Sirtuin assay of purified Hos2 preparations using resveratrol: UPDATE 1

<p>Fluorimetric Sirtuin1 like deacetylase assay with rHos2 protein using Fluor de Lys®−Sirt1 substrate in presence of Sirtuin1 activator Reserveratrol. This experiment is carried out in presence of HDAC inhibitor Trichostatin to inhibit any residual Histone deacetylase activity. Updated from: http://dx.doi.org/10.6084/m9.figshare.841658.</p

Deacetylase activities of rHos2 and dose response curves of rHos2 inhibition by standard HDAC inhibitors: UPDATE 1

<p>Dose response curve of rHos2 enzyme inhibition by MS275: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of MS-275.</p> <p>Dose response curve of rHos2 enzyme inhibition by SAHA: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of suberoylanilide hydroxamic acid (SAHA).</p> <p>Dose response curve of rHos2 enzyme inhibition by TSA: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of trichostatin.</p> <p>rHos2 conc_Enzyme activity: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate. Updated from: http://dx.doi.org/10.6084/m9.figshare.841655</p

Tubulin deacetylation and rHos2 protein blots

<p>Western blot analysis of rHos2 protein polyclonal anti-sera: Western blot analysis of rHos2 protein using polyclonal antisera raised from mice.</p> <p>Tubulin Deacetylation Blot data_F1000: Western blot analysis for tubulin Deacetyation by rHos2 protein.</p

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

A plate map for the MIC in a 384-well plate.

<p>A total of 30 test compounds/plate (2 compounds/row) could be tested as a 10-concentration dose response (10c-DR as A2–11 to P2–11 and A14–23 to N14–23). <b>Quality controls (QC)</b>: Columns 1 and 13 = media controls (MC), columns 12 and 24 = culture controls (CC). The 31^st and 32^nd rows = reference drug controls (e.g., isoniazid in rows O&P14 to O&P23) and rows MN14 to MN23 were used for additional reference drug QC, if required. Culture addition was performed with the Multidrop-Combi liquid handling system, and incubation was performed at 37°C for 3–4 days (Msm) and 3–4 weeks (Mtb). Post-incubation, the MIC data were recorded using a spectrophotometer (Spectramax^384Plus) at O.D._600nm.</p

MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.

<p>MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.</p

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

Optimisation of assay conditions.

<p>Initial assay optimization was done in Msm: <b>a. Minimum culturable volume in Msm</b>: Msm culture was dispensed in 1.56-μl aliquots (1.56μlX64) multiple times, up to 50μl twice (50μlX2), on 24-well agar beds and conventional plates. The sum of the cfu obtained for each volume was plotted. The parallel lines of the cfu data plotted against the plating volumes confirmed that there was no variation in the net bacterial counts obtained from plating different volumes. Hence, the 25-μl volume was selected. <b>b. Drug carry-over assay in Msm</b>: Untreated and isoniazid-treated samples yielded a similar number of cfus. Isoniazid exposure yielded similar bacterial counts from both the conventional and 24-well spot-assay over a range of concentrations up to 16 μg/ml.</p

Spot-assay correlation in Mtb and the Manhattan analysis.

<p>The spot-assay was replicated in Mtb for RIOE. <b>a. An MBC correlation between the spot-assay and the conventional method</b>: A strong positive correlation (r² = 0.808). <b>b & c: The Manhattan analysis</b> of isoniazid-treated reference controls exhibited variation within the 2-fold in the spot-assay (<b>b</b>) and conventional MBC methods (<b>c</b>).</p

The schematics of spotting from an MIC plate to a 24-well plate.

<p><b>a</b>. Culture aspiration from a MIC plate using filter-tips fixed at alternate positions of a 12-channel Pipetman. <b>b</b>. <b>Twenty-four-well plate</b>: Row-wise dispensing into a 24-well plate. Two compounds/row, with a 10c-DR each: e.g., Row A: Compound#1 = 2–11 wells and Compound#2 = 14–23 wells (1 & 13 = media control, 12 & 24 = culture control). One plate of 24 wells = data for 2 compounds (10c-DR) for 1 row of MIC plate: The left half of the 24-well plate contains the 1^st compound (#2 to 11), and the right half contains the 2^nd compound (#14 to 23). <b>c. A picture of a spot-assay in a 24-well plate</b>: colony morphology and countable colonies (e.g., rifampicin on the left half and isoniazid on the right half of the plate).</p