anti cftr rabbit polyclonal igg antibody  (Alomone Labs)

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 2. Effect of ethanol on CFTR expression A, representative histological images show CFTR expression after treatment of the pancreatic ducts with ethanol (EtOH; 30, 50 and 100 mM) for 12 h. Pancreas slices were excited at 405 nm (Hoechst 33342) and 488 nm (Alexa Fluor 488), and emissions were collected at 405–465 and 488–550 nm, respectively. B, summary bar charts show the mean fluorescence intensity in the ductal cells normalized to the ductal area and expressed in arbitrary units. Data are presented as the mean ± SD. ∗P < 0.001 (30 mM EtOH), P < 0.001 (50 mM EtOH) and P < 0.001 (100 mM EtOH) vs. apical staining intensity of control. ∗∗P = 0.012 (50 mM EtOH) and P < 0.001 (100 mM EtOH) vs. cytoplasm staining intensity of control. ns = no significance, P = 0.781 (30 mM EtOH vs. cytoplasm staining intensity of control) (n = 5–8 per group).

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 3. Effect of VX-809 on CFTR expression Representative histological images show CFTR expression after treatment of the pancreatic ducts with ethanol (EtOH; 30 mM) and VX-809 (1, 3, 5 and 10 μM) for 12 h (A) or VX-809 (10 μM) for 2, 5 and 10 h (C). Pancreas slices were excited at 405 nm (Hoechst 33342) and 488 nm (Alexa Fluor 488), and emissions were collected at 405–465 and 488–550 nm, respectively. Scale bar = 20 μm. Summary bar charts show the dose-dependent (B) and time-dependent (D) effect of VX-809. Mean fluorescence intensity in the ductal cells was normalized to the ductal area and expressed in arbitrary units. Data are presented as the mean ± SD. ∗P < 0.001 (30 mM EtOH) vs. apical staining intensity of control (n = 5–8 per group).

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 4. Effect of VX-770 on CFTR expression Representative histological images show CFTR expression after treatment of the pancreatic ducts with ethanol (EtOH; 30 mM) and VX-770 (1, 3, 5 and 10 μM) for 12 h (A) or VX-770 (10 μM) for 2, 5 and 10 h (C). Pancreas slices were excited at 405 nm (Hoechst 33342) and 488 nm (Alexa Fluor 488), and emissions were collected at 405–465 and 488–550 nm, respectively. Scale bar = 20 μm. Summary bar charts show the dose-dependent (B) and time-dependent (D) effect of VX-809. Mean fluorescence intensity in the ductal cells was normalized to the ductal area and expressed in arbitrary units. Data are presented as the mean ± SD. ∗P < 0.001 (30 mM EtOH), P < 0.001 (1 μM VX-770), P < 0.001 (3 μM VX-770), P = 0.0078 (2 h VX-770) and P = 0.0014 (5 h VX-770) vs. apical staining intensity of control (n = 5–8 per group).

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 5. Effect of VX-809/VX-770 combination treatment on CFTR expression. Representative histological images show CFTR expression after treatment of the pancreatic ducts with ethanol (EtOH; 30 mM) and a combination of VX-809 and VX-770 (1, 3, 5 and 10 μM) (A) or with different concentrations of EtOH (50 and 100 mM) and a combination of VX-809 and VX-770 (10 μM) (C). Pancreas slices were excited at 405 nm (Hoechst 33342) and 488 nm (Alexa Fluor 488), and emissions were collected at 405–465 and 488–550 nm, respectively. Scale bar = 20 μm. Summary bar charts show the effect of the combination at 30 mM (B) and at 50 and 100 mM (D) EtOH. Mean fluorescence intensity in the ductal cells was normalized to the ductal area and expressed in arbitrary units. Data are presented as the mean ± SD. ∗P < 0.001 (30 mM EtOH), P < 0.001 (50 mM EtOH), P < 0.001 (100 mM EtOH) and P < 0.001 (100 mM EtOH + 10 μM combination) vs. apical staining intensity of control. P = 0.012 (50 mM EtOH) and P = 0.009 (100 mM EtOH) vs. cytoplasm staining intensity of control (n = 5–8 per group).

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 6. Effect of ethanol and CFTR modulators on the activity of CFTR A, current–voltage (I–V) relationships were obtained by measuring forskolin-stimulated CFTR currents over a 4 ms period starting 495 ms into the voltage pulse. B, representative whole cell currents recorded before and after stimulation with 5 μM forskolin. C, summary of current densities (measured at +60 mV) in forskolin-stimulated cells. The current densities have been normalized to the cell input capacitance (pA/pF). Data are presented as the mean ± SD. ∗P < 0.001 (30 mM EtOH), P < 0.001 (10 μM VX-770) and P < 0.001 (10 μM combination) vs. control. ∗∗P < 0.001 (10 μM VX-770 + 30 mM EtOH), P < 0.001 (10 μM VX-809 + 30 mM EtOH) and P < 0.001 (10 μM combination + 30 mM EtOH) vs. ethanol (n = 3–5 per group).

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Activity Assay, Control

Journal: The Journal of Physiology

Article Title: Restoring CFTR function with Orkambi decreases the severity of alcohol‐induced acute pancreatitis

doi: 10.1113/jp287289

Figure Lengend Snippet: Figure 9. Activity and expression of CFTR in acute pancreatitis A, current–voltage (I–V) relationships were obtained by measuring forskolin-stimulated CFTR currents over a 4 ms period starting 495 ms into the voltage pulse. B, summary of current densities (measured at +60 mV) in forskolin-stimulated cells. The current densities have been normalized to the cell input capacitance (pA/pF). Data are presented as the mean ± SD. ∗P < 0.001 (Orkambi) and P < 0.001 (AP) vs. Phys. Sal. (control). ∗∗P < 0.001 (AP + Orkambi) vs. AP (n = 4–6 per group). C, representative immunohistochemical staining shows the presence of CFTR in pancreatic samples from the control (phys. sal) and EtOH-treated (AP) animals with or without Orkambi treatment. Scale bar = 50 μm. D, summary bar chart shows the effect of AP with or without Orkambi treatment on the expression of CFTR. Mean fluorescence intensity in the ductal cells was normalized to the ductal area and expressed in arbitrary units. Data represent the mean ± SD for 5–8 specimens/five animals each group. ∗P = 0.05 (Orkambi) and P < 0.001 (AP) vs. apical staining intensity of Phys. Sal. (control). ∗∗P = 0.028 vs. apical staining intensity of AP. ∗∗∗P < 0.001 vs. cytoplasm staining intensity of Phys. Sal (control). Phys sal., physiological saline; AP, acute pancreatitis.

Article Snippet: After being blocked with 1% normal goat serum for 60min, the sections were incubated with anti-CFTR rabbit polyclonal IgG antibody (catalog. no. ACL-006; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

Techniques: Activity Assay, Expressing, Control, Immunohistochemical staining, Staining, Fluorescence, Saline

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) Principal Component Analysis (PCA) plot of RNA-seq data showing the global gene expression profiles of bronchial brushing tissues from healthy controls vs STAT3-HIES donors (7 donors/group). Expression of mRNAs of (B) Club cell markers SCGB1A1, SCGB3A1, KRT7, (C) ionocyte marker FOXI1, and (D) epithelial ion channels ANO1 (TMEM16A), SLC26A9, SCNN1A, B, and G were quantified by bulk RNA-seq in control vs STAT3-HIES bronchial brushing tissues. (E) CFTR protein from primary control and STAT3-HIES HBE cells (4 donors/group) was measured by western blot followed by densitometry quantification. * and Φ represent band C and B of CFTR protein, respectively. Actin expression served as loading control. (F-H) Control and STAT3-HIES HBE cells (4 donors/group) were differentiated under ALI conditions prior to exposure with IL1β. (F) Expressions of SCGB1A1, SCGB3A1and FOXI1 mRNAs were determined by TaqMan assays and (G) the SCGB1A1 + cell frequencies presented in cultures were quantified by whole mount IF staining and morphometrics. (H) Expression of ion channel genes ANO1 (TMEM16A), SCNN1A, B, and G mRNAs was determined by TaqMan assays. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor, except in (A-D) . ALI: air-liquid interface; HBE: human bronchial epithelial: IF: immunofluorescence.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Gene Expression, Expressing, Marker, Control, Western Blot, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) Total RNA was freshly isolated from bronchial brushing tissues of healthy control and STAT3-HIES donors (7 donors/group), and CFTR mRNA was measured by bulk RNA-seq. (B) Primary HBE cells isolated from control and STAT3-HIES bronchial brushing tissues (4 donors/group) were expanded and differentiated under ALI conditions prior to IL1β exposure. CFTR mRNA and activity were measured by TaqMan assays and Ussing chamber assay, respectively. (C) Normal HBE cells (n=9 donors) were transduced with lentiviruses expressing control vector, wild type STAT3 (WT-OE) and mutant STAT3 (R382W-OE) and cultured under ALI prior to IL1β exposure followed by bulk RNA-seq. CFTR activity in response to forskolin were measured and quantified. (D) Normal HBE cells (n=5 donors) were electroporated with control or STAT3 CRISPR/Cas9, followed by ALI culture prior to IL1β exposure. CFTR mRNA and activity of each culture were quantified. (E) Linear regression test was performed to STAT3 and CFTR mRNAs of well-differentiated normal HBE cells from 44 donors (measured by RNA-seq), the Ln transformed Pearson correlation coefficient (r) was reported in the graph. Symbols with same colors in the same panel indicate multiple cultures originated from the same donor except in ( A , E ). ALI: air-liquid interface; HBE: human bronchial epithelial; OE: overexpression; Ln: natural logarithm; TMP: transcripts per million.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Isolation, Control, RNA Sequencing, Activity Assay, Boyden Chamber Assay, Transduction, Expressing, Plasmid Preparation, Mutagenesis, Cell Culture, CRISPR, Transformation Assay, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) cDNA sequences of wild type STAT3 (STAT3 WT-OE) and STAT3 R382W mutation were cloned to pLVX-EF1α-IRES-Puro lentiviral vector. The R283W mutation was confirmed by Sanger sequencing. (B) Normal HBE cells (n=3 donors) transduced with control, STAT3 WT-OE and R382W-OE lentiviruses were cultured under ALI conditions. STAT3 protein expression at 1 and 4 weeks of ALI were detected by western blot and quantified by densitometry. The TATA box binding protein (TBP) served as a loading control. (C) Normal HBE cells (n=3 donors) were transduced with control, WT-OE and R382W-OE lentiviruses. The subcellular localization of p-STAT3 (upper panel of each section) in response to IL1β was detected by IF staining. The white dash-lines in the lower panels provided a visual guide for the spatial distribution of p-STAT3 within the subcellular architecture. (D-E) STAT3 WT-OE and R382W-OE lentiviruses were transduced into H441 cells. (D) The subcellular localization of p-STAT3 in H441 was detected by IF staining, and (E) expression of p-STAT3, STAT3 and TBP was detected by western blot in the whole cell lysates and in nuclear vs cytoplasmic fractions. (F) The western blot signals were semi-quantified by densitometry for calculation of (i) the ratio of p-STAT3/STAT3 in whole cell lysates (WCL), (ii) the fold change of p-STAT3/STAT3 in response to IL6 and (iii) the ratio of p-STAT3 in nuclear vs cytoplasmic fraction in response to IL6 in H441 cells. Experiments were repeated in 4 independent cultures. (G) CFTR protein in control, WT-OE and R382W-OE lentiviruses infected HBE cultures was detected by western blot. The image is representative of HBE cells from 4 donors. The signals on the western blot were semi-quantified by densitometry. (Hi,iii,iv) SCGB1A1, SCGB3A1 and FOXI1 mRNAs were quantified by RNA-seq. (Hii) The frequency of SCGB1A1 + cells was quantified by whole mount staining and morphometrics. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor. IF: immunofluorescence; ALI: air-liquid interface; HBE: human bronchial epithelial; WCL: whole cell lysates, OE: overexpression.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Sequencing, Transduction, Control, Cell Culture, Expressing, Western Blot, Binding Assay, Staining, Infection, RNA Sequencing, Immunofluorescence, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) STAT3 CRISPR/Cas9 ribonucleoprotein complex was electroporated into HBE cells. Genomic DNA was isolated for Sanger sequencing and analyzed by Synthego online analysis tool. Expression of (B) STAT3 and (C) CFTR protein in control vs STAT3 KO HBE was detected by western blot. The images are representatives of western blots of HBE cells from 5 donors. (D) Primary STAT3-HIES HBE cells (from 2 donors) were transduced with control or STAT3 WT-OE lentiviruses followed by ALI culture. Expression of STAT3 and CFTR mRNAs was determined by TaqMan assays. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor regardless of shapes. KO: knockout; HBE: human bronchial epithelial; ALI: air-liquid interface; OE: overexpression.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: CRISPR, Isolation, Sequencing, Expressing, Control, Western Blot, Transduction, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) Normal HBE cells (n=6 donors) were exposed to IL1β for 8 and 24 hours, and IL6 mRNA fold change was compared to 0 hour by TaqMan assays. (B) Normal HBE cells were treated with or without IL6R monoclonal antibody Tocilizumab (TCZ) prior to exposure to IL1β or IL6 for 8 hours. p-STAT3 and STAT3 proteins were detected by western blot. The image is representative HBE cells from 8 donors. (C) CFTR mRNA in response to IL6 exposure was measured by TaqMan assays in well differentiated normal HBE cultures (n=5 donors). (D) Expression of p-STAT3 and STAT3 proteins was detected by western blot in normal HBE cells treated with GP130 inhibitor SC144 (10µM) or DMSO (vehicle) prior to expose to IL1β (1ng/ml) for 8 hours. The image is representative of HBE cells from 3 donors with 2 cultures/condition/donor. (E) p-STAT3, STAT3 proteins were detected by western blot in normal HBE cells pre-treated with JAK inhibitor Tofacitinib (TFB) prior to IL1β exposure. The image is a representative of HBE cells from 8 donors. (F) JAK1,2,3 mRNAs in normal HBE cells (n=6 donors) were determined by bulk RNA-seq and. (G) JAK1 CRISPRs were composed of combination of 3 individual CRISPRs shown in the table, and Indel%, Knockout-Score were calculated in targeted normal HBE. (H) Expression of JAK1, p-STAT3, STAT3 and TBP proteins was determined by western blot in HBE cells targeted by control or JAK1 CRISPR/Cas9 exposed to vehicle or IL1β. JAK1 expression was determined by western blot and semi-quantified by densitometry. The image is representative of HBE cells from 5 donors. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor. KO: knockout; ALI: air-liquid interface; HBE: human bronchial epithelial.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, RNA Sequencing, Knock-Out, Control, CRISPR

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) Normal HBE cells (n=4 donors) were exposed to IL1β, and apical and basolateral secretion of IL6 was measured by ELISA at 0, 8 and 24 hours after treatment. (B) Expression of p-STAT3 and STAT3 proteins were detected by western blot and their ratios were calculated in the normal HBE cells (n=7 donors) exposed with IL1β for 0, 8 and 24 hours, and CFTR mRNA (n=10 donors) was measured up to 72 hours. (C) To block IL6 signaling, Tocilizumab (TCZ) was administered to normal HBE cells (n=8 donors) prior to exposure to IL6 or IL1β. p-STAT3 and STAT3 proteins were detected, and their ratios were calculated. The CFTR mRNA fold changes in response to IL1β with or without TCZ was detected by TaqMan assays (n=7 donors). (D) To inhibit GP130 functions, SC144 was administered to the normal HBE cells (n=3 donors) prior to exposure to IL1β; p-STAT3 and STAT3 proteins were detected and their ratios were calculated. The CFTR mRNA change in response to IL1β was measure in normal HBE cells (n=9 donors) treated with SC144 at 0 (DMSO), 1 and 10 µM. (E) To inhibit JAK activity, Tofacitinib (TFB, 1µM) was administered to normal HBE cells (n=8 donors) prior to exposure to IL1β. The ratio of p-STAT3 to STAT3 proteins and CFTR mRNA (n=6 donors) was measured. (F) Normal HBE cells (n=5 donors) were electroporated with control or JAK1 CRISPR/Cas9, followed by ALI culture prior to exposure to IL1β. The ratio of p-STAT3 to STAT3 was calculated after western blot and densitometry quantification and CFTR mRNA was measured. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor, regardless of shapes. ALI: air-liquid interface; HBE: human bronchial epithelial.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Blocking Assay, Activity Assay, Control, CRISPR

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) MUC5B and MUC5AC mRNAs of bronchial brushing tissue in vivo were quantified by bulk RNA-seq (7 donors/group). Normal HBE cells were transduced with (B) control, STAT3 WT-OE and STAT3 R382W-OE lentiviruses (n=6 donors), or (C) electroporated with control or STAT3 CRISPR/Cas9 (n=5 donors), followed by ALI culture. Apical secretion of MUC5B and MUC5AC proteins was detected by mucin agarose western blot and quantified by densitometry, and (D) the expression of XBP1S mRNA was determined by quantitative RT-PCR in the STAT3 R382W-OE and STAT3-KO HBE cells, respectively. (E, F) Linear regression tests were performed on CFTR mRNA with MUC5B and MUC5AC mRNAs (determined by bulk RNA-seq) in the normal HBE cells (n=9) transduced with (E) WT-OE vs (F) R382W-OE lentiviruses at baseline or after exposure to IL1β. (G) Apical (i) solids contents, (i.e., dry weight) and (ii) fluid (wet weight) secretions were measured in the control, WT-OE and R382W-OE HBE using microbalance. (iii) To visualize the effect of R382W-OE on apical fluid secretion, the culture plate was tilted to pool secretions at the Transwell’s bottom edge. The accumulated ASL meniscus volumes were highlighted by green dash lines in WT-OE vs R382W-OE HBE cells with or without IL1β treatment. (iv) Mucus concentrations (%solids) were calculated based on the ratio of dry to wet weight. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor, regardless of the shapes, except in (A) . HBE: human bronchial epithelia; ALI: air-liquid interface; Ln: natural logarithm; TPM: transcripts per million; ASL: airway surface liquid; OE: overexpression.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: In Vivo, RNA Sequencing, Transduction, Control, CRISPR, Western Blot, Expressing, Quantitative RT-PCR, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) MUC5B and MUCAC mRNAs were measured by TaqMan assays in control vs STAT3-HIES primary HBE cells (n=4 donors/group) cultured in vitro with or without IL1β exposure. (B) MUC5B and MUC5AC mRNAs were measured by bulk RNA-seq in the normal HBE cells (n=9 donors) expressing control, STAT3 WT-OE and STAT3 R382W-OE lentiviruses under ALI conditions prior to IL1β exposure. (C) MUC5B and MUC5AC mRNAs were measured in STAT3-KO (by CRISPR/Cas9) HBE (n=5 donors) cultured under ALI conditions prior to IL1β exposure. (D) BPIFB1 mRNA was measured in R382W-OE or STAT3 KO HBE. (E) SPDEF and FOXA3 mRNAs were quantified by bulk RNA-seq in normal HBE cells expressing control, WT-OE and R382W-OE lentiviruses, and treated with or without IL1β. (F) Basal and IL1β-induced CFTR, MUC5B, and MUC5AC mRNAs were quantified by TaqMan assays in normal well differentiated HBE from 41 donors. The correlations between CFTR (x-axis) and MUC5B or MUC5AC (shown in blue or red, respectively, both on the y-axis) mRNAs in normal HBE at (i) baseline and (ii) exposure to IL1β were demonstrated by linear regression. Symbols with the same colors in the same panel indicate multiple cultures originated from the same donor, regardless of the shapes, except in (F) . HBE: human bronchial epithelia; ALI: air-liquid interface; OE: overexpression.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Control, Cell Culture, In Vitro, RNA Sequencing, Expressing, CRISPR, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: Normal HBE cells (from 9 donors) were transduced with control, STAT3 WT-OE, and STAT3 R382W-OE lentiviruses and cultured under ALI conditions prior to IL1β exposure. STAT3-KO HBE cells were generated by CRISPR/Cas9 and ALI cultured prior to IL1β exposure. (A) BPIFA1 mRNA was measured in R382W-OE and STAT3-KO, and their control HBE cells by RNA-seq and TaqMan assays, respectively. (B) Basolateral secretions of CXCL1 and CSF3 proteins were measured by mesoscale multiplex. (C) IL17C, (D) S100A8, S100A9, NOX1, and DUOX2 mRNAs were measured by bulk RNA-seq in the R382W-OE vs WT-OE and control HBE cells. (E) DEFB4A, (F) S100A8, S100A9, NOX1, and DUOX2 mRNAs were measured in IL1R1-KO (targeted by IL1R1 CRISPR/Cas9) HBE cells (from 4 donors). (G) The correlations between STAT3 and IL1R1 mRNAs (measured by bulk RNA-seq) in normal well differentiated HBE cells (n=44) were tested by linear regression. (H) A proposed model of STAT3 regulation of innate epithelial defense through IL1R1-dependent and -independent pathways: At baseline, STAT3 is required for producing antimicrobial molecules BPIFA1, LTF, CCL20, LCN2 and chemokines CXCL1, and CSF3. STAT3 is required for IL1R1 expression in HBE cells. Therefore, STAT3 controls expression of IL1β-inducible host defense molecules, including β-defensin, NOX1, DUOX2, IL17C, and genes regulate ASL pH, including CFTR, pendrin and other ion transporters. (I) Na + -dependent, HCO 3 - electrogenic cotransporter (NBCe1), SLC4A4 mRNA was detected by bulk RNA-seq in normal HBE cells (n=9 donors) transduced with control, WT-OE, and R382W-OE lentiviruses exposed with or without IL1β. ASL: airway surface liquid; HBE: human bronchial epithelial; KO: knock-out; OE: overexpression; Ln: natural logarithm; TMP: transcripts per million.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Transduction, Control, Cell Culture, Generated, CRISPR, RNA Sequencing, Multiplex Assay, Expressing, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) A group of selective genes involved in the Notch pathway that regulate the transition from Club/secretory cells to deutrosomal/multiciliated cell lineage during normal epithelial cell ciliogenesis (referenced by the published longitudinal airway epithelial cell mucociliary differentiation scRNA-seq data) were compared in STAT3 WT-OE vs STAT3 R382W-OE HBE cells (from 9 donors). Gene names were labeled in green vs red colors to indicate their expression status of induction vs inhibition, respectively, during this transitional period. The data presented in light blue areas were plotted to left y axis and data presented in pink areas were plotted to the right y axis. Expression of (B) Notch pathway activators NOTCH1, NOTCH3, (C) secretory cell markers SCGB1A1, CFTR, MUC5B, and (D) basal cell marker TP63 and suprabasal KRT13 in response to LY450139 treatment (250nM and 500nM) or vehicle (DMSO) was determined by TaqMan assay in STAT3 R382W-OE HBE cells (n=9 donors). Symbols with the same color in the same panel indicate cultures originated from the same donor HBE: human bronchial epithelial; ALI: air-liquid interface; OE: overexpression.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Labeling, Expressing, Inhibition, Marker, TaqMan Assay, Over Expression

Journal: bioRxiv

Article Title: Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

doi: 10.1101/2024.08.14.607930

Figure Lengend Snippet: (A) In normal airway epithelial cells, STAT3 regulates basal CFTR-mediated fluid and mucin secretion, and JAK1/STAT3-mediated multiciliogenesis necessary for homeostatic mucociliary clearance (MCC) and normal airway surface liquid (ASL) pH. (B) In response to injuries (caused by infections, aspiration, and the resultant inflammation), STAT3 is activated by JAK1-mediated IL-1 signaling to increase CFTR-mediated fluid and mucin secretion, enhancing ASL volume while moderately decreasing mucus concentration compared to baseline. STAT3 is essential for IL1R1 expression, thus regulating the production and function of IL1R1-dependent and independent host defense molecules in response to IL1β. These include the secretion of antimicrobial peptides (AMP), reactive oxygen species (ROS), CFTR/pendrin-mediated bicarbonate (HCO3 - ) for ASL alkalization, and cytokines/chemokines critical for immune cell recruitment. These activities support coordinated MCC and microbicidal activities, clearing infections from the airways. (C) STAT3-HIES airway epithelial cells fail to maintain CFTR activity and mucin secretion, resulting in diminished ASL volume at baseline. Insufficient CFTR function, along with abnormal ion exchangers’ expression, leads to ASL acidification, increasing the vulnerability of STAT3-HIES epithelial cells. (D) Upon injuries, STAT3-HIES epithelial cells fail to adequately increase fluid and mucin secretion by secretory cells. The defective ciliated cell regeneration fails to provide motor function for MCC, resulting in low ASL volume, low MUC5B content, poor-transportable mucus layer, and static MCC. Decreased secretion of AMP/ROS, HCO3 - (for ASL alkalization), and cytokines/chemokines necessary for antimicrobial activity increase the susceptibility to infection on airway surface. (E) Impaired MCC during the acute phase leads to ongoing infection and inflammation, progressively increasing mucus concentration and leading to mucus hyperconcentration and plugging that produces local hypoxia in STAT3-HIES airways. Due to defective ciliation, the ATP-mediated autoregulation of mucus concentration via the mucus-cilia mechanism and motor function needed for mucus transport are lacking. Regional hypoxia promotes a vicious cycle by activating ENaC-mediated fluid/Na+ absorption and inhibiting ciliogenesis, that in turn further increasing mucus concentration and impairing MCC, resulting in chronic/persistent bacterial and fungal infection, and bronchiectasis over time.

Article Snippet: The lysates were clarified, and CFTR protein was immunoprecipitated using a rabbit anti-CFTR polyclonal antibody (UNC CFTR-antibody 155) and Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Expressing, Activity Assay, Infection

Journal: eBioMedicine

Article Title: Female mice display sex-specific differences in cerebrovascular function and subarachnoid haemorrhage-induced injury

doi: 10.1016/j.ebiom.2024.105058

Figure Lengend Snippet: CFTR expression and function in female cerebral arteries. (a) CFTR mRNA expression in female cerebral arteries is plotted over Zeitgeber time (n = 3 samples from 3 mice at each Zeitgeber time point). Data are double-plotted for visualization purposes; white shading indicates “lights on” and dark shading indicates “lights off”. CFTR mRNA expression exhibits a statistically significant circadian rhythm, according to JTK_CYCLE analysis. (b) A comparison of CFTR protein expression in cerebral arteries isolated from female mice at Zeitgeber Time 11 (ZT11; n = 9 samples from 9 mice) versus Zeitgeber Time 23 (ZT23; n = 9 samples from 9 mice). (c) The effect of CFTR channel inhibition (100 nmol/L CFTR (inh)-172 for 30 min in vitro ) on myogenic tone in female cerebral arteries isolated at ZT11 (n = 6 arteries from 3 mice; paired comparisons). (d) Myogenic tone and phenylephrine-stimulated vasoconstriction in female cerebral arteries isolated at ZT11 from mice treated with lumacaftor (3 mg/kg/day; n = 5 arteries from 3 mice) or vehicle (n = 6 arteries from 4 mice) for 2 days. (e) Myogenic tone in female cerebral arteries isolated at ZT23 from mice treated with 3 mg/kg/day lumacaftor (n = 5 arteries from 3 mice), 30 mg/kg/day lumacaftor (n = 7 arteries from 4 mice) or vehicle (n = 10 arteries from 5 mice) for 2 days. All data are means ± standard deviation. In panel a, the Bonferroni-adjusted JTK_CYCLE p value is shown below the graph. Statistical comparisons in panel b used an independent t-test; panel c used paired t-tests; and panel d used either an independent t-test (20 mmHg and 1–10 nmol/L) or a Mann–Whitney U test (40–80 mmHg and 100 nmol/L-10 μmol/L). Statistical comparisons in panel e used either a Kruskal–Wallis test (20 mmHg) or a One-Way ANOVA (40–80 mmHg). Comparison test p-values are shown above the error bars.

Article Snippet: The 100–280 kDa weight range of the blot was incubated with rabbit polyclonal anti-CFTR primary antibody (1:1000 in 5% milk/PBST; Alomone Labs cat# ACL-006, RRID:AB_2039804), while the remaining range (35–100 kDa) was incubated with mouse monoclonal anti-α-tubulin primary antibody (1:5000 in 5% milk/PBST; Cell Signalling Technology cat# 3873, RRID:AB_1904178).

Techniques: Expressing, Comparison, Isolation, Inhibition, In Vitro, Standard Deviation, MANN-WHITNEY

Journal: Journal of medicinal chemistry

Article Title: Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

doi: 10.1021/acs.jmedchem.3c00608

Figure Lengend Snippet: Figure 2. Discovery of the first RNF5 inhibitor, the 1,2,4-thiadiazolylidene inh-2 by Sondo et al.40 and chemical modification campaign around the 1,2,4-thiadiazol-5-ylidene scaffold. (A) Chemical structures and F508del-CFTR corrector activity of the RNF5 inhibitor inh-2 and the RNF5 activator analog-1. NA = not active. (B) Proposed binding mode of inh-2 into the RNF5 pocket. Blue dashed lines indicate the π−π stacking interactions, while yellow dashed lines indicate the hydrogen bonds. (C) Overview of the optimization strategy of inh-2 for SAR exploration.

Article Snippet: CFBE41o− cells stably expressing wt- or F508del-CFTR were grown to confluence on 60 mm diameter dishes and treated for 24 h with the indicated compounds in the absence or in the presence of MG-132 (10 μM) in the last 4 h. CFTR immunoprecipitation was performed as previously reported.40 In brief, cells were lysed and, after centrifugation, the protein concentration in the supernatant was calculated using the BCA assay (Euroclone, Pero (MI), Italy) following supplier’s instructions An aliquot of supernatant corresponding to 600 μg of protein was incubated for 1 h with 2 μg/sample of rabbit polyclonal anti-CFTR antibody (Alomone Labs, Jerusalem, Israel), with rocking at room temperature (RT).

Techniques: Modification, Activity Assay, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

doi: 10.1021/acs.jmedchem.3c00608

Figure Lengend Snippet: Figure 5. Bar graph showing CFTR activity in CFBE41o−cells following 24 h treatment with vehicle alone or with analogs of inh-2 at the indicated concentrations. The vehicle alone (DMSO) and corrector VX-809 (1 μM) were used as negative and positive controls, respectively.

Article Snippet: CFBE41o− cells stably expressing wt- or F508del-CFTR were grown to confluence on 60 mm diameter dishes and treated for 24 h with the indicated compounds in the absence or in the presence of MG-132 (10 μM) in the last 4 h. CFTR immunoprecipitation was performed as previously reported.40 In brief, cells were lysed and, after centrifugation, the protein concentration in the supernatant was calculated using the BCA assay (Euroclone, Pero (MI), Italy) following supplier’s instructions An aliquot of supernatant corresponding to 600 μg of protein was incubated for 1 h with 2 μg/sample of rabbit polyclonal anti-CFTR antibody (Alomone Labs, Jerusalem, Israel), with rocking at room temperature (RT).

Techniques: Activity Assay

Journal: Journal of medicinal chemistry

Article Title: Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

doi: 10.1021/acs.jmedchem.3c00608

Figure Lengend Snippet: Figure 8. Evaluation of effects of putative RNF5 inhibitors on ATG4B-mediated basal autophagy. (A) Representative confocal microscopy images of F508del-CFTR expressing CFBE41o−cells treated with the indicated compounds and loaded with MDC. Scale bar = 50 μm. (B) The bar graph shows the quantification of the number of spots (resembling autophagic vesicles) in cells treated with the indicated compounds, normalized for the control condition. Data are expressed as means ± SEM, n = 4−6. Asterisks indicate statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: CFBE41o− cells stably expressing wt- or F508del-CFTR were grown to confluence on 60 mm diameter dishes and treated for 24 h with the indicated compounds in the absence or in the presence of MG-132 (10 μM) in the last 4 h. CFTR immunoprecipitation was performed as previously reported.40 In brief, cells were lysed and, after centrifugation, the protein concentration in the supernatant was calculated using the BCA assay (Euroclone, Pero (MI), Italy) following supplier’s instructions An aliquot of supernatant corresponding to 600 μg of protein was incubated for 1 h with 2 μg/sample of rabbit polyclonal anti-CFTR antibody (Alomone Labs, Jerusalem, Israel), with rocking at room temperature (RT).

Techniques: Confocal Microscopy, Expressing, Control

Journal: Journal of medicinal chemistry

Article Title: Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

doi: 10.1021/acs.jmedchem.3c00608

Figure Lengend Snippet: Figure 9. Putative RNF5 inhibitors improve mutant CFTR rescue by approved correctors by decreasing CFTR ubiquitylation. (A) Bar graph showing CFTR activity in CFBE41o−cells following 24 h treatment with vehicle alone or with inh-2 (5 μM) and its analogue 16 (5 μM) as single agents or combined with correctors VX-809 (3 μM), VX-445 (3 μM), or VX-661 + VX-445 (10 μM + 3 μM). (B) Bar graph showing CFTR activity in CFBE41o−cells transfected with NT or RNF5 siRNA and treated for 24 h with DMSO, or analogue 16 (5 μM), or VX-661 + VX-445 (10 μM + 3 μM), or their combination. The effect of CFTR siRNA is shown as additional control of transfection efficiency. Asterisks indicate statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05, while n.s. indicates “not significant”. (C) Biochemical analysis of CFTR ubiquitylation and expression pattern in CFTR immunoprecipitates from CFBE41o−cells after 24 h treatment with analogue 16 (5 μM), VX-661 + VX-445 (10 μM + 3 μM), and their combination in the absence or in the presence of MG-132 (10 μM; last 4 h) to block proteasomal degradation. Images for CFTR and Ub blots of F508del- and wt-CFTR samples are different exposures of the same membranes. (D−F) Analysis of intensity profiles of CFTR and ubiquitin (D) in the absence or (E and F) in the presence of MG-132.

Article Snippet: CFBE41o− cells stably expressing wt- or F508del-CFTR were grown to confluence on 60 mm diameter dishes and treated for 24 h with the indicated compounds in the absence or in the presence of MG-132 (10 μM) in the last 4 h. CFTR immunoprecipitation was performed as previously reported.40 In brief, cells were lysed and, after centrifugation, the protein concentration in the supernatant was calculated using the BCA assay (Euroclone, Pero (MI), Italy) following supplier’s instructions An aliquot of supernatant corresponding to 600 μg of protein was incubated for 1 h with 2 μg/sample of rabbit polyclonal anti-CFTR antibody (Alomone Labs, Jerusalem, Israel), with rocking at room temperature (RT).

Techniques: Mutagenesis, Activity Assay, Transfection, Control, Expressing, Blocking Assay, Ubiquitin Proteomics