Journal: bioRxiv

Article Title: Endosomal Chemokine Receptor Signalosomes Regulate Central Mechanisms Underlying Cell Migration

doi: 10.1101/2022.09.27.509755

Figure Lengend Snippet: Endosomal G i/o activation by internalized CCR7. (A) Confocal microscopy imaging displaying HEK293-CCR7 cells transiently expressing the early endosomal marker Rab5a-RFP. The cells were treated with either 100 nM CCL19-Alexa488, 100 nM CCL21-Alexa488, or Ni-NTA-Alexa488 for 15 min followed by extensive washout to remove extracellular chemokines. The cells were imaged 15-30 min after this washout step. (B) Schematic representation of the EbBRET-based assay to monitor proximity between RlucII-miniGi and the endosomal marker rGFP-Rab5 upon CCR7 activation from endosomes. (C) EbBRET measurements from CCR7-expressing HEK293 cells co-transfected with RlucII-miniGi and rGFP-Rab5 upon stimulation with 100 nM CCL19, 100 nM CCL21, or vehicle control. Data represents the mean ±SE from N=3 independent experiments. One-way ANOVA with Turkey’s multiple comparison post hoc test was applied to determine statistical differences between the measurements (** p < 0.01; *** p < 0.001). (D) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells cotransfected with the plasma membrane marker RFP-Lck and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 10 min. (E) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the endosomal marker RFP-EEA1 and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 30 min.

Article Snippet: RFP-Lck (C-tRFP-Lck cloned into PCMV6-AC-RFP expression vector) and RFP-EEA1 (TagRFP-T-EEA1 cloned into pEGFP-C1 vector) were purchased from Addgene (respectively #RC100049 and #42635).

Techniques: Activation Assay, Confocal Microscopy, Imaging, Expressing, Marker, Transfection