Journal: American Journal of Cancer Research

Article Title: Small molecule TSC01682 inhibits osteosarcoma cell growth by specifically disrupting the CUL4B-DDB1 interaction and decreasing the ubiquitination of CRL4B E3 ligase substrates

doi:

Figure Lengend Snippet: TSC01682 significantly inhibited osteosarcoma cell growth through decreasing protein levels of CRL4B components. (A and B) TSC01682 treatment significantly inhibited osteosarcoma cell growth. Saos2 (A) and MG63 (B) cells were grown in DMEM medium supplemented with different concentrations (0, 1, 2, 4, 8, 16, 32 and 64 μM) of TSC01682 and TSC01131 for 5 days, respectively. Cell viability was determined using an MTT assay. Cell viability without treatment was defined as 100%, and cell viability under other conditions was normalized to that of untreated cells. TSC01131 vs DMSO (#P < 0.05, ##P < 0.01 and ###P < 0.001); TSC01682 vs DMSO (*P < 0.05, **P < 0.01 and ***P < 0.001); and TSC01131 vs TSC01682 (°P < 0.05). (C and D) The effects of TSC01682 and TSC01131 on protein levels of CRL4B components and their substrates. Saos2 (C) and MG63 cells (D) were treated with 2 μM and 20 μM TSC01682 and TSC01131 for 12 h at 37°C, followed by an examination of the protein levels of CUL4B, DDB1, DCAF11, DCAF13, p21, and PTEN. GAPDH was used as a loading control.

Article Snippet: Cell culture and transfection Two osteosarcoma cell lines, Saos2 (#HTB-85) and MG63 (#CRL-1427), and one osteoblast cell line, hFOB1.19 (#CRL-11372), one colorectal carcinoma cell line HCT-116 (CCL-247), one ovarian cancer cell line SKOV3 (#HTB-77), one gastric cancer cell line MKN45 (#CRL-1739) and one normal colon cell line CCD-18Co (#CRL-1459) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: MTT Assay, Control

Journal: American Journal of Cancer Research

Article Title: Small molecule TSC01682 inhibits osteosarcoma cell growth by specifically disrupting the CUL4B-DDB1 interaction and decreasing the ubiquitination of CRL4B E3 ligase substrates

doi:

Figure Lengend Snippet: TSC01682 significantly inhibited the ubiquitination of p21 and PTEN. A. TSC01682 treatment significantly decreased the CUL4B-DDB1 interaction in Saos2 cells. Saos2 cells were treated with 20 μM TSC01682 or TSC01131 for 12 h, respectively. The resulting cells were subjected to immunoprecipitation assays using anti-CUL4B and anti-DDB1 antibodies. The input and output proteins were detected using anti-CUL4B and anti-DDB1 antibodies, respectively. GAPDH and IgG were used as loading controls. B and C. TSC01682 significantly inhibited the ubiquitination of p21 and PTEN in vivo. Saos2 cells were cotransfected with pCDNA3-2 × Flag-PTEN + pCDNA3-3 × HA-ubiquitin or pCDNA3-2 × Flag-p21 + pCDNA3-3 × HA-ubiquitin and incubated at 37°C for 48 h. Subsequently, cells were treated with 20 μM TSC01682 or TSC01131 for 12 h. The resulting cells were lysed and immunoprecipitated with an anti-Flag antibody to detect loading levels of p21 and PTEN. The blots were also probed with an anti-HA antibody to detect ubiquitinated p21 or PTEN.

Article Snippet: Cell culture and transfection Two osteosarcoma cell lines, Saos2 (#HTB-85) and MG63 (#CRL-1427), and one osteoblast cell line, hFOB1.19 (#CRL-11372), one colorectal carcinoma cell line HCT-116 (CCL-247), one ovarian cancer cell line SKOV3 (#HTB-77), one gastric cancer cell line MKN45 (#CRL-1739) and one normal colon cell line CCD-18Co (#CRL-1459) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, In Vivo, Incubation

Journal: American Journal of Cancer Research

Article Title: Small molecule TSC01682 inhibits osteosarcoma cell growth by specifically disrupting the CUL4B-DDB1 interaction and decreasing the ubiquitination of CRL4B E3 ligase substrates

doi:

Figure Lengend Snippet: TSC01682 treatment decreased cell proliferation, colony formation, and cell invasion. (A and B) TSC01682 treatment inhibited osteosarcoma cell proliferation. Saos2 (A) and MG63 (B) cells were grown in DMEM medium supplemented with 20 μM TSC01682 and TSC01131 for 5 days. Cells were collected each day and cell viability was assessed using an MTT assay. TSC01131 vs DMSO (#P < 0.05 and ##P < 0.01); TSC01682 vs DMSO (*P < 0.05, **P < 0.01 and ***P < 0.001); and TSC01131 vs TSC01682 (°P < 0.05). (C and D) TSC01682 treatment inhibited colony formation of osteosarcoma cells. Saos2 and MG63 cells were seeded on six-well plates at a density of 300 cells per well. Cells were grown in serum-free DMEM medium supplemented with 10 μM TSC01131 or TSC01682 and then continuously cultured for two weeks. Colonies were stained with 0.5% crystal violet and photographed (C), and colony numbers were quantified (D). *P < 0.05 and ***P < 0.001. (E and F) TSC01682 inhibited osteosarcoma cell invasion. A total of 1 × 105 cells in each treatment were applied to examine their invasion ability using a Boyden chamber assay. The invading cells were stained with 0.1% crystal violet and photographed (E), and cell numbers were quantified (F). *P < 0.05 and ***P < 0.001.

Article Snippet: Cell culture and transfection Two osteosarcoma cell lines, Saos2 (#HTB-85) and MG63 (#CRL-1427), and one osteoblast cell line, hFOB1.19 (#CRL-11372), one colorectal carcinoma cell line HCT-116 (CCL-247), one ovarian cancer cell line SKOV3 (#HTB-77), one gastric cancer cell line MKN45 (#CRL-1739) and one normal colon cell line CCD-18Co (#CRL-1459) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: MTT Assay, Cell Culture, Staining, Boyden Chamber Assay

Journal: American Journal of Cancer Research

Article Title: Small molecule TSC01682 inhibits osteosarcoma cell growth by specifically disrupting the CUL4B-DDB1 interaction and decreasing the ubiquitination of CRL4B E3 ligase substrates

doi:

Figure Lengend Snippet: TSC01682 inhibited osteosarcoma tumor growth in vivo. (A) TSC01682 treatment eliminated tumors in vivo. Saos2 cells were injected intradermally into the flanks of nude mice. On day 20 after injection, mice with similar tumor volumes (approximately 350 mm3) were randomly divided into three groups (n = 5 in each group) and then injected with DMSO, 20 μM TSC01131 and 20 μM TSC01682, respectively. Tumor volumes were measured with calipers at 5-day intervals. TSC01131 vs DMSO (#P < 0.0, ##P < 0.01 and ###P < 0.001); TSC01682 vs DMSO (*P < 0.05, **P < 0.01 and ***P < 0.001); and TSC01131 vs TSC01682 (°P < 0.05). (B-D) TSC01682 treatment affected the protein levels of CRL4B components in vivo. The nude mice (WT), untreated tumors (untreated), tumors treated with TSC01682 and tumors treated with TSC01131 were lysed and subjected to western blotting to examine the protein levels of CUL4B, DDB1, DCAF11, DCAF13, p21, and PTEN. GAPDH was used as a loading control (B). The relative protein signals were quantified and shown in (C) and (D). *P < 0.05, **P < 0.01 and ***P < 0.001. (E) IHC staining. The bone-adjacent tissues from nude mice (WT), untreated tumors (untreated), tumors treated with TSC01682 and tumors treated with TSC01131 were subjected to IHC staining with anti-CUL4B, anti-DCAF11, and anti-PTEN antibodies, respectively. Scale bars = 100 µm. The positive signaling numbers were counted. *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: Cell culture and transfection Two osteosarcoma cell lines, Saos2 (#HTB-85) and MG63 (#CRL-1427), and one osteoblast cell line, hFOB1.19 (#CRL-11372), one colorectal carcinoma cell line HCT-116 (CCL-247), one ovarian cancer cell line SKOV3 (#HTB-77), one gastric cancer cell line MKN45 (#CRL-1739) and one normal colon cell line CCD-18Co (#CRL-1459) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: In Vivo, Injection, Western Blot, Control, Immunohistochemistry

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Monolayers of mouse endothelial cells (SVEC4-10) in transwell plates were treated with 40μg/mL EVs derived from non-infected or Mtb- infected RAW 264.7 macrophages. 4kD FITC-labeled dextran (A) or 70kD Rhodamine-labeled dextran (B) was added to top chamber and culture medium was taken from bottom chamber at various time points and the dextran concentration quantified. All the data points were generated from three independent replicates. *p<0.05 when compared to untreated endothelial cells (RC). UnEV: EVs from non-infected Raw264.7 cells, RvEV: EVs from H37Rv-infected cells. (C) Endothelial cell monolayers were stimulated with EVs derived from non-infected or Mtb -infected macrophages for 3 hours or left untreated. CFSE-labeled mouse BMMs were added to the SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged 4 hrs after their addition to the SVEC-10 cells (representative images of two independent experiments). (D) The number of BMMs in seven randomly selected fields were counted for each condition and the total number of cells calculated. Shown is the average from two experimental replicates + SD with asterisk (*) indicating a p value < 0.05 when compared to untreated endothelial cells (RC).

Article Snippet: SVEC4-10 cells were seeded on the top chamber of the Transwell plate (Costar, Corning, NY; 0.4μm pore size polycarbonate membrane filter) coated with 6–10 μg/cm 2 of type 1 collagen (Sigma) and cultured for 6 days.

Techniques: Derivative Assay, Infection, Labeling, Concentration Assay, Generated

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: (A) SVEC4-10 cell monolayers were left untreated or stimulated for 3 hrs with EVs derived from non-infected or Mtb -infected mice. CFSE-labeled mouse BMMs were added to SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged. The number of BMMs in seven randomly selected fields were counted and the total number of cells for each condition defined. The data is the average of three independent mouse Mtb infections +SD with (*) indicating a p value < 0.05 compared to RC. (B) Quantitative RT-PCR was performed on endothelial cells that were left untreated or treated for 4 hours with EVs derived from uninfected or Mtb -infected macrophages. Total RNA was extracted followed by cDNA synthesis. Fold change of gene expression was calculated by comparative Ct method. Data is from two independent mouse Mtb infections. (C) Scatter plots of flow cytometry analysis of CCL2 expression. Endothelial cells were left untreated or treated for 16 hours with EVs derived from non-infected or Mtb -infected macrophages. Permeabilized cells were stained with PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gate was set for isotype control and was maintained for all subsequent analysis. RC: untreated cells. Un-EV: serum-derived EVs from uninfected mice, D7-EV, D14-EV, D21-EV: serum derived EVs from mice infected for 7, 14 and 21 days respectively. Data is representative of the CCL2 expression from two independent experiments.

Article Snippet: SVEC4-10 cells were seeded on the top chamber of the Transwell plate (Costar, Corning, NY; 0.4μm pore size polycarbonate membrane filter) coated with 6–10 μg/cm 2 of type 1 collagen (Sigma) and cultured for 6 days.

Techniques: Derivative Assay, Infection, Labeling, Quantitative RT-PCR, Expressing, Flow Cytometry, Staining

Journal: Journal of Neuroinflammation

Article Title: Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury

doi: 10.1186/s12974-018-1282-6

Figure Lengend Snippet: NLRP3 inflammasome pathway expressed in PC12 and bEnd3 cells in the transwell co-culture system after OGD/R was induced by BV2 cells. a The expression levels of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 in BV2, PC12, and bEnd3 cells in different groups, as measured by western blot. b The changes of NLRP3, ASC, and cleaved caspase-1 in BV2 cells in the transwell co-culture with PC12 cells at different time points after reoxygenation, as measured by western blot. c The changes of NLRP3, ASC, and cleaved caspase-1 in PC12 cells in the transwell co-culture with BV2 cells at different time points after reoxygenation, as measured by western blot. d The changes of NLRP3, ASC, and cleaved caspase-1 in PC12 cells with isolated cultured between the two groups, as measured by western blot. e The changes of NLRP3, ASC, and cleaved caspase-1 in bEnd3 cells among the different groups, as measured by western blot. * p < 0.05, ** p < 0.01. OGD/R: oxygen-glucose deprivation/reoxygenation. The OGD continued for 4 h, followed by reoxygenation

Article Snippet: The transwell co-culture system was conducted as previously reported [ ]: BV2 cells were cultured on the upper compartment of a two-chamber transwell system (0.4-mm pore size of polycarbonate membrane coated with poly-L-lysine; Corning, Corning, NY, USA), and PC12 or bEnd3 cells were grown on the bottom well of the chamber, cultured in DMEM/F12 medium [ ].

Techniques: Co-Culture Assay, Expressing, Western Blot, Isolation, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury

doi: 10.1186/s12974-018-1282-6

Figure Lengend Snippet: The stimuli released from co-cultured BV2 cells originated from NLRP3 inflammasome signaling pathway in BV2 cells. a The expression levels of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 in PC12 and bEnd3 cells in the transwell co-cultures in different groups, as measured by western blot. OGD/R treatment of transwell co-culture system was conducted at 24 h after siRNA transfection in BV2 cells. b The changes of NLRP3, ASC, and cleaved caspase-1 in PC12 cells among the different groups, as measured by western blot. c The changes of NLRP3, ASC, and cleaved caspase-1 in bEnd3 cells among the different groups, as measured by western blot. d The expression levels of IL-1β and IL-18 in PC12 cells among the different groups, as measured by RT-PCR and ELISA. e The expression levels of IL-1β and IL-18 in bEnd3 cells among the different groups, as measured by RT-PCR and ELISA. * p < 0.05, ** p < 0.01. OGD/R: oxygen-glucose deprivation/reoxygenation. The OGD continued for 4 h, followed by reoxygenation

Article Snippet: The transwell co-culture system was conducted as previously reported [ ]: BV2 cells were cultured on the upper compartment of a two-chamber transwell system (0.4-mm pore size of polycarbonate membrane coated with poly-L-lysine; Corning, Corning, NY, USA), and PC12 or bEnd3 cells were grown on the bottom well of the chamber, cultured in DMEM/F12 medium [ ].

Techniques: Cell Culture, Expressing, Western Blot, Co-Culture Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury

doi: 10.1186/s12974-018-1282-6

Figure Lengend Snippet: The main source of NLRP3 inflammasomes expressed in PC12 or bEnd3 cells in transwell co-culture system was from their own productions. a The expression levels of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 in PC12 and bEnd3 cells in different groups, as measured by western blot. OGD/R treatment in isolated cultures or transwell co-cultures was conducted at 24 h after siRNA transfection in PC12 or bEnd3 cells. b The changes of NLRP3, ASC, and cleaved caspase-1 in PC12 cells among the different groups, as measured by western blot. c The changes of NLRP3, ASC, and cleaved caspase-1 in bEnd3 cells among the different groups, as measured by western blot. * p < 0.05, ** p < 0.01. OGD/R: oxygen-glucose deprivation/reoxygenation. The OGD continued for 4 h, followed by reoxygenation

Article Snippet: The transwell co-culture system was conducted as previously reported [ ]: BV2 cells were cultured on the upper compartment of a two-chamber transwell system (0.4-mm pore size of polycarbonate membrane coated with poly-L-lysine; Corning, Corning, NY, USA), and PC12 or bEnd3 cells were grown on the bottom well of the chamber, cultured in DMEM/F12 medium [ ].

Techniques: Co-Culture Assay, Expressing, Western Blot, Isolation, Transfection

Journal: Journal of Neuroinflammation

Article Title: Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury

doi: 10.1186/s12974-018-1282-6

Figure Lengend Snippet: NLRP3 knockdown in BV2 cells could inhibit the apoptosis of PC12 cells in the transwell co-cultures after OGD/R. a, b The rate of apoptotic PC12 cells among the different groups, as measured by flow cytometry. OGD/R treatment in isolated cultures or transwell co-cultures was conducted at 24 h after siRNA transfection. * p < 0.05, ** p < 0.01. OGD/R: oxygen-glucose deprivation/reoxygenation. The OGD continued for 4 h, followed by reoxygenation

Article Snippet: The transwell co-culture system was conducted as previously reported [ ]: BV2 cells were cultured on the upper compartment of a two-chamber transwell system (0.4-mm pore size of polycarbonate membrane coated with poly-L-lysine; Corning, Corning, NY, USA), and PC12 or bEnd3 cells were grown on the bottom well of the chamber, cultured in DMEM/F12 medium [ ].

Techniques: Knockdown, Flow Cytometry, Isolation, Transfection

Journal: Journal of Neuroinflammation

Article Title: Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury

doi: 10.1186/s12974-018-1282-6

Figure Lengend Snippet: Mitochondrial protector could rescue the NLRP3 inflammasome pathway expressed in PC12 and bEnd3 cells in the transwell co-cultures after OGD/R. a The expression levels of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 in PC12 and bEnd3 cells among the different groups, as measured by western blot. b The changes of NLRP3, ASC, and cleaved caspase-1 in PC12 cells among the different groups, as measured by western blot. c The changes of NLRP3, ASC, and cleaved caspase-1 in bEnd3 cells among the different groups, as measured by western blot. d The expression levels of IL-1β and IL-18 in PC12 cells among the different groups, as measured by RT-PCR and ELISA. e The expression levels of IL-1β and IL-18 in bEnd3 cells among the different groups, as measured by RT-PCR and ELISA. The 100 μm diazoxide was applied to the cells when got reoxygenation. * p < 0.05, ** p < 0.01. OGD/R: oxygen-glucose deprivation/reoxygenation. The OGD continued for 4 h, followed by reoxygenation

Article Snippet: The transwell co-culture system was conducted as previously reported [ ]: BV2 cells were cultured on the upper compartment of a two-chamber transwell system (0.4-mm pore size of polycarbonate membrane coated with poly-L-lysine; Corning, Corning, NY, USA), and PC12 or bEnd3 cells were grown on the bottom well of the chamber, cultured in DMEM/F12 medium [ ].

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay