Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 8. β1-integrin is an SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with len- tiviruses carrying scrambled or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by Western blot of the lysate and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as a percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) Rep- resentative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N = 32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N = 29 neurons; SNX17-shRNA: 0.839 ± 0.040, N = 28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N = 28 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *P < 0.05. Error bars are SEM. (E) Validation of an shRNA clone (V2LMM_39157, Horizon Discovery) to knockdown rat ITGB1. pGIPZ scrambled non- target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5 d post-infection, cells were treated with 1 μg/ml of dox- ycycline to promote the expression of eGFP or ITGB1-GFP. 24 h later, extracts were generated and analyzed by Western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. Treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N = 31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N = 33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N = 26 neurons; ctrl-

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 9. β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP < 30) or from 30 to 90 min after cLTP (cLTP > 30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP, or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N = 19 neurons; isotype ctrl cLTP < 30 min: 16.790 ± 1.106, N = 9 neurons; isotype ctrl cLTP > 30 min: 16.900 ± 0.954, N = 10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 18.110 ± 1.247, N = 6 neurons; β1-integrin blocking neurons cLTP > 30 min: 13.170 ± 0.458, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N = 19 neurons; isotype ctrl cLTP < 30 min: 3.333 ± 0.800, N = 9 neurons; isotype ctrl cLTP > 30 min: 3.110 ± 0.740, N = 10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 3.952 ± 1.214, N = 6 neurons; β1-integrin blocking cLTP > 30 min: 1.096 ± 0.302, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with β1- integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N = 28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N = 25 neurons; β1-integrin blocking: 0.621 ± 0.015, N = 31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (G) Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N = 28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N = 25 neurons; β1-integrin blocking: 0.931 ± 0.054, N = 31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 10. Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender. com.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

doi: 10.1038/ncomms12502

Figure Lengend Snippet: ( a ) integrin beta-1 is increased in activated (A) compared with quiescent (Q) rat HSCs by qRT–PCR. ( b ) Immunofluorescence of rat AHSCs shows integrin beta-1 costained in cells with pro-fibrotic markers α-SMA (green; DAPI as blue nuclear counterstain) and SOX9 (red). ( c , d ) Activated mouse HSCs (‘Control') show decreased protein levels for α-SMA, COL1 and SOX9 by immunoblotting following the loss of integrin beta-1 (‘ Itgb1 -null'). Quantification from n ≥3 experiments in c and example immunoblots shown in d . ( e ) α-SMA is lost by immunofluorescence from activated mouse HSCs (‘Control') following integrin beta-1 inactivation (‘ Itgb1 -null'). DAPI (blue) used as nuclear counterstain. ( f , g ) Proliferation measured by BrdU incorporation of activated mouse HSCs (‘Control') declines following integrin beta-1 inactivation (‘ Itgb1 -null'). Quantification from n ≥3 experiments in f with example immunofluorescence in g . DAPI, blue nuclear counterstain. ( h , j ) Migration of activated mouse HSCs (‘Control') over 24 h is almost entirely attenuated following the loss of integrin beta-1 (‘ Itgb1 -null'). Quantification from n =3 biological replicates with 30–77 cells in each experiment in h with an individual example of migratory tracks (in μm) shown in i , j . ( k , l ) Contractile properties of activated mouse HSCs (‘Control') are markedly attenuated after inactivation of integrin beta-1 (‘ Itgb1 -null'). Quantification of gel contraction from n =3 experiments is shown in k in the presence or absence of mitomycin-C (Mito-C) and example images shown in l . Scale bars, 50 μm. Two-tailed unpaired t -test was used for statistical analysis. Data are shown as means±s.e.m. * P <0.05, ** P <0.01, ‡ P <0.001. C, control-activated HSCs; N, integrin beta-1-null HSCs.

Article Snippet: Following a 10 min block with 10% mouse serum and CD16/32, cells were incubated with a phycoerythrin-conjugated anti-integrin beta-1 (12-0291, 1:200) or isotype control (12-4888, 1:200; both from eBioscience) at 4 °C for 20 min in the dark .

Techniques: Quantitative RT-PCR, Immunofluorescence, Control, Western Blot, BrdU Incorporation Assay, Migration, Two Tailed Test

Journal: Nature Communications

Article Title: PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

doi: 10.1038/ncomms12502

Figure Lengend Snippet: ( a ) Heatmap and cluster analysis showing gene expression changes (1.5-fold; P <0.05) in biological duplicates of activated (A) control (‘Cnt') HSCs and following integrin beta-1 inactivation (‘Null'). Quiescent (Q) HSCs are also included for comparison. Seven clusters were identified based on upregulated (red), downregulated (blue) and intermediate (yellow) gene expression. ( b , c ) Functional annotation by gene ontology for enrichment in Cluster 3. Proportions are shown in b . Individual categories and the genes underlying them are shown in c .

Article Snippet: Following a 10 min block with 10% mouse serum and CD16/32, cells were incubated with a phycoerythrin-conjugated anti-integrin beta-1 (12-0291, 1:200) or isotype control (12-4888, 1:200; both from eBioscience) at 4 °C for 20 min in the dark .

Techniques: Gene Expression, Control, Comparison, Functional Assay

Journal: Nature Communications

Article Title: PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

doi: 10.1038/ncomms12502

Figure Lengend Snippet: ( a,b ) Quantified increase in total MYL9 and YAP-1 protein levels on activation of rat HSCs from n ≥3 experiments ( a ) with representative immunoblot shown in b . ( c–e ) Total YAP-1 and MYL9 are diminished in activated mouse HSCs (‘Control') following integrin beta-1 loss (‘ Itgb1 -null'). Quantification from n =3 experiments shown in c with representative immunoblot in d . In the immunofluorescence in e , note the rounded inactivated appearance of the Itgb1 -null cells. The remaining total YAP-1 signal is more cytoplasmic (see h , i ). Scale bar, 50 μm. ( f , g ) Itga11 knockdown in activated rat HSCs using two different siRNA oligos. Data for each oligo are shown relative to its own scrambled control in either black or grey ( n =3 for each). ( f ) Detection of Myl9 transcripts was diminished to an almost identical extent as for Itga11 . ( g ) Protein detection of MYL9 and total YAP-1 was also diminished following Itga11 knockdown. ( h , i ) The proportion of phosphorylated YAP (PYAP, inactive form), is increased following integrin beta-1 loss (‘ Itgb1 -null') from activated mouse HSCs (‘Control'; n =3 experiments) and localises more predominantly to the cytoplasm ( i ). DAPI, blue nuclear counterstain, is shown. Scale bar, 50 μm. ( j ) Luciferase activity (in relative light units; RLU) following co-transfection of constructs containing the wild-type ( MYL9- TEAD) or mutated ( MYL9- ΔTEAD) TEAD motif from the 3′-untranslated region of the MYL9 gene with empty vector (Control) or YAP expression vector. Results are normalized to a Renilla vector and expressed relative to the control MYL9 luciferase construct without YAP. ( k ) Transcript levels by qRT–PCR following inhibition of YAP-TEAD interaction using VP in activated rat HSCs expressed relative to DMSO control. Two-tailed unpaired t -test was used for statistical analysis. Data are shown as means±s.e.m. * P <0.05, ** P <0.01, † P <0.005, ‡ P <0.001.

Article Snippet: Following a 10 min block with 10% mouse serum and CD16/32, cells were incubated with a phycoerythrin-conjugated anti-integrin beta-1 (12-0291, 1:200) or isotype control (12-4888, 1:200; both from eBioscience) at 4 °C for 20 min in the dark .

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Knockdown, Luciferase, Activity Assay, Cotransfection, Construct, Plasmid Preparation, Expressing, Quantitative RT-PCR, Inhibition, Two Tailed Test

Journal: Nature Communications

Article Title: PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

doi: 10.1038/ncomms12502

Figure Lengend Snippet: ( a – f ) Quantification of immunoblots showing: ( a ) increased protein levels of PAK-1 and PAK-3 on activation of rat HSCs compared with quiescent (Q) cells; ( b ) decreases in both PAK-1 and PAK-3 following the loss of integrin beta-1 (‘ Itgb1 -null') in activated mouse HSCs (AHSCs). Levels in mouse QHSCs are shown for comparison. ( c ) Expression of PAK-1 and PAK-3 proteins are similarly diminished following Itga11 knockdown using two independent siRNAs in activated mouse HSCs relative to control (scrambled siRNA). ( d , e ) Decreases in the levels of activated HSC markers, SOX9, COL1 and phosphoMyl9 (PMYL9) following PAK-1 abrogation by siRNA1 in activated rat HSCs relative to their respective scrambled control levels (‘Control'). Similar decreases were observed with a second independent siRNA (siRNA2). Representative immunoblot is shown in e . ( f ) Decreases in COL1 and SOX9 following the inhibition of group I PAKs using IPA3 treatment in rat and human-activated HSCs compared with DMSO control. ( g ) IPA3 treatment of activated rat HSCs disrupts the actin cytoskeleton (phalloidin staining in green). Note, the rounded cell appearance following IPA3 treatment similar to and . Scale bar, 50 μm. ( h ) Relative expression levels by qRT–PCR following VP or IPA3 treatment of activate rat HSCs for three genes identified as markers of HSC inactivation . As expected, Col1a1 levels were decreased in response to both treatments. For experiments in a – h , n =3 or 4. Two-tailed unpaired t -test was used for statistical analysis. Data are shown as means±s.e.m. * P <0.05, ** P ≤0.01, † P <0.005, ‡ P ≤0.001.

Article Snippet: Following a 10 min block with 10% mouse serum and CD16/32, cells were incubated with a phycoerythrin-conjugated anti-integrin beta-1 (12-0291, 1:200) or isotype control (12-4888, 1:200; both from eBioscience) at 4 °C for 20 min in the dark .

Techniques: Western Blot, Activation Assay, Comparison, Expressing, Knockdown, Control, Inhibition, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

doi: 10.1038/ncomms12502

Figure Lengend Snippet: ( a ) Expression analysis by qRT–PCR of in vivo activated HSCs extracted from wild-type mice following CCl 4 injections compared with olive oil control. ( b , c ) FACS analysis for integrin beta-1 on in vivo activated HSCs extracted from Pdgfrb-BAC-eGFP mice following CCl 4 -induced fibrosis compared with olive oil control. Fluorescence intensity is shown using a phycoerythrin (PE)-conjugated antibody to integrin beta-1 (integrin beta-1-PE) and isotype control (Iso-PE) ( b ). Graphical representation of integrin beta-1 fluorescence demonstrating increased integrin beta-1 on unpermeabilized live HSCs the context of liver fibrosis ( c ). All experiments are n =4. Two-tailed unpaired t -test was used for statistical analysis. Data in bar charts show means±s.e.m. * P <0.05, ** P <0.01.

Article Snippet: Following a 10 min block with 10% mouse serum and CD16/32, cells were incubated with a phycoerythrin-conjugated anti-integrin beta-1 (12-0291, 1:200) or isotype control (12-4888, 1:200; both from eBioscience) at 4 °C for 20 min in the dark .

Techniques: Expressing, Quantitative RT-PCR, In Vivo, Control, Fluorescence, Two Tailed Test

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 1 S100A9 promotes prostate cancer cell invasion and β1 integrin expression through interaction with TLR4. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (A) PC-3 and DU-145 cells invasion was measured by transwell invasion assay. (B) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (C, D) Cell extracts were immunoprecipitated (IP) with control mouse IgG, mouse anti-S100A9 antibody. Immunoblot (IB) was used to detect S100A9, TLR4 and RAGE. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA. (E) TLR4 expression was examined by Western blot after 48 h siRNA transfection. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA followed by stimulation with S100A9. (F) Tumor cell invasion was measured by transwell invasion assay. (G) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Rabbit anti-human antibodies against S100A9 (72590), TLR4 (2246), integrin β1 (34971), FAK (3285), p-FAK (3281), RAGE (6996), NF-κB (8242), Phospho-NF-κB p65 (Ser536) antibody (3031) were purchased from Cell Signaling Technology (Danvers, MA, USA). β1 integrin inhibitory antibody MAB 13 (552828) and the isotype control antibody rat IgG2aκ (553927) were purchased from (BD Biosciences-CN, Shanghai, China).

Techniques: Expressing, Transwell Invasion Assay, Western Blot, Immunoprecipitation, Control, Transfection

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 2 NF-κB mediates S100A9-induced prostate cancer cell β1 integrin up-regulation. PC-3 and DU-145 cells transfected with or without TLR4 siRNA or control siRNA, were transfected with NF-κB-luciferase reporter plasmid, and treated with S100A9 (20 µg/ml) for 48 h. (A, B) Activity of NF-κB was detected by measuring the relative activity of luciferase. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ ml) for 48 h. (C) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Rabbit anti-human antibodies against S100A9 (72590), TLR4 (2246), integrin β1 (34971), FAK (3285), p-FAK (3281), RAGE (6996), NF-κB (8242), Phospho-NF-κB p65 (Ser536) antibody (3031) were purchased from Cell Signaling Technology (Danvers, MA, USA). β1 integrin inhibitory antibody MAB 13 (552828) and the isotype control antibody rat IgG2aκ (553927) were purchased from (BD Biosciences-CN, Shanghai, China).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Western Blot

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 3 S100A9 promotes prostate cancer cell invasion via integrin β1/FAK signaling. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (A) Fibronectin expression was determined by Western blot. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 48 h. (B) Supernatant fibronectin (FN) concentration was determined by ELISA. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 30 min. (C) The phosphorylation of FAK was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 30 min, the expression of integrin β1 (D) or phosphorylation of FAK (E) was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (F) The invasion activity were measured by transwell invasion assay. (G) PC-3 and DU- 145 cells were treated with β1 integrin functional blocking antibody MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 30 min and the phosphorylation of FAK was measured by Western blot. (H) PC-3 and DU-145 cells were treated with MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 48 h for invasion. (I) PC-3 and DU-145 cells were pretreated for 30 min with FAK inhibitor, PF562271 (100 nM) followed by stimulation with S100A9 (20 µg/ml) for 48 h for invasion. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Rabbit anti-human antibodies against S100A9 (72590), TLR4 (2246), integrin β1 (34971), FAK (3285), p-FAK (3281), RAGE (6996), NF-κB (8242), Phospho-NF-κB p65 (Ser536) antibody (3031) were purchased from Cell Signaling Technology (Danvers, MA, USA). β1 integrin inhibitory antibody MAB 13 (552828) and the isotype control antibody rat IgG2aκ (553927) were purchased from (BD Biosciences-CN, Shanghai, China).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Transfection, Control, Activity Assay, Transwell Invasion Assay, Functional Assay, Blocking Assay

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 4 S100A9 induces prostate cancer cell metastasis in vivo. DU-145 cells were transfected with pcDNA3.1 or pc DNA-S100A9 plasmid. (A, B) The expression or secretion of S100A9 was determined by Western blot and ELISA. The cells were injected to nude mice via tail vein. 30 days after inoculation, nude mice were sacrificed. (C) Expressions of S100A9 and integrin β1 in xenograft tumors were detected by immunohistochemistry. (D) Expressions of FAK, p-FAK, NF-kB p65 and p-NF-kB p65 in xenograft tumors were determined by Western blot. (E) Micrometastatic tumors in the lungs of mouse xenografts were counted and subjected to H and E staining. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M.*p<0.05.

Article Snippet: Rabbit anti-human antibodies against S100A9 (72590), TLR4 (2246), integrin β1 (34971), FAK (3285), p-FAK (3281), RAGE (6996), NF-κB (8242), Phospho-NF-κB p65 (Ser536) antibody (3031) were purchased from Cell Signaling Technology (Danvers, MA, USA). β1 integrin inhibitory antibody MAB 13 (552828) and the isotype control antibody rat IgG2aκ (553927) were purchased from (BD Biosciences-CN, Shanghai, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemistry, Staining

Journal: Bioactive Materials

Article Title: Construction of functional neural network tissue combining CBD-NT3-modified linear-ordered collagen scaffold and TrkC-modified iPSC-derived neural stem cells for spinal cord injury repair

doi: 10.1016/j.bioactmat.2024.01.012

Figure Lengend Snippet: CBD-NT3-modified LOCS and TrkC-modified iPSC-derived NSCs constructs a functional neural network tissue. (A–B) The expression of cholinergic neuron marker ChAT and glutaminergic neuron marker VGLUT1 in each group in vitro. (C) The expression of mature neuron marker MAP2 in each group in vitro. (D) Synapse formation was determined by immunofluorescence detection of SYN- and PSD95-positive cells. (E) The expression of astrocyte marker GFAP in each group in vitro. (F) The deposition of laminin and the expression of integrin β1-positive cells reflect the interaction between the cells and LOCS (G-J) The cells in each group were also significantly different in morphology, as observed using SEM. Cells made contact with each other through axon-like protrusions, forming a network structure similar to that of neural links (White arrows indicate axon-like protrusions). (Scale bars: 20 μm in A-F).

Article Snippet: The following antibodies were used for immunofluorescence: Nestin (Proteintech, 29285-1-AP, 1:200), Sox2 (Proteintech, 11064-1-AP, 1:200), TrkC (Proteintech, 66380-1-Ig, 1:100), HNA (Abcam, ab191181, 1:100), GFAP (Proteintech, 16825-1-AP, 1:200), Tuj-1 (Abcam, ab18207, 1:100), Map-2 (Proteintech, 17490-1-AP, 1:100), NeuN (Abcam, ab177487, 1:100), MBP (Proteintech, 10458-1-AP, 1:100), Syn (Proteintech, 17785-1-AP, 1:100), PSD95 (Proteintech, 20665-1-AP, 1:100), 5-HT (Sigma-Aldrich, S5545, 1:1000), ChAT (Proteintech, 20747-1-AP, 1:200), VGLUT1 (Proteintech, 55491-1-AP, 1:200), CD68 (Abcam, ab283654, 1:100), NG2 (Abcam, ab275024, 1:100), Laminin (Proteintech, 23498-1-AP, 1:200), Integrin beta 1 (Abcam, ab179471, 1:100).

Techniques: Modification, Derivative Assay, Construct, Functional Assay, Expressing, Marker, In Vitro, Immunofluorescence

Journal: Bioactive Materials

Article Title: Construction of functional neural network tissue combining CBD-NT3-modified linear-ordered collagen scaffold and TrkC-modified iPSC-derived neural stem cells for spinal cord injury repair

doi: 10.1016/j.bioactmat.2024.01.012

Figure Lengend Snippet: Functional neural network tissue transplantation improvs microenvironment of SCI area. (A–E) GFAP-positive cells demonstrated the extent of the SCI area and astrocyte regeneration in each group. (F-H) HNA-positive cells demonstrated the survival of the transplanted cells in the SCI area of the rats in each group. (I–M) CD68-positive cells determined inflammation in SCI area of each group. (N–R) NG2 determined scar formation in SCI area of each group. (S-W) The deposition of laminin and the expression of integrin β1-positive cells reflected functional neural network tissue transplantation can substantially improve the microenvironment of the SCI area and promote extracellular matrix remodeling. (Scale bars: 500 μm in A-H, 50 μm in I-W and all high-power fields).

Article Snippet: The following antibodies were used for immunofluorescence: Nestin (Proteintech, 29285-1-AP, 1:200), Sox2 (Proteintech, 11064-1-AP, 1:200), TrkC (Proteintech, 66380-1-Ig, 1:100), HNA (Abcam, ab191181, 1:100), GFAP (Proteintech, 16825-1-AP, 1:200), Tuj-1 (Abcam, ab18207, 1:100), Map-2 (Proteintech, 17490-1-AP, 1:100), NeuN (Abcam, ab177487, 1:100), MBP (Proteintech, 10458-1-AP, 1:100), Syn (Proteintech, 17785-1-AP, 1:100), PSD95 (Proteintech, 20665-1-AP, 1:100), 5-HT (Sigma-Aldrich, S5545, 1:1000), ChAT (Proteintech, 20747-1-AP, 1:200), VGLUT1 (Proteintech, 55491-1-AP, 1:200), CD68 (Abcam, ab283654, 1:100), NG2 (Abcam, ab275024, 1:100), Laminin (Proteintech, 23498-1-AP, 1:200), Integrin beta 1 (Abcam, ab179471, 1:100).

Techniques: Functional Assay, Transplantation Assay, Expressing