Figure 2.
Effect of siRNA KD of RUNX1, CAV1, and FLOT1 in HEL cells on uptake and retention of albumin. (A) Effect of siRNA KD of RUNX1, CAV1, and RUNX1 + CAV1 on uptake and retention of albumin was assessed by flow cytometry. PMA-treated HEL cells were transfected with siRNA oligos (100 nM) targeting RUNX1, CAV1, or combination of both for 48 hours. They were then incubated with 30 μg/mL albumin Alexa Fluor 488 for the indicated period, fixed, washed, and albumin uptake was assessed using flow cytometry. MFI is shown (mean ± SEM of 3 experiments). Control siRNA (black line); RUNX1 siRNA (red); CAV1 siRNA (orange); RUNX1 + CAV1 siRNA (green). P values shown are for comparisons with control siRNA by Student t test. Representative flow cytometry histograms at 0 and 24 hours are shown on the right. (B) Immunoblots showing RUNX1, Cav1, and Flot1 with actin as loading control in control cells and after KD of RUNX1, CAV1, or both. The quantification is shown on the right (mean ± SEM, n = 3). P values are for comparisons by Student t test. (C) Effect of siRNA KD of RUNX1, FLOT1, or combination of both on uptake of albumin by flow cytometry. Following siRNA KD, HEL cells were incubated with 30 μg/mL of albumin Alexa 488 for up to 24 hours, fixed, and analyzed by flow cytometry. Albumin uptake is expressed as MFI (mean ± SEM, n = 3). P values shown are for comparisons with control siRNA by Student t test. Control siRNA (black), RUNX1 siRNA (red), FLOT1 siRNA (orange), and RUNX1 + FLOT1 siRNA (green). Representative flow cytometry histograms are shown on the right. (D) Immunoblots showing relative levels of clathrin, dynamin-2, and ARF6 after RUNX1 KD in HEL cells. The relative protein expression is shown below (n = 3). (E) HEL-cell albumin levels by immunoblotting after siRNA RUNX1 KD. HEL cells were incubated with RUNX1 siRNA for 24 hours in culture media containing fetal bovine serum. At 24 hours, the cells were washed with buffer and albumin levels assessed in cell lysates. Shown are a representative immunoblots with quantification of albumin and RUNX1 on the right (n = 3 experiments). (F) HEL-cell albumin levels over 24 hours in control cells and RUNX1-deficient cells. HEL cells were incubated with RUNX1 or control siRNA for 24 hours in culture media containing FBS. At 24 hours, cells were washed and suspended in buffer without FBS. Albumin levels were assessed using immunoblotting in cell lysates at intervals shown. Shown is a representative immunoblot and quantification (n = 3 experiments).

Effect of siRNA KD of RUNX1, CAV1, and FLOT1 in HEL cells on uptake and retention of albumin. (A) Effect of siRNA KD of RUNX1, CAV1, and RUNX1 + CAV1 on uptake and retention of albumin was assessed by flow cytometry. PMA-treated HEL cells were transfected with siRNA oligos (100 nM) targeting RUNX1, CAV1, or combination of both for 48 hours. They were then incubated with 30 μg/mL albumin Alexa Fluor 488 for the indicated period, fixed, washed, and albumin uptake was assessed using flow cytometry. MFI is shown (mean ± SEM of 3 experiments). Control siRNA (black line); RUNX1 siRNA (red); CAV1 siRNA (orange); RUNX1 + CAV1 siRNA (green). P values shown are for comparisons with control siRNA by Student t test. Representative flow cytometry histograms at 0 and 24 hours are shown on the right. (B) Immunoblots showing RUNX1, Cav1, and Flot1 with actin as loading control in control cells and after KD of RUNX1, CAV1, or both. The quantification is shown on the right (mean ± SEM, n = 3). P values are for comparisons by Student t test. (C) Effect of siRNA KD of RUNX1, FLOT1, or combination of both on uptake of albumin by flow cytometry. Following siRNA KD, HEL cells were incubated with 30 μg/mL of albumin Alexa 488 for up to 24 hours, fixed, and analyzed by flow cytometry. Albumin uptake is expressed as MFI (mean ± SEM, n = 3). P values shown are for comparisons with control siRNA by Student t test. Control siRNA (black), RUNX1 siRNA (red), FLOT1 siRNA (orange), and RUNX1 + FLOT1 siRNA (green). Representative flow cytometry histograms are shown on the right. (D) Immunoblots showing relative levels of clathrin, dynamin-2, and ARF6 after RUNX1 KD in HEL cells. The relative protein expression is shown below (n = 3). (E) HEL-cell albumin levels by immunoblotting after siRNA RUNX1 KD. HEL cells were incubated with RUNX1 siRNA for 24 hours in culture media containing fetal bovine serum. At 24 hours, the cells were washed with buffer and albumin levels assessed in cell lysates. Shown are a representative immunoblots with quantification of albumin and RUNX1 on the right (n = 3 experiments). (F) HEL-cell albumin levels over 24 hours in control cells and RUNX1-deficient cells. HEL cells were incubated with RUNX1 or control siRNA for 24 hours in culture media containing FBS. At 24 hours, cells were washed and suspended in buffer without FBS. Albumin levels were assessed using immunoblotting in cell lysates at intervals shown. Shown is a representative immunoblot and quantification (n = 3 experiments).

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