2.1 Cell culture and antibodies
Human epithelial colorectal cells (HT29) were purchased from National Centre for Cell Science (NCCS) Pune India, High glucose DMEM and McCoy’s 5A medium, Fetal bovine serum, 100x anti-anti solution was purchased from Gibco (USA), TPVG (1x) was purchased from Himedia labs Mumbai, India, ERK1/2, Beta-Actin(sc-47778) were purchased from SantaCruz biotech (USA), p38α (#9218), Heat Shock proteins was procured from Cell Signalling technology (USA). Alexa Fluor 488 was purchased from Jackson Immuno Research Laboratories (USA), Monarch® Genomic DNA Kit (T3010S) was purchased from BioLabs, New England. TUNEL assay kit (ECKA334) and p-ERK1/2 (E-AB-70292) and JNK (E-AB-60070) were purchased from Elabscience (USA). PMSF (Sigma-329986). FemtoLeucent (786003) enhanced chemiluminiscence kit was purchased from G-Biosciences. ProLong™ Gold Mounting medium with DAPI was purchased from Fisher scientific (USA). Caffeic Acid Phenethyl Ester (FC19630) was purchased from BioSynth CarboSynth (UK) and TCI chemicals (Japan), DCFH (4091-99-0) was purchased from Cayman Chemicals company MI (USA).
2.2 Serum starvation
The serum starvation protocol was done with slight changes in previously executed method of Nair . HT29 cells were grown in 6 well plates or 90mm sterile culture dishes, on complete first cell division in 21 hours’ media (10% FBS + DMEM) was replaced with 3% media until the end of treatment period.
2.3. Cell viability assay
Cell viability assessment was performed by widely used colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay . Briefly 5×103 cells were seeded evenly into flat bottom 96 well plate. At 70–80% confluence, cells were treated with variable concentration (10-100µM) of CAPE for 24 and 48hrs ± serum supplementation. At the end of the treatment period, drug laden media was aspirated followed by 90µl of serum free media + 10µl of 5mg/ml of MTT reagent. Incubated for 3–4 hours in dark at 37⁰c, with visible formazan crystals, media with MTT reagent was removed, followed by 100µl DMSO to each well, placed on orbital shaker for 3–5 minutes and reading was taken at 540nm with BioRad iMark™ Elisa plate reader.
2.4 Morphological studies
HT29 Cells were seeded on 22mm round coverslip in 6-well plate, after treatment with CAPE, media was removed and washed twice with fresh 1x PBS, morphological studies were recorded with Motic inverted microscope AE-31 with 20x magnification.
2.5 Cell Migration Assay
Cell migration was performed as previously done by Li . Cells were seeded in 6 well plate left until it reached 80–90% confluence, followed by an artificial wound or scratch across the monolayer of the well in plus (+) contour. Images of control and CAPE treated cells were taken on 0 hour, 24 hours and 48-hour interval with Motic AE31 microscope and scratch dimensions were analysed by ImageJ software.
2.6 Clonogenic assay
This assay was performed with slight modifications as previously described [40, 41]. Briefly, around 100–200 cells were seeded into a 12 well plate left to grow for 7 days in growth medium, media was changed every 48hrs. The cells were treated with CAPE and post incubation time, the cells were washed with 1x PBS twice then fixed with ice cold methanol left at RT for 10–15 minutes, stained with 0.5% crystal violet solution, followed by multiple washes with distilled water until excessive dye washed off. The plates were then left to air dry. Colonies were analysed macro and microscopically, colonies were counted by particle extension in ImageJ software.
2.7 Assessment of intracellular reactive oxygen species
Determination of ROS was done by method previously described by Kim . Briefly, HT29 cells with or without serum were seeded in coverslips in a 6 well plate and treated with CAPE for definite time points. A working concentration of 10mM of DCFH in DMSO was prepared fresh before use and was added into each well and incubated at 37°C for 30 min in dark. thereafter washed with PBS. The cells were imaged in a fluorescence microscope broad range FITC filter with excitation wavelength of 485 nm and an emission wavelength of 530 nm.
2.8 Analysis of Cell cycle by FACS
Cell cycle analysis was carried out by method of Yao  with slight variation in fixation procedure. Cells were seeded into a 6-well plate and treated with CAPE, washed once with PBS, centrifuged at 2000 rpm and discarded the supernatant and fixed cells by dropwise ice cold absolute ethanol, stored at -80⁰C for overnight for optimal fixation. Subsequently ethanol was replaced with 1x PBS, 0.15 TritonX 100, and 10mg/ml RNase-A, cocktail and 10µl of 1mg/ml stock of Propidium iodide was added to each tube incubated for 10-15mins in dark. FACS data was acquired by Beckman Coulter CytoFlex and analysed by CytExpert software.
2.9 DNA fragmentation assay
HT29 cells grown in 60mm culture plates followed by treatment with CAPE for 24 and 48 hours. Genomic DNA extraction was done according to manufacturer’s protocol. Briefly 3×106 cells were collected by centrifugation at 1000×1g for 1 minute, followed by 1x Proteinase K, then 100µl of DNA lysis buffer was added to each tube, Incubated at 56ºC for 5 minutes, transferred to column inserted tubes, centrifuged at 1000×1g for 1 minute for optimal binding of genomic DNA, then centrifuged at 14000 rpm for 10 minutes, discarded the elute, and transferred column to fresh tube, centrifuged at 14000 rpm then transferred to DNAse free tubes with pre warmed DNA elution buffer, after final centrifugation at 14000 rpm gDNA was collected and quantified by A260/A280 by spectrophotometer and then ran with a 1.5% agarose gel electrophoresis for 1 hour.
2.9 Tunel Assay
Tunel assay was performed according to manufactures instructions. Briefly, HT29 cells were grown on coverslips in 6 well plates, after treatment period cells were washed twice with 1x PBS for 5 minutes, followed by fixation with 4% neutral buffered formalin for 30 minutes, washed with PBS 3x, 100ul of 1x Proteinase-K was added to all control and treated samples, Washed cells with PBS 3x for 5 minutes each, added Tunel buffer and TdT enzyme mixture, Finally added 50µl of enhanced FITC-fluorescein mix and mounted on new slides with ProLong™ Gold mounting medium. The image acquisition was done using Zeiss confocal microscope with FITC and DAPI filter.
Immunofluorescence was done with modification as per the method of Çağatay  Cells were seeded in 6 well plates on round cover slips, after experimental period, media was aspirated replaced with 2 x washes with PBS, fixed with ice cold methanol or 4% neutral buffered formalin and stored at -20⁰ for future use. The fixed cells were blocked with 5% bovine serum albumin dissolved in PBST, followed by incubation with P38α antibody 1:300 overnight at 4°C, then slides were washed with PBST 3x for 5 minutes each, later incubated for 1 hour with Alexa Fluor® 488 conjugated secondary antibody for one hour, washed with PBST and mounted after evaporating extra moisture with ProLong Gold Mounting medium enhanced with DAPI. The images were captured using Nikon fluorescence microscope and merged using ImageJ software overlay extension.
2.11 Immunoblot analysis
Control and treated HT29 cells were harvested either by cell scrapper or trypsinization at end of the treatment period, followed by centrifugation at 1500rpm for 5 minutes, the pellet was washed 3 times with 1X PBS, supernatant was discarded and 500µl of RIPA lysis buffer fortified with 1mM PMSF protease inhibitor. Subsequently, cell samples were further centrifuged at 12000rpm at 4°C for 30 minutes. Supernatant was collected in a fresh tube and protein was quantified using Bradford’s reagent with biophotometer (Eppendorf). 30–40µg of control and treated protein samples were run in the SDS-PAGE in BioRad Mini-Protean®, apparatus which was further transferred to Amersham Protran nitrocellulose membrane (Merck- GE10600002). Further the membranes were washed with PBST (1x PBS + 0.1% Tween 20) V/v and blocked with BLOT-QuickBlocker™ (G-Biosciences 786 − 011) for one hour at room temperature, followed by 4-12Hr incubation at 4°C with primary antibodies, then 4x wash with PBST once completed secondary antibody incubation for one hour, finally 3x wash with PBST, the blots were developed using femtoleucent ECL solution in dark and scanned using CanoScan LiDE-400.
Cells were lysed for 30 min on ice in IP lysis buffer (10 mm Tris-HCl, pH 8, 150 mm NaCl, 5 mm EDTA, pH 8, 0.5% (v/v) Triton X-100,) and 10ul RNAse A 10mg/ml. cell lysates of control and treated HT29 cells poured into the fresh tubes along with Protein A/G PLUS-Agarose (SantaCruz Biotechnology SC-2003) and 1mg/ml per tube of p-38α MAPK antibody in centrifuge tube placed on axis rotator overnight at 4°C, subsequently unbound fractions were removed by a washing with IP lysis buffer fortified with sodium ortho-vanadate followed by centrifugation at 12000 rpm for 5mins the bound factions were collect from supernatant and unbound factions in pellet were discarded, Further the samples were used to run SDS-PAGE and western blotting.
2.13 Bioinformatics studies
The structural activity relationship and molecular docking study was carried out using Glide/Schrödinger software between CAPE and 3D structures of HSP90 and p23 (PDB ID. 7RY1, IEJ) compared docking score and binding energy with widely known HSP inhibitor Geldanamycin.
The statistical analysis was done using GraphPad Prism v 9, software. One-way ANOVA followed by students t-test whenever applicable Tukey’s was used. P values less than 0.05 were considered as statistically significant. Data are represented as mean ± SD. and mean ± CV unless stated otherwise