, ***, p<0.001. Data represent mean ±SEM.

Article Snippet: In some experiments, neutralizing antibody to SLIT3 (5 μg/ml, R & D Systems) was added to cultures along with the osteoclast conditioned medium.

Techniques: In Vitro, Recombinant, Control, Cell Culture, Staining, ALP Activity Assay, Gene Expression, Real-time Polymerase Chain Reaction, Activity Assay, Blocking Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rac1 inhibition via Srgap2 restrains inflammatory osteoclastogenesis and limits the clastokine, SLIT3

doi: 10.1002/jbmr.3945

Figure Lengend Snippet: Protein levels of SLIT3 in Srgap2 deficient and Rac1-activated osteoclasts. (A) SLIT3 was detected by Western blotting of lysates from Srgap2-cKO and control BMMs cultured with M-CSF and RANKL for up to 4 days. Srgap2 was detected to confirm the genotype and β-Actin was used as an internal control. (B) SLIT3 levels were measured in the conditioned medium from control and Srgap2-cKO cells that were treated with M-CSF and RANKL for 5 days. (C) SLIT3 proteins were detected by Western blotting in wild type BMMs transduced with empty vector (EV), Myc-tagged constitutively active Rac1 or Myc-tagged constitutively active Cdc42. Transduced BMMs were cultured with M-CSF and RANKL for 4 days. Transduced genes were confirmed by Western blotting with anti-Myc antibody and β-Actin was used as a loading control. (D) BMM cells from control and Srgap2-cKO mice were cultured with or without NSC23766, a specific Rac1 inhibitor, for 4 days in the presence of M-CSF and RANKL. (E) SLIT3 levels in the serum from control and Srgap2-cKO mice. N=12–13. ***, p<0.001.

Article Snippet: In some experiments, neutralizing antibody to SLIT3 (5 μg/ml, R & D Systems) was added to cultures along with the osteoclast conditioned medium.

Techniques: Western Blot, Control, Cell Culture, Transduction, Plasmid Preparation

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Slit3 repression correlated with HCC tumor growth. a Tumor size of HCC patients cohort 1 ( n = 40) with high or low Slit3 expression. b Relative Slit3 expression inversely correlated with tumor size of HCC patients cohort 1 ( n = 40). c Tumor size of HCC patients cohort 2 ( n = 25) with high or low Slit3 expression. d Relative Slit3 expression inversely correlated with tumor size of HCC patients cohort 2 ( n = 25)

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Slit3 negatively regulated HCC cell growth. a Expression of Slit3 in HCC cell-lines. b and c Stable Slit3 repressed clones (shSlit3) of LM3 and PLC cells displayed induced cell proliferation when compared with the control cells (shCTL) after 72 h. d Stable Slit3 overexpressed clones (Slit3) of Hep3B cells displayed impaired cell proliferation when compared with the control cells (pcDNA) after 72 h. The protein expression level was quantified by ImageJ software and normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Clone Assay, Control, Software

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Molecular mechanism associated with stable Slit3 repression. Protein expression of certain proteins associated with cell cycle progression (cyclin D3), apoptosis (survivin) and GSK3β/β-catenin pathway (β-catenin and phosopho-GSK3β (ser9) in LM3 and PLC Slit3 shRNA transfectants (shSlit3) and shRNA control (shCTL). The protein expression level was quantified by ImageJ software and normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, shRNA, Control, Software

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Slit3 negatively regulated HCC cell growth in vivo. a Stable Slit3 repressed clones (LM3-shSlit3) displayed induced tumor growth when compared with the control cells (LM-shCTL) 6 weeks post-injection of cells subcutaneously into flank region of nude mice ( n = 5). b Stable Slit3 overexpressed clones (Hep3B-Slit3) displayed impaired tumor growth when compared with the control cells (Hep3B-pcDNA) 6 weeks post-injection of cells subcutaneously into flank region of nude mice ( n = 5). c Immunohistochemical staining of Slit3, CD31, p27, β-catenin and phosopho-GSK3β (ser9) in tumor formed by LM3-shCTL and LM3-shSlit3 stable cells. d Immunohistochemical staining of Slit3, CD31, p27, β-catenin and phosopho-GSK3β (ser9) in tumor formed by Hep3B-pcDNA and Hep3B-Slit3 stable cells. Representative results from two mice of each group were shown

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: In Vivo, Clone Assay, Control, Injection, Immunohistochemical staining, Staining

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Stable repression of Slit3 expression enhanced chemoresistance in HCC cells. (Left panel) LM3-shSlit3 and shCTL cells and (right panel) PLC-shSlit3 and shCTL cells were treated with ( a ) 10 μM sorafenib, ( b ) 10 μM oxaliplatin and ( c ) 100 μM 5-FU for 72 h. The number of cells was determined by MTT assay and expressed as percentage of that under vehicle control treatment. When compared with shCTL cells, shSlit3 cells showed significantly stronger resistance to the treatment of all chemotherapeutic drugs. Each experiment was performed in triplicate and data was obtained from three independent experiments

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, MTT Assay, Control

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Molecular mechanism associated with Slit3 repression-induced chemoresistance. Protein expression of β-catenin, cyclin D3 and survivin in LM3 and PLC Slit3 shRNA transfectants (shSlit3) and shRNA control (shCTL) following 72-h vehicle control (C), 10 μM sorafenib (SOR), 10 μM oxaliplatin (OXA) and 100 μM 5-FU (5FU) treatment. The protein expression level was quantified by ImageJ software and normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, shRNA, Control, Software

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Down-regulation of β-catenin diminished the effects of Slit3-repression. Stable PLC Slit3-repressed cells (PLC-shSlit3) and control clone (PLC-shCTL) were transiently transfected with siRNA control (siCTL) or β-catenin siRNA (si-β-catenin) to investigate the effect of β-catenin down-regulation on Slit3-regulated effects. a PLC-shSlit3 siCTL cells showed significantly higher proliferation rate than PLC-shCTL siCTL cells whereas such induction was significantly impaired in PLC-shSlit3 si-β-catenin cells. b to d The induced chemoresistance in PLC-shSlit3 siCTL cells when compared with PLC-shCTL siCTL cells upon treatment of sorafenib, oxaliplatin and 5-FU were impaired in PLC-shSlit3 si-β-catenin cells. e The inductions in protein expression of phospho-GSK3β, cyclin D3 and survivin in PLC-shSlit3 siCTL cells when compared with PLC-shCTL siCTL were all reduced in PLC-shSlit3 si-β-catenin cells

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Control, Transfection, Expressing

Journal: BMC Cancer

Article Title: Suppression of Slit3 induces tumor proliferation and chemoresistance in hepatocellular carcinoma through activation of GSK3β/β-catenin pathway

doi: 10.1186/s12885-018-4326-5

Figure Lengend Snippet: Clinicopathological correlation of Slit3 expression in HCC patients

Article Snippet: Antibody against Slit3 was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Over Expression, Infection