Figure 4.
Effect of siRNA KD of RUNX1, CAV1, or both on albumin colocalization with Cav1, Flot1, RAB11, and LAMP2 in HEL cells by immunofluorescence microscopy. (A) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin colocalization with Cav1 and Flot1. Control cells and those with siRNA KD of RUNX1, CAV1, or the combination were incubated with 30 μg/mL albumin Alexa 488 for 30 and 120 minutes, fixed and immobilized on poly-L-lysine-coated coverslips. Albumin is shown in green fluorescence. HEL cells were additionally stained with anti-Cav1 (red) or anti- Flot1 (blue) antibodies to assess colocalization as seen in merged images, evaluated by Epifluorescence microscope (EVOS FL Autoimaging). Because HEL cells had minimal endogenous Cav1, it was enhanced to assess colocalization with albumin. At 30 minutes, in control cells albumin (green) was colocalized with Cav1 (red), as shown in yellow in merged images, but not with Flot1 (blue). With RUNX1 KD, albumin colocalization with Cav1 was decreased with increased colocalization with Flot1. With CAV1 KD alone no albumin was discernible. At 120 minutes, with RUNX1 KD there was a decrease in albumin colocalization with Cav1 and an increase with Flot1. (B) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin (green) colocalization with RAB11 (red), a marker for recycling endosomes. At 30 or 120 minutes, there was low colocalization of albumin (green) with RAB11 (red) in control cells or on RUNX1 KD. On CAV1 KD alone or together with RUNX1 KD there was negligible albumin in the cells. Representative immunoblots showing RAB11, RUNX1, and actin levels and densitometric quantification are shown. Data are shown as mean ± SEM (n = 4). (C) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin (green) colocalization with lysosomal marker LAMP2 (red). At 30 minutes, there was some colocalization of albumin with LAMP2 in control cells and on RUNX1 KD. At 120 min the colocalization of albumin with LAMP2 appeared more prominent with RUNX1 KD than in control cells. Representative immunoblot showing protein expression of LAMP2, RUNX1, and actin, and densitometric quantification. Shown as mean ± SEM (n = 3).

Effect of siRNA KD of RUNX1, CAV1, or both on albumin colocalization with Cav1, Flot1, RAB11, and LAMP2 in HEL cells by immunofluorescence microscopy. (A) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin colocalization with Cav1 and Flot1. Control cells and those with siRNA KD of RUNX1, CAV1, or the combination were incubated with 30 μg/mL albumin Alexa 488 for 30 and 120 minutes, fixed and immobilized on poly-L-lysine-coated coverslips. Albumin is shown in green fluorescence. HEL cells were additionally stained with anti-Cav1 (red) or anti- Flot1 (blue) antibodies to assess colocalization as seen in merged images, evaluated by Epifluorescence microscope (EVOS FL Autoimaging). Because HEL cells had minimal endogenous Cav1, it was enhanced to assess colocalization with albumin. At 30 minutes, in control cells albumin (green) was colocalized with Cav1 (red), as shown in yellow in merged images, but not with Flot1 (blue). With RUNX1 KD, albumin colocalization with Cav1 was decreased with increased colocalization with Flot1. With CAV1 KD alone no albumin was discernible. At 120 minutes, with RUNX1 KD there was a decrease in albumin colocalization with Cav1 and an increase with Flot1. (B) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin (green) colocalization with RAB11 (red), a marker for recycling endosomes. At 30 or 120 minutes, there was low colocalization of albumin (green) with RAB11 (red) in control cells or on RUNX1 KD. On CAV1 KD alone or together with RUNX1 KD there was negligible albumin in the cells. Representative immunoblots showing RAB11, RUNX1, and actin levels and densitometric quantification are shown. Data are shown as mean ± SEM (n = 4). (C) Representative images showing the effect of KD of RUNX1 or CAV1 alone or in combination on albumin (green) colocalization with lysosomal marker LAMP2 (red). At 30 minutes, there was some colocalization of albumin with LAMP2 in control cells and on RUNX1 KD. At 120 min the colocalization of albumin with LAMP2 appeared more prominent with RUNX1 KD than in control cells. Representative immunoblot showing protein expression of LAMP2, RUNX1, and actin, and densitometric quantification. Shown as mean ± SEM (n = 3).

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