rabbit anti-carbonic anhydrase ix caix  (Novus Biologicals)

Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: Expression of HIF-1α and CAIX in MIA PaCa-2 and PANC-1 post MZM treatment. ( A ) Basal levels of mRNA transcript for HIF-1α and CAIX in MIA PaCa-2 and PANC-1 cells at 3, 18 and 24 h under hypoxia and normoxia. ( B ) Basal expression of HIF-1α and CAIX protein in MIA PaCa-2 and PANC-1 cells at 24 and 48 h. ( C , D ) Expression of HIF-1α and CAIX in MIA PaCa-2 cells treated with MZM at increasing concentrations (0.1–3 mM) for 24 and 48 h under hypoxia and normoxia. ( E , F ) Expression of HIF-1α and CAIX in PANC-1 cells treated with MZM at increasing concentrations (0.1–3 mM) for 24 and 48 h under hypoxia and normoxia.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: Inhibition of HIF-1α and CAIX in PDAC cells. ( A–D ) Under hypoxia , mRNA transcripts levels of HIF-1α and CAIX in MIA PaCa-2 ( A and B) and PANC-1 ( C and D) post treatment with MZM alone or in combination with GEM. The relative expression of genes was calculated with the 2 –∆∆Ct method, using β-actin as housekeeping gene for normalization. Statistical differences between the groups were determined by one-way ANOVA and post hoc multiple variances using Tukey test. The statistically significant difference is represented as * p < 0.05, ** p < 0.01 and *** p < 0.001 for control v/s specific group. ( E–H ) Western blot showing HIF-1α and CAIX protein expression levels in MIA PaCa-2 ( E and F) and PANC-1 ( G and H) post treatment with MZM alone or in combination with GEM (400 nM) at 24 and 48 h under normoxia (21% O 2 ) , and hypoxia condition (1% O 2 ) . Numbers on top indicate fold difference in expression which was determined after densitometry analysis using Image J software. β-actin was used as loading control.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Inhibition, Expressing, Control, Western Blot, Software

Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: ( A ) Inhibition of carbonic anhydrase IX as a surrogate marker of hypoxia, acidosis, and glycolytic metabolism by MZM in PC. Left side of figure indicates hypoxia related upregulation of CAIX and the related effects of this spike. The right of the figure indicates the inhibitory potential of MZM against all the markers studied and therefore all the pathways affected. CFA, colony forming assay, WHA , wound healing assay; PDX, patient derived xenograft, GEM, Gemcitabine. ( B ) Proposed cellular mechanism of action for MZM in PDAC cells. The illustration depicts proposed mechanism of MZM against carbonic anhydrase IX (CAIX) as a potential target in KRAS mutated pancreatic cancer. Under hypoxia, activated CAIX alters pH of intracellular milieu after intake of bicarbonate ions, generation of proton pool, increased uptake of glucose and accumulation of lactate. The altered pH favours stimulation of oncogenic signals by pro survival pathways which cater PC cells to proliferate, survive, invade, and metastasize under acidic environment. Pharmacological inhibition of CAIX by MZM modulates pericellular and intracellular activity owing to hypoxia, acidosis and glycolytic flux and further reduces tumor growth in GEM resistant PC cells. Abbreviations: MZM, Methazolamide; GEM, Gemcitabine, PDAC, Pancreatic ductal adenocarcinoma, NHE1, Sodium/proton exchanger 1; GLUT, Glucose transporter; MCT4, Monocarboxylate transporter 4; PTEN, Phosphatase and tensin homolog; NBC, Sodium bicarbonate cotransporter; IGF, Insulin growth factor; HGF, Hepatocyte growth factor; EGF, Epithelial growth factor; VEGF, Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor; ATPase, Adenosine triphosphatase; HRE, Hypoxia response element; Hif-1α, Hypoxia inducible factor; MDR1, Multidrug resistance protein 1; RTK, Receptor tyrosine kinase; PHD1/2, Hypoxia-inducible factor prolyl hydroxylase 1/2; VHL, Von Hippel-Lindau Syndrome; LDHA, Lactate dehydrogenase A. Green upside arrow indicates upregulation under hypoxia. Red downside arrow indicates downregulation of effect mediated by MZM. pHe, extracellular pH; pHi, intracellular pH; red star indicates activated KRAS pathway.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Inhibition, Marker, Wound Healing Assay, Derivative Assay, Activity Assay