blockit alexa fluor red fluorescent oligo  (Thermo Fisher)

Journal: Nature Communications

Article Title: KCNJ15 /Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis

doi: 10.1038/ncomms9532

Figure Lengend Snippet: ( a , b ) Efficient knockdown of KCNJ15 shown with Western blotting, red arrow pointing to a non-specific band. Kir4.2/GAPDH ratio is used to quantify the protein level. n =3. ( c , d ) Migration trajectories and quantification of directional migration (directedness values (cos θ )) demonstrated that KCNJ15 knockdown abolished galvanotaxis and cells completely lost migration direction in an EF. Black and red lines indicate trajectories of cells migrated toward cathode and anode side, respectively. n =100 cells for each group, confirmed in two other replicates. ( e ) KCNJ15 knockdown did not affect cell migration speed whether in an EF or not (compare the trajectories in c). There are no statistically significance between each group. n =100 cells for each group, confirmed in two other replicates. ( f , g ) Directional cell migration in scratch assay were identical between KCNJ15 knockdown and scrambled RNAi treatment. Wound closure is represented as % of open area. When the error bars are not seen, the bars are smaller than the symbols. Wound was made using a pipette tip. There are no statistically significance between two groups. n =3. Scale bar in f , 200 μm. ( h ) KCNJ15 knockdown abolished cathodal distribution of Akt-PH-EGFP, a reporter for PIP 3 localization. hTCEpi cells were transfected with siRNA and pcDNA3-Akt-PH-EGFP plasmid DNA. Fluorescence of Akt-PH-EGFP was recorded by fluorescence microscope. Arrow indicates PIP 3 accumulation in cathode-facing side of control cells. Scale bar, 50 μm. Cells were transfected with siRNA against KCNJ15 or control oligo, and incubated for 48 h. EF=200 mV mm −1 . Statistical analyses were performed by Student's t -test. Data represented as mean±s.e.m. * P <0.05; ** P <0.01. NS, not significant.

Article Snippet: EpiLife culture medium with Ca 2+ (60 μM), EpiLife defined growth supplement, DMEM, foetal bovine serum (FBS), non-essential amino-acids solution ( × 100), penicillin/streptomycin, BlockiT red fluorescent oligo and Lipofectamine 2000 reagent were purchased from Life Technologies Inc. (Carlsbad, CA, USA).

Techniques: Knockdown, Western Blot, Migration, Wound Healing Assay, Transferring, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Control, Incubation

Journal: Nature Communications

Article Title: KCNJ15 /Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis

doi: 10.1038/ncomms9532

Figure Lengend Snippet: ( a ) hTCEpi cells migrate toward cathode, and HaCaT (human keratinocyte cells) and MDA-MB-231 (human breast cancer cells) cells migrate toward anode. Knockdown of KCNJ15 abolished directional migration in all three types of cells. At 48 h after transfection, cells were seeded onto galvanotaxis chamber. Positive directedness values indicate cathodal migration, whereas negative directedness value indicates anodal migration. ( b ) KCNJ15 knockdown did not have significant effects on migration speed. hTCEpi cells, HaCaT and MDA-MB-231 were transfected with siRNA against KCNJ15 or control oligo. Cell numbers analysed, 100 hTCEpi cells, 60 HaCaT cells and 80 MDA-MB-231 cells. Results confirmed in two separate experiments. Statistical analysis was performed by the Student's t -test. Data represented as mean±s.e.m. * P <0.05. ** P <0.01. EF=200 mV mm −1 . NS, no significance.

Article Snippet: EpiLife culture medium with Ca 2+ (60 μM), EpiLife defined growth supplement, DMEM, foetal bovine serum (FBS), non-essential amino-acids solution ( × 100), penicillin/streptomycin, BlockiT red fluorescent oligo and Lipofectamine 2000 reagent were purchased from Life Technologies Inc. (Carlsbad, CA, USA).

Techniques: Knockdown, Migration, Transfection, Control

Journal: Nature Communications

Article Title: KCNJ15 /Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis

doi: 10.1038/ncomms9532

Figure Lengend Snippet: ( a , b ) Intracellular polyamines are required for cells to sense extracellular EFs. Depletion of polyamines with DENSPM abolished galvanotaxis. Migration trajectories of control cells and DENSPM treated cells with or without EF. Red lines indicate trajectories of cells that migrated toward anode side. hTCEpi cells were treated with 25 μM DENSPM for 2 days. DENSPM activates SPM/SPD catabolizing enzyme SAT/SSAT which catabolizes SPM/SPD to N 1 -acetyl SPM/SPD and reduces intracellular polyamine. ( c ) Depletion of polyamines affected cell migration speed. ( d , e ) Increased intracellular polyamines significantly enhanced galvanotaxis of U251 cells ( d ) or HaCaT cells ( e ). Knockdown of KCNJ15 cancelled PUT-enhanced galvanotaxis. Cells were transfected with control oligo or KCNJ15 siRNA, and treated with or without PUT (100 μM) for 2 days. ( f ) Lentivirus-mediated expression of WT and E157N Kir4.2 proteins. hTCEpi cells transduced with lentivirus. The Expression of WT and polyamine-binding defective mutant (E157N) Kir4.2 proteins was confirmed by western blotting. ( g , h ) Expression of polyamine-binding defective mutant of KCNJ15 (E157N) significantly decreased directedness but had little effect on cell motility. hTCEpi cells were infected with recombinant lentivirus and incubated for 2 days. Directedness and speed were evaluated. ( i , j ) An applied EF-induced asymmetry of intracellular polyamines. Representative image of the polyamine staining ( i ). Scale bar in i , 50 μm. Intensities of polyamines staining in cathode-facing side were divided by those in anode-facing side (right side divided by left side in no EF cells) ( j ), Polyamine distribution in response to EF. hTCEpi cells were subjected to EF (200 mV mm −1 ) for 0, 10, 30 or 60 min. Intracellular polyamines were stained with anti-polyamine antibody. Arrows in i indicate polyamine accumulation in cathode-facing side of hTCEpi cells. Statistics: b and c : n =100 cells for each group, confirmed in three independent experiments. d : n =50, e : n =120, g and h : n =120, j : n =16–23. All confirmed in two to three separate experiments. EF=200 mV mm −1 . Statistical analyses were performed by Student's t -test ( b – e , g , h ), or analysis of variance followed by Student's t -test ( j ). Data represented as mean±s.e.m. * P <0.05. ** P <0.01. NS, no significance.

Article Snippet: EpiLife culture medium with Ca 2+ (60 μM), EpiLife defined growth supplement, DMEM, foetal bovine serum (FBS), non-essential amino-acids solution ( × 100), penicillin/streptomycin, BlockiT red fluorescent oligo and Lipofectamine 2000 reagent were purchased from Life Technologies Inc. (Carlsbad, CA, USA).

Techniques: Migration, Control, Knockdown, Transfection, Expressing, Transduction, Binding Assay, Mutagenesis, Western Blot, Infection, Recombinant, Incubation, Staining