Tubastatin

Human embryonic kidney cells, HEK293T, human cervical carcinoma cells, HeLa, human breast cancer cells, MDA-MB 231, human lung adenocarcinoma cellsA549, chronic myeloid leukemia cells, K562, and human colon cancer cells, HCT11, were obtained from National Centre for Cell Sciences (NCCS), Pune, India. Cell line authentication by STR analysis was performed for 10 genetic loci for all the cell lines by Life code, Genomic technologies Pvt. Ltd., and tested for mycoplasma negative by performing PCR (mycoplasma specific genes) using EZdetect PCR kit for mycoplasma detection (CCK009). All the cells were cultured in high glucose DMEM medium containing 10% foetal bovine serum and 1% Penicillin/streptomycin (Himedia). Cells were seeded in 60 mm dishes and upon attaining 80–90% confluency, treatment was given with PCI-34051(Cayman Chemicals) (10 μM/20 μM) [12 (link)] and tubastatin (5 μM) for 24 h [12 (link)] or both in combination (PCI-34051 and tubastatin) and Forskolin (Sigma) (10 μM) for 45 min independently along with untreated control as described earlier [13 (link)].

Tubacin, Tubastatin, and MC1568 were purchased from Sigma-Aldrich (St.Louis, MO). ACY1215 was purchased from ChemieTek　(Indianapolis, IN). Human recombinant TGF-β1 was purchased from R&D systems (Minneapolis, MN). Bleomycin was purchased from TEVA Pharmaceutical Industries (Petach Tikva, Israel). The following primary antibodies were purchased from the following companies: Santa Cruz (Dallas, TX; HDAC6 [H-300], HIF-1α), Abcam (Cambridge, MA; type-1 collagen), Sigma-Aldrich (α-SMA, acetylated α-tubulin), Cell Signaling Technology (Danvers, MA; β-actin, Smad2, phosphorylated Smad2, Smad3, phosphorylated Smad3, Akt, phosphorylated Akt (Ser473), PHLPP, Erk, phosphorylated Erk, p38, phosphorylated p38, HDAC6 [for the mouse samples], α-tubulin, LC3B). Secondary antibodies used were anti-mouse or anti-rabbit IgG HRP-linked antibodies (Cell Signaling Technology).