Journal: Immunology and Cell Biology

Article Title: Partial loss of actin nucleator actin‐related protein 2/3 activity triggers blebbing in primary T lymphocytes

doi: 10.1111/imcb.12304

Figure Lengend Snippet: Evaluation of the effects of Arp3‐knockdown (KD) on F‐actin, proliferation, cytotoxic phenotype and apoptotic status. (a) Representative immunoblot of Arp3 ( ACTR 3 ) expression levels in cells transduced with anti‐Actr3 short hairpin RNA (sh RNA) , control sh RNA and non‐transduced wild‐type ( WT ) cytotoxic effector T lymphocytes (CTLs ) lysed at day 5 post‐harvest. See also Supplementary figure a. (b) The column graph shows the densitometry analysis of the Arp3 protein band. Arp3 expression signal was normalized against WT expression level and corrected against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH ) signal ( n = 4 independent experiments, ** P = 0.0014, also see Figure a, b). (c, d) Total F‐actin content in the Arp3‐KD. (c) Flow cytometry plot demonstrating phalloidin staining of F‐actin; KD in red, WT in black, control in gray. (d) Quantification of total F‐actin in Arp3‐KD CTL s on day 7 post‐isolation, 48 h post‐fluorescence activated cell sorting ( n = 3 independent experiments, **** P < 0.0001). The relative fluorescence expressions were normalized to WT control. (e) A representative FACS plot shows transduction efficiency of splenocytes harvested from Lifeact‐ GFP × OT ‐I mice on day 5 post‐transduction ( n = 4 independent experiments). (f) FACS histograms of transduced T cells (red) and WT [non‐transduced cells (gray) acting as an internal control] stained with anti‐ CD 8α, anti‐Vα 2 , anti‐ CD 44, anti‐ CD 25, anti‐ CD 69 and anti‐ CD 62L antibodies as indicated (data from day 6 following T cell isolation; representative results of three independent experiments). (g) Growth curve of FACS ‐sorted transduced CTLs and WT control over 5 days (counting commenced 48 h post‐thawing, day 6 post‐harvest). See also Supplementary figure c, d. (h) Quantification of cell numbers compared with WT cells on day 5 post‐ FACS sort ( n = 3 independent experiments, * P = 0.03). (i) Carboxyfluorescein diacetate succinimidyl ester (CFSE) profiles of control transduced and Arp3‐KD CTLs for 3 days post‐staining on days 6–9 post‐harvest (representative of three independent experiments between days 6 and 11 post‐harvest, see also Supplementary figure b for CFSE labeled prior to cytokine stimulation of splenocytes). (j) Representative flow cytometry plots illustrating Annexin V staining in vector control, Arp3‐KD and non‐transduced internal control. (k) The mC herry expression ratio between transduced and control (non‐transduced) Annexin V + cells (data pooled from two independent experiments; days 6–8 post‐harvest). ns denotes not significant. Data are represented as mean ± s.d. ; statistical significance was calculated between control and KD cells using the unpaired Student's t ‐test. DAPI, 4,6‐diamidino‐2‐phenylindole; FSC, forward scatter; GFP, green fluorescent protein.

Article Snippet: In short, freshly isolated, unstimulated splenocytes or transduced T cells (5 × 10 4 cells) were labeled with 5 μ m of carboxyfluorescein diacetate succinimidyl ester dye (CellTrace CFSE, Thermo Fisher Scientific).

Techniques: Knockdown, Western Blot, Expressing, Transduction, shRNA, Control, Flow Cytometry, Staining, Isolation, Fluorescence, FACS, Cell Isolation, Labeling, Plasmid Preparation