HTRF Phospho-HSP27 (Ser78) Detection Kit, 500 Assay Points

Induction of phospho-HSP27 (Ser78) in A549 and Hela cells

A549 and Hela cells were plated in a 96-well plate (12,500 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with a dose-response of Anisomycin, a p38 MAPK activator, for 30 min. After culture medium removal, cells were lysed with 50 µl of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate (dilution 1/2 and 1/8 for Phospho-HSP27 Ser78, dilution 1/4 and 1/8 for Total HSP27 and neat and 1/2 for Phospho-p38 MAPK Thr180/Tyr182 for A549 and Hela samples respectively) were transferred into a HTRF 384-well low volume white microplate then 4 µL of the HTRF Phospho-HSP27 (Ser78) or HTRF Total HSP27 (Revvity, #64HSPTPEG/PEH/PEY) or HTRF Phospho-p38 MAPK Thr180/Tyr182 (Revvity, #64P38PET/PEG/PEH) detection reagents were added. HTRF signal for three kits was recorded using Envision Nexus reader after a 4h-incubation at room temperature. In parallel, cell viability was assessed. To do this, 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added (Revvity #6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at room temperature.

As expected, Anisomycin, a p38 MAPK activator, induced a dose-dependent increase in p38 MAPK phosphorylation on Threonine180/Tyrosine 182 and HSP27 phosphorylation on Serine 78, without any significant effect on the expression level of the total HSP27 protein.  In addition, Anisomycin did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

Inhibition of phospho-HSP27 (Ser78) in A549 cells

A549 cells were plated in a 96-well plate (12,500 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with a dose response of PF-3644022, a MAPKAPK2 inhibitor, for 2 h then co-treated with 500 nM Anisomycin, a p38 MAPK activator, for 30 min. After culture medium removal, cells were lysed with 50 µl of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate (dilution 1/2 for Phospho-HSP27 Ser78 and dilution 1/4 for Total HSP27) were transferred into a HTRF 384-well low volume white microplate then 4 µL of the HTRF Phospho-HSP27 (Ser78) or HTRF Total HSP27 (Revvity, #64HSPTPEG/PEH/PEY) detection reagents were added. HTRF signal for both kits was recorded after a 4h-incubation at room temperature. In parallel, cell viability was assessed. To do this, 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added (Revvity, #6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at room temperature.

As expected, PF-3644022, a MAPKAPK2 inhibitor, repressed HSP27 phosphorylation on Serine 78 induced by Anisomycin, a p38 MAPK activator, leading to a dose-dependent decrease of Phospho-HSP27 (Ser78) signal, without any significant effect on the expression level of the total HSP27 protein. In addition, these co-treatment did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

Specificity of phospho-HSP27 (Ser78) assay using HAP1 KO cells

The human phospho-HSP27 Serine 78 expression level was assessed using the HTRF Phospho-HSP27 (Ser78) detection kit in HAP1 Wild Type (WT) cells (Revvity, #C631) and in two HAP1 knock-out (KO) HSP27 cell lines (Revvity, #HZGHC004943c012 and HZGHC004943c001).

HAP1 WT and KO cells were plated in a 96-well plate (20,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with 1 µM Anisomycin, a p38 MAPK activator, for 30 min. After culture medium removal, cells were lysed with 50 µl of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate were transferred into a HTRF 384-well low volume white microplate then 4 µL of the HTRF Phospho-HSP27 (Ser78) detection reagents were added. HTRF signal was recorded using HTRF reader after an overnight-incubation at room temperature.

As depicted in figure, HTRF signal was exclusively detected in the HAP1 WT cell line, with basal signal levels increasing following Anisomycin treatment. Conversely, near-background signals were observed in both HAP1 KO HSP27 cell lines, demonstrating the specificity of the HTRF Phospho-HSP27 (Ser78) assay for the detection of the HSP27 protein.

Assessment of phospho-HSP27 (Ser78) level in various human cell lines

A549, Hela and MCF7 cells were plated in a 96-well plate (12,500 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with 1 µM Anisomycin, a p38 MAPK activator, for 30 min. After culture medium removal, cells were lysed with 50 µl of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate (dilution 1/2, 1/8 and 1/32 for A549, Hela and MCF7 samples respectively) were transferred into a HTRF 384-well low volume white microplate then 4 µL of the HTRF Phospho-HSP27 (Ser78) detection reagents were added. HTRF signal was recorded using Envision Nexus reader after a 4h-incubation at room temperature.

Treatment with Anisomycin induced an increase of HTRF Phospho-HSP27 (Ser78) signal in each tested cell lines.

HTRF Phospho HSP27 (Ser78) assay compared to Western Blot

A549 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then, cells were treated with 1 µM Anisomycin, a p38 MAPK activator, for 30 min. After culture medium removal, cells were lysed with supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking.  Equal amounts of cell lysates (16 µL) were used for a side-by-side comparison of Western Blot and HTRF techniques.

 

Using the HTRF Phospho-HSP27 (Ser78) assay, 120 cells/well were enough to detect a significant signal, while 3,750 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Phospho-HSP27 (Ser78) assay is 32 times more sensitive than Western Blot technique.

HSP27 signaling pathway

HSP27, also known as HSPB1, is an ATP-independent chaperone. It plays a role in cell resistance to stress factors such as heat shock, hypoxia, and oxidative stress. However, during disease, HSP27’s function is hijacked, leading to its overexpression in cancer cells. This increased expression or HSP27 phosphorylation (triggered by MK-2 and MK-3, p70S6K, PKG, and PKC) contributes to tumor progression and treatment resistance, making HSP27 an interesting therapeutic target.