Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 1. sFRP1 was abundantly produced in nerve ECM following injury and associated with nerve degeneration (A) The isolation of sciatic nerve samples and proteomic analysis process. (B) The clustering distribution of injured and uninjured nerve samples as plotted by PCA analysis. (C) Differentially expressed proteins between injured and uninjured nerve samples are displayed in volcano plot. N = 3 mice. Proteins regulated over 1.5-fold changes (adj. p < 0.05) are highlighted in blue (downregulated) and red (upregulated). (D) GO enrichment analysis indicating the classification of differentially expressed proteins related to the biological process category. (E) Differentially expressed proteins in the GO category of ECM are displayed as a heatmap. (F) Western blotting analysis demonstrating increased production of sFRP1 in the injured nerve tissue. (G) Quantification of sFRP1 protein level in sciatic nerves isolated from uninjured and injured mice as indicated by western blot analysis. N = 3 mice. (H) Representative TEM, HE, and TB images of injured nerves isolated from mice treated with WAY-316606 and PBS at 3 weeks post injury. N = 6 mice. (I and J) Quantification of myelinated axon diameter and g-ratio as indicated in TEM images. (K) Quantification of myelinated axon density as indicated in HE-stained images. (L) Representative IHC images of human nerves stained for sFRP1 at 12 h after injury. Statistical significance was determined using two-tailed unpaired Student’s t tests; **p < 0.01; *p < 0.05; ns, no difference. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Produced, Isolation, Western Blot, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 3. Mice with deletion of sFRP1 in SCs profoundly reduced macrophage infiltration and improved nerve regeneration (A) Sfrp1flox/flox mice were bred with PlpcreErt1 mice to generate tamoxifen-inducible SC-specific sFRP1 knockout (Sfrp1flox/floxPlpcreErt1) and littermate control (Sfrp1flox/flox) mice. (B and C) Representative SCG10 immunostaining and related quantification of sciatic nerves at 14 days post transection. N = 6 mice. The dashed line indicates the transection site. Scale bar, 500 mm. (D and E) Representative F4/80 immunostaining (red) of sciatic nerves taken from the injury site, 1,000, 2,000, and 3,000 mm distal to the injury site and related quantification of infiltrated macrophages. Scale bar, 100 mm. N = 6 mice. (F and G) Western blot analysis and related quantification of TNF-a level in injured nerves at 24 h post transection. N = 3 mice. (H and I) Triple staining of CCL2 (green), F4/80 (red), and NeuN (pink) on sciatic DRG sections from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice and related quantification of CCL expression level in DRGs. N = 6 mice. No significant difference of CCL2 expression is observed between DRGs of Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice. (J–L) Representative TUBB3 immunostaining (green) of sciatic DRG neurons isolated from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice (n = 6 mice) and related quantification. DRG neurons were cultured in vitro for 4 days or 7 days. (M and N) Representative immunostaining and related quantification of ATF3 (red) and the neuronal marker NeuN (green) in sciatic DRGs at 24 h after nerve injury. N = 6 mice. Scale bar, 50 mm. Statistical significance in (C) and (E) was analyzed by two-way ANOVA followed by Sidak’s post hoc analysis. Statistical significance was determined using two- tailed unpaired Student’s t tests; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Knock-Out, Control, Immunostaining, Western Blot, Staining, Expressing, Isolation, Cell Culture, In Vitro, Marker, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 4. SFRP1 induces the F4/80+ CD86+ proinflammatory macrophage phenotype and inhibits oxidative metabolism (A and B) The axon length of sciatic DRG neurons demonstrates no significant difference in response to sFRP1 treatment. N = 6 biological replicates. (C) Representative TEM images reveal that the morphology and structure of mitochondria were well preserved in sFRP1-treated neurons. (D and E) Representative TEM images and related quantification of nerve transections (N = 6 mice). The suppressing effect of sFRP1 on axon regrowth is alleviated in a macrophage-deficient condition. (F) Double staining of IL-1b (red) and TNF-a (green) on sFRP1-treated BMDMs. (G) sFRP1-induced phenotypic switch is revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. (H and I) Quantification of the percentage of IL-1b and TNF-a-positive cells as reflected by Figure 4F. Biological replicates n = 3 with two technical replicates each. (J) Double staining of Arg-1 (red) and Wnt3a (green) on sFRP1 and PBS-treated BMDMs. (K) The internalizing capacity of BMDMs was measured by incubating with 100 mg/mL pHrodo BioParticles (green). BMDMs were visualized by F4/80 (red) staining.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Double Staining, Staining

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 6. Depletion of HSP90 in macrophages attenuated neuroinflammation and nerve degenerative changes exerted by sFRP1 (A) Hsp90aaflox/+ mice were bred with Lyz2-cre mice to generate macrophage-specific HSP90-deficient (Hsp90aaflox/+Lyz2-cre) and littermate control (Hsp90aaflox/+) mice. (B and C) Representative IF images of SCG10 staining and related quantification of sciatic nerves at 2 weeks post injury. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (D and E) Representative IF images of F4/80 staining (red) of sciatic nerves and related quantification of macrophages at 2 weeks post injury. Scale bar, 100 mm. N = 6 mice. (F–I) Double staining of TNF-a (red) and IL-1b (green) on nerve longitudinal sections and related quantification. (J–L) Representative TUBB3 staining (green) and related quantification of sciatic DRG neurons isolated from Hsp90aaflox/+ and Hsp90aaflox/+Lyz2-cre mice after 4 days and 7 days of culture. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (C) and (E), and using two-tailed unpaired Student’s t tests in (F), (G), (K), and (L); **p < 0.01; ***p < 0.001; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Control, Staining, Double Staining, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 7. SFRP1-neutralizing antibody treatment improved axon regeneration in vivo and in vitro (A and B) Representative SCG10 immunostaining and related quantification of murine injured nerves at 2 weeks after nerve transection. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (C) Schematic diagram of DRG neuron and macrophage microfluidic coculture chamber assay. (D) Representative optical images of macrophages in the neuron-macrophage coculture chambers. (E and F) Representative TUBB3 immunofluorescent images of neurons in the neuron-macrophage co-culture chambers and related quantification of average axon length in microfluidic channels. Biological replicates n = 3 with two technical replicates each. (G) Schematic diagram of DRG neuron and macrophage direct coculture assay. (H and I) Representative IF images stained for TUBB3 (green) on sciatic DRG neurons, and quantification of average axon length per cell in the direct coculture dishes. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (B) and (I) and using two-tailed unpaired Student’s t tests in (F); ***p < 0.001; **p < 0.01; *p < 0.05. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: In Vivo, In Vitro, Immunostaining, Boyden Chamber Assay, Co-Culture Assay, Co-culture Assay, Staining, Two Tailed Test

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 3. Ferroptosis-inducing and immunostimulatory capabilities of the biomimetic hydrogel. (a) Schematic diagram for the tumor cell/immune cell co- incubation system in transwell plates. The tumor cells (B16F10, 4T1) or immune cells were inoculated in the bottom chamber of the 24-well transwell culture plate, while the hydrogel soaking solution was placed in the upper chamber. (b) Changes of GPX4 activity in B16F10 cells after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (c) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (d) CLSM imaging of lipid ROS generation in B16F10 cells after different treatments. Higher green fluorescence intensity indicates greater lipid ROS production. (e) Quantitative fluorescence analysis of lipid ROS levels in panel D (n = 4). (f) Flow cytometric analysis on the hydrogel-mediated ferroptosis levels of B16F10 cells after different treatments. (g) ATP levels in the supernatants of cell culture after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (h) CLSM imaging of CRT expression in B16F10 cells after different treatments. Stronger red fluorescence indicates higher expression levels. (i) Quantitative fluorescence analysis of CRT expression levels in panel H (n = 4). (j) CLSM imaging of cellular HMGB1 abundance after different treatments. Lower red fluorescence indicates greater HMGB1 release into the extracellular compartment. (k) Quantitative fluorescence analysis of HMGB1 release in panel J (n = 4). (l) Flow cytometric analysis on the treatment-induced maturation of BMDCs. (m) Flow cytometric analysis on the activation status of macrophages by monitoring the CD80 expression levels. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: Incubation, Activity Assay, Control, Imaging, Cell Culture, Expressing, Activation Assay

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 4. Gel@RSL3 + GM-CSF + aPD-L1 activates immune response in vitro. (a-d) Flow cytometric analysis of the activation status of DCs (CD11c + /MHC II + ), M1 macrophages (F4/80 + /CD80 + ) and T cells (CD8 + /CD3 + and CD8a + /IFN- 𝛾+ ) in the co-incubation system of splenic immune cells and B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM-CSF (n = 4). (e) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (f) PD-L1 expression in tumor cells with after the hydrogel-mediated ferroptosis-immunotherapy. Group set-up for panel e-f: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM- CSF). (g-h) Flow cytometric analysis of the expression levels of effector T cell marker CD4 + /CD8 + and CD8a + /IFN- 𝛾+ in T cells co-incubated with B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. (i) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (j) Evaluation on the GSH levels in B16F10 cells after different treatments (n = 4). (k) Western blot analysis of the expression level of CRT, HMGB1 and SLC7A11 in different groups. (l) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (m) MDA levels in B16F10 cells after different treatments (n = 4). Group set-up for panel I-M: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD- L1). (n) Flow cytometric analysis on the death rate of B16F10 cells after different treatments, including (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001, ∗ ∗ ∗ ∗ indicates significance at P < 0.0001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vitro, Activation Assay, Incubation, Control, Co-Culture Assay, Expressing, Marker, Western Blot

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 5. Antitumor effect of biomimetic hydrogel in vivo. (a) Schematic illustration of the treatment scheme of the B16F10-luc tumor-bearing mice (n = 7). (b) Treatment procedures on the tumor-bearing mice. (c) In vivo bioluminescence images of B16F10-luc tumor-bearing mice throughout the treatment period with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (d) Tumor size changes during the incubation period after different treatments. (e) Survival analysis of mice after different treatments. (f) Body weight changes after treatment with different samples. (g) Evaluation on the GPX4 activity in tumor tissues after different treatments (n = 4). (h) Western blot analysis on the expression of CRT, HMGB1 and SLC7A11 in B16F10-luc tumors after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (i) MDA levels in tumor tissue after different treatments (n = 4). (j) H&E and TUNEL staining of tumor tissue samples after treatment (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vivo, Control, Incubation, Activity Assay, Western Blot, Expressing, TUNEL Assay, Staining

Journal: NPJ biofilms and microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Fig. 2 Liver transcriptome analysis of L. reuteri-treated darkness rats. Heatmaps displaying 36 highly expressed genes (a) and 76 lowly expressed genes (b) in the liver of darkness rats compared with control and DL.reuteri rats through DEG analysis using Ballgown software (| fold change | > 0.6 in the log2 ratio value, raw P < 0.05). c Top terms from GO analysis (above) and terms from KEGG analysis (below) of the 112 differential genes in the DEG analysis. The P values of GO and KEGG analyses were determined on the DAVID website. d Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm in the DEG analysis. e Spearman rank correlations between module eigengenes (ME) and clinical biochemical index in the WGCNA analysis (|ρ | > 0.3, *P < 0.05). f Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm of purple module genes (102) in the WGCNA analysis. Circle, genes; Square, KEGG terms; Hexagon, GO terms. The bigger the square or hexagon, the more genes involved. g Crosstalk among different groups of genes identified the possible target genes taking essential roles in the lipid metabolism of L. reuteri-treated darkness rats. h mRNA abundances of Galr1, Galr2, Nr1d1, Nr1d2, Insig2, Srebf1, Lxra, and Rxra in rat liver detected by qPCR. β-Actin was used as a loading control for qPCR analyses. i Serum galanin concentration detected by ELISA. Statistical analysis (h, i) was performed with one-way ANOVA followed by Newman–Keuls multiple comparison test. n = 8 per group. Data present means ± SEM. *P < 0.05, **P < 0.01.

Article Snippet: Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2023) 47 GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), Fig. 8 Proposed mechanisms for the amelioration of L. reuteri on dyslipidemia in circadian dysrhythmia-induced PCOS-like rats.

Techniques: Control, Software, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: NPJ biofilms and microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Fig. 8 Proposed mechanisms for the amelioration of L. reuteri on dyslipidemia in circadian dysrhythmia-induced PCOS-like rats. Left, circadian dysrhythmia due to constant darkness resulted in dyslipidemia and reproductive hallmarks of PCOS in rats. Elevated galanin-GALR1 induced by darkness exposure functioned as an upstream factor of PI3K/AKT pathway and further suppressed NR1D1-induced SREBF1 transcription and translation, thus inducing hepatic lipid accumulation in PCOS-like rats. Right, L. reuteri supplementation ameliorated dyslipidemia and reproductive hallmarks in circadian dysrhythmia-induced PCOS-like rats. L. reuteri restructured microbiome-metabolome network in darkness rats ameliorating the abundance of Lactobacillus, Clostridium sensu stricto 1, Ruminococcaceae UCG-010, and Family XIII AD3011 group, followed by varied serum levels of cortisol, cis-9-palmitoleic acid, 13-methylmyristic acid, capric acid, and dUMP. Notably, capric acid mediated the inhibition of L. reuteri on hepatic GALR1-PI3K/AKT-NR1D1-SREBP1 pathway, which eventually alleviated dyslipidemia.

Article Snippet: Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2023) 47 GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), Fig. 8 Proposed mechanisms for the amelioration of L. reuteri on dyslipidemia in circadian dysrhythmia-induced PCOS-like rats.

Techniques: Inhibition

Journal: Journal of radiation research

Article Title: Combining network pharmacology and in vitro and in vivo experiments to study the mechanism of Keluoxin in the treatment of radiation nephropathy†.

doi: 10.1093/jrr/rrad050

Figure Lengend Snippet: Fig. 1. The network of Keluoxin in the treatment of radiation nephropathy. (A) Venn diagram of the intersecting targets of Keluoxin and radiation nephropathy. (B) The compound-target network of Keluoxin. (C) The interaction network of 50 key target proteins. (D) The compound-target-pathway network.

Article Snippet: Foetal bovine serum, phosphate buffer solution (PBS), DMEM, a penicillin and streptomycin mixture, Cell Counting Kit 8 (CCK-8) kits and lactate dehydrogenase (LDH) kits were purchased from Beijing Solabo Technology Co, Ltd. creatinine (CR), blood urea nitrogen (BUN), IL-6, TNF-α, TGF-β and IFN-γ kits were purchased from Shanghai Biyuntian Technology Co, Ltd. Trypsin, JAK1, JAK2, STAT1, STAT3 and HIF-1α antibodies were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: Journal of radiation research

Article Title: Combining network pharmacology and in vitro and in vivo experiments to study the mechanism of Keluoxin in the treatment of radiation nephropathy†.

doi: 10.1093/jrr/rrad050

Figure Lengend Snippet: Fig. 4. Effect of Keluoxin on lipid peroxidation of TCMK-1 after X-ray irradiation. (A, B) Effect of serum-containing Keluoxin on MDA and GSH levels after X-ray irradiation. After 10 Gy irradiation, the expression of MDA increased and the expression of GSH decreased in TCMK-1 cells, with statistical differences (all compared with CON groups). And medicated serum-containing Keluoxin can inhibit these changes.

Article Snippet: Foetal bovine serum, phosphate buffer solution (PBS), DMEM, a penicillin and streptomycin mixture, Cell Counting Kit 8 (CCK-8) kits and lactate dehydrogenase (LDH) kits were purchased from Beijing Solabo Technology Co, Ltd. creatinine (CR), blood urea nitrogen (BUN), IL-6, TNF-α, TGF-β and IFN-γ kits were purchased from Shanghai Biyuntian Technology Co, Ltd. Trypsin, JAK1, JAK2, STAT1, STAT3 and HIF-1α antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Irradiation, Expressing

Journal: Journal of radiation research

Article Title: Combining network pharmacology and in vitro and in vivo experiments to study the mechanism of Keluoxin in the treatment of radiation nephropathy†.

doi: 10.1093/jrr/rrad050

Figure Lengend Snippet: Fig. 3. Effect of different doses of X-ray on the activity of TCMK-1 cells. (A) TCMK-1 cell mortality after different doses of X-ray irradiation. Measure cell mortality 48 h after X-ray irradiation. As the X-ray dose increases, the cell mortality rate gradually increases. (B) TCMK-1 cell viability at 12, 24 and 48 h after different doses of X-ray irradiation. Measure cell mortality 12, 24 and 48 h after X-ray irradiation. As the X-ray dose increases, the cell survival rate gradually decreases. The most significant decrease in cell viability was observed 48 h after 10 Gy X-ray irradiation. (C) Effect of medicated serum containing Keluoxin on TCMK-1 cell viability. As the X-ray dose increases, the cell survival rate gradually decreases and medicated serum-containing Keluoxin can inhibit this decline (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Article Snippet: Foetal bovine serum, phosphate buffer solution (PBS), DMEM, a penicillin and streptomycin mixture, Cell Counting Kit 8 (CCK-8) kits and lactate dehydrogenase (LDH) kits were purchased from Beijing Solabo Technology Co, Ltd. creatinine (CR), blood urea nitrogen (BUN), IL-6, TNF-α, TGF-β and IFN-γ kits were purchased from Shanghai Biyuntian Technology Co, Ltd. Trypsin, JAK1, JAK2, STAT1, STAT3 and HIF-1α antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Activity Assay, Irradiation

Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 2. Effects of serum and cell density on ciliogenesis in mouse embryonic fibroblasts (MEFs). (A) MEFs (MEF-WT and MEF-STIM1/) were seeded at low or high density, followed by incubation with or without serum for 24 h. Immunofluorescence images of the cilia marker ARL13B (green) and nuclei (blue). (B) Percentages of ciliated cells are presented as means SEM of the n = 3 independent experiments. **P < 0.01; ***P < 0.001 by one-way ANOVA. (C, D) MEF-STIM1/ cells were incubated in culture medium without serum with (C) 1 lM ionomycin or (D) 0.1 lM BAPTA-AM at low density for 1 h. Images show immunofluorescence staining for ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (C) n = 3 and (D) n = 4 independent experiments. *P < 0.05; **P < 0.01 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Incubation, Marker, Staining

Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 4. Effects of serum and cell density on ciliogenesis in Hs 578T cells. (A) Hs 578T cells were seeded at a low or high density, followed by incubation with or without serum for 24 h. Immunofluorescence images of the cilia marker ARL13B (green) and nuclei (blue). (B) Percentages of ciliated cells are presented as means SEM of the n = 3 independent experiments. *P < 0.05; ***P < 0.001 by one-way ANOVA. (C, D) Hs 578T cells were incubated in culture medium without serum with (C) 1 lM ionomycin or (D) 0.1 lM BAPTA-AM at low density for 1 h. Images show immu- nofluorescence staining for ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (C) n = 5 and (D) n = 6 independent experiments. *P < 0.05; ***P < 0.001 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Incubation, Marker, Staining

Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 5. STIM1 negatively regulates ciliogenesis in Hs 578T cells. (A) Hs 578T cells were transfected with mOrange-STIM1 to overexpress STIM1 proteins, followed by incubation without serum at low density. Immunofluorescence images of STIM1 (red), ARL13B (green), and nuclei (blue). (B) Hs 578T cells were transfected with shSTIM1-GFP to knock down STIM1 proteins, followed by incubation with 10% FBS at low density. Immunofluorescence images of shSTIM1 (green), ARL13B (red), and nuclei (blue). (C) Western blots show STIM1 expression after siRNA targeting STIM1. (D) Hs 578T cells were transfected with siSTIM1 for 72 h. Immunofluorescence images of ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (A) n = 6, (B) n = 4, (C) = 3 and (D) n = 3 independent experiments. **P < 0.01 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Transfection, Incubation, Knockdown, Western Blot, Expressing