Journal: Nature Cell Biology

Article Title: Aggresome assembly at the centrosome is driven by CP110–CEP97–CEP290 and centriolar satellites

doi: 10.1038/s41556-022-00869-0

Figure Lengend Snippet: ( a ) RPE-1 cells were transiently transfected with GFP-HTT97Q and stained for pHSP27 or Ub + . DNA was stained with DAPI. Scale bar 10 μm. ( b ) Immunoblot of soluble and insoluble fractions from WT and PCM1 KO cells transfected with GFP-HTT25Q. GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( c ) Cells were transfected with GFP-HTT97Q for 5 hours, then treated with DMSO or nocodazole for 5 hours before being fixed and stained for GFP and α-tubulin. Scale bar 10 μm. ( d ) Immunoblot of soluble and insoluble fractions from cells prepared as in c . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( e ) WT and STIL KO U-2 OS cells were transiently transfected with GFP-HTT97Q then stained for GFP, CEP135 and α-tubulin. Scale bar 10 μm. ( f ) Immunoblot of soluble and insoluble fractions from cells prepared as in e . GAPDH and Histone H3 were used controls for the soluble and insoluble fractions, respectively. ( g ) HEK293T-FLAG-miniTurbo-CP110 cells were treated and stained as indicated. Scale bar 10 μm, 2 μm. ( h ) Immunoblot of HEK293T-FLAG-miniTurbo-CP110 cells treated and probed as indicated. α-tubulin was used as a loading control. ( i ) The number of preys from DMSO- or MG132-treated groups. Preys were defined as detailed in the Methods. ( j ) Functional enrichment analysis was performed with the preys from i . using g:Profiler and the KEGG database. ( k ) Spectral counts from the genes which are implicated in Parkinson’s disease, proteasome function or Huntington’s disease are shown. Genes which are related to the respective pathways are marked in green. Unprocessed immunoblots are provided as source data. For i , j , k : the raw mass spectrometry data and analysis giving rise to these panels is available in Supplementary Table .

Article Snippet: To produce lentivirus, 4 × 10 6 HEK293T (female, human kidney; ATCC, ACS-4500) cells were seeded in a T-75 flask and transfected with the relevant lentiviral transfer vector, 3 μg psPAX2 and 2 μg pCMV-VSV-G using Lipofectamine 3000 (Invitrogen).

Techniques: Transfection, Staining, Western Blot, Control, Functional Assay, Mass Spectrometry

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: A–D Representative whole‐cell currents in HEK 293 cells expressing TRPV1 (A), ASIC3 (B), P2X2 (C), or P2X4 (D) in response to capsaicin (A), acidic pH (B), or ATP (C, D) in the absence of presence of borneol ( n = 5 for each channel). E The effect of different concentrations of borneol on the enzymatic activity of human COX‐2. The number of independent measurements is marked on top of each bar. F Averaged intracellular Ca 2+ increases in cultured mouse DRG neurons in response to consecutive applications of 200 μM borneol, 200 μM menthol, and 67 mM KCl. A total of 81 in 1,689 neurons from four mice were found to be borneol‐sensitive and were included in the analysis. G Representative intracellular Ca 2+ signals in HEK 293 cells expressing human TRPM8 (hTRPM8) in response to different concentrations of borneol. After each application of borneol, Ca 2+ ionophore ionomycin was applied to calibrate Ca 2+ response. RFU: relative fluorescence unit. H Dose–response curves of the borneol‐ or menthol‐induced increase in intracellular Ca 2+ in hTRPM8‐expressing HEK 293 cells. Smooth curves are fit to the Hill equation with an EC 50 of 65 μM and a Hill coefficient of 2.0 for borneol ( n = 15) and an EC 50 of 13 μM and a Hill coefficient of 2.0 for menthol ( n = 6 at concentrations of 0.1, 0.3, and 1 μM; n = 9 at concentration of 3 μM; n = 13 at concentrations of 10, 30, 100, and 300 μM). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. I Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in hTRPM8‐expressing HEK 293 cells ( n = 6). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing, Activity Assay, Cell Culture, Fluorescence, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Representative intracellular Ca 2+ signals in HEK 293 cells transfected with empty vector in response to borneol and subsequent applied Ca 2+ ionophore ionomycin ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing hTRPM8 in response to different concentrations of menthol and the subsequently applied Ca 2+ ionophore ionomycin.

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: EMBO Molecular Medicine

Article Title: A clinical and mechanistic study of topical borneol‐induced analgesia

doi: 10.15252/emmm.201607300

Figure Lengend Snippet: Quantification of consecutively applied 100 μM menthol‐ and 600 μM borneol‐induced hTRPM8 currents. Currents were normalized to 100 μM menthol‐induced currents at +80 mV ( n = 6). Representative intracellular Ca 2+ signals in HEK 293 cells expressing mouse TRPM8 in response to different concentrations of borneol. Dose–response curves of borneol‐induced increase in intracellular Ca 2+ in mouse TRPM8‐expressing HEK 293 cells. The smooth curve is a fit to the Hill equation with an EC 50 of 116 μM ( n = 12). The data were normalized to ionomycin‐induced intracellular Ca 2+ increases. Time course of menthol‐ and subsequently applied borneol‐induced whole‐cell currents in mouse TRPM8‐expressing HEK 293 cells ( n = 5). Data information: All the data are presented as the mean ± standard error of the mean (SEM).

Article Snippet: Intracellular calcium imaging of HEK 293 cells was performed using the FlexStation 3 microplate reader (Molecular Devices).

Techniques: Expressing