( E ) Expression of Cxcl12 and Il - 7 in the bone marrow from Bbs4 + / + Bbs18 + / + ( WT ) mice ( black circles , n = 7 mice ) , Bbs4 + / KO ( Het , green circle , n = 3 ) , Bbs18 + / KO ( Het , blue circle , n = 3 ) , Bbs4KO / KO ( green square , n = 5 ) , and Bbs18KO / KO ( blue square , n = 2 ) was analyzed by RT - qPCR in five independent experiments . The expression was normalized to Gapdh . The statistical significance of the difference between the BBSome - deficient mice ( Bbs4KO / KO and Bbs18KO / KO ) and controls were calculated using two - tailed Mann - Whitney test . Median is shown .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(E) Expression of Cxcl12 and Il-7 in the bone marrow from Bbs4+/+ Bbs18+/+ (WT) mice (black circles, n=7 mice), Bbs4+/KO (Het, green circle, n=3), Bbs18+/KO (Het, blue circle, n=3), Bbs4KO/KO (green square, n=5), and Bbs18KO/KO (blue square, n=2) was analyzed by RT-qPCR in five independent experiments. The expression was normalized to Gapdh. The statistical significance of the difference between the BBSome-deficient mice (Bbs4KO/KO and Bbs18KO/KO) and controls were calculated using two-tailed Mann-Whitney test. Median is shown.


( F ) MEF cell lines were derived from a single Bbs4 + / + ( WT ) or a single Bbs4KO / KO embryos . The expression of Cxcl12 in these cell lines was analyzed by RT - qPCR . The expression was normalized to Gapdh and to Bbs4 + / + MEFs ( = 1 ) for each experiment . Three biological replicates . One sample t test was used for the statistical analysis .

O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

(F) MEF cell lines were derived from a single Bbs4+/+ (WT) or a single Bbs4KO/KO embryos. The expression of Cxcl12 in these cell lines was analyzed by RT-qPCR. The expression was normalized to Gapdh and to Bbs4+/+ MEFs (=1) for each experiment. Three biological replicates. One sample t test was used for the statistical analysis.


( G ) Expression of Cxcl12 and canonical - WNT responsive gene Alpl in untreated ( NT ) ST2 cells , or ST2 cells treated with WNT3A or WNT5A was analyzed by RT - qPCR . The expression was normalized to Gapdh and untreated ST2 sample ( = 1 ) for each experiment . Four biological replicates were performed . One sample t test was used for the statistical analysis .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Expression of Cxcl12 and canonical-WNT responsive gene Alpl in untreated (NT) ST2 cells, or ST2 cells treated with WNT3A or WNT5A was analyzed by RT-qPCR. The expression was normalized to Gapdh and untreated ST2 sample (=1) for each experiment. Four biological replicates were performed. One sample t test was used for the statistical analysis.


( A Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters White blood cell count n = 41 for parameters Neutrophils , Lymphocytes , Monocytes , Eosinophils Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis in (

O O O O O B-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O

(A Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters White blood cell count n=41 for parameters Neutrophils, Lymphocytes, Monocytes, Eosinophils Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in (A


( B Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

(B Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . In ( C ) , the percentage of patients or controls having CRP ≥ 5 were compared using Fisher ' s exact test with post - hoc Sidak correction for multiple comparisons , BBS patients n = 42 .

O O O B-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. In (C), the percentage of patients or controls having CRP≥5 were compared using Fisher's exact test with post-hoc Sidak correction for multiple comparisons, BBS patients n=42.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters Mean cell volume , Hemoglobin n = 39 for parameters Red Blood cells , Mean cell Hemoglobin and red cell distribution width ( RDW ) . Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis

O O O B-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Mean cell volume, Hemoglobin n=39 for parameters Red Blood cells, Mean cell Hemoglobin and red cell distribution width (RDW). Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in


Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters Platelets ; Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis in F ) .

O O O B-TISSUE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Platelets; Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in F).


G ) Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

G) Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


D Microscale thermophoresis ( MST ) curves for binding of peptides to labelled GyraseA14 . The circles indicate the experimental values and the solid lines indicate the fitted fluorescence values obtained upon analysis using the MO . Affinity Analysis software ( version 2 . 2 . 5 , NanoTemper Technologies ) . Data information : In panels D , data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panel D , the data are presented as mean ± SD values obtained from all the replicates .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Microscale thermophoresis (MST) curves for binding of peptides to labelled GyraseA14. The circles indicate the experimental values and the solid lines indicate the fitted fluorescence values obtained upon analysis using the MO.Affinity Analysis software (version 2.2.5, NanoTemper Technologies). Data information: In panels D, data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panel D, the data are presented as mean ± SD values obtained from all the replicates.


B , In vitro bactericidal action of the CP1 - WT peptide on E . coli Top10 cells . B Growth profile of E . coli Top10 in the presence of increasing concentrations of the CP1 - WT peptide . Data information : data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panels B data are presented as mean ± SD values obtained from all the replicates . In panel B , N . T . - No treatment ; 4 . 5 % DMSO ( solvent control ) ; CP1 - WT ( test peptide ) ; CP1 - Mut ( negative control peptide ) and Cip - 10µg / mL ciprofloxacin ( positive control ) .

O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O

B, In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. B Growth profile of E. coli Top10 in the presence of increasing concentrations of the CP1-WT peptide. Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels B data are presented as mean ± SD values obtained from all the replicates. In panel B, N.T. - No treatment; 4.5% DMSO (solvent control); CP1-WT (test peptide); CP1-Mut (negative control peptide) and Cip - 10µg/mL ciprofloxacin (positive control).


C In vitro bactericidal action of the CP1 - WT peptide on E . coli Top10 cells . C Estimation of Minimum Inhibitory Concentration ( MIC ) , indicated in terms of OD600 and Minimum Bactericidal Dose ( MBD ) , indicated in terms of CFU / mL , show that the CP1 - WT peptide has similar MIC and MBD values of 40µM ( ~ 130µg / mL ) . Data information : data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panels C , data are presented as mean ± SD values obtained from all the replicates .

O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. C Estimation of Minimum Inhibitory Concentration (MIC), indicated in terms of OD600 and Minimum Bactericidal Dose (MBD), indicated in terms of CFU/mL, show that the CP1-WT peptide has similar MIC and MBD values of 40µM (~130µg/mL). Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels C, data are presented as mean ± SD values obtained from all the replicates.


A - D Peptide - induced membrane damage was studied by confocal microscopy and measured by propidium iodide ( PI ) influx ( % PI uptake ) in ( A ) S . Typhimurium , ( B ) A . baumannii , ( C ) S . aureus and ( D ) P . aeruginosa . Data information : In panels A - D , n = 5 biological replicates ; scale bars : 5µm . Representative images are shown . In the bar graphs , data are presented as mean ± SD values of percentage of propidium iodide uptake ( % PI uptake ) , averaged across all the replicates . 3 % , 7 % , 8 % and 3 % DMSO , and 80µM , 50µM , 50µM and 80µM of the peptides in the respective DMSO concentrations were used to treat S . Typhimurium , A . baumannii , S . aureus and P . aeruginosa respectively .

O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O

A-D Peptide-induced membrane damage was studied by confocal microscopy and measured by propidium iodide (PI) influx (% PI uptake) in (A) S. Typhimurium, (B) A. baumannii, (C) S. aureus and (D) P. aeruginosa. Data information: In panels A-D, n = 5 biological replicates; scale bars: 5µm. Representative images are shown. In the bar graphs, data are presented as mean ± SD values of percentage of propidium iodide uptake (% PI uptake), averaged across all the replicates. 3%, 7%, 8% and 3% DMSO, and 80µM, 50µM, 50µM and 80µM of the peptides in the respective DMSO concentrations were used to treat S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively.


E - H Minimum Inhibitory Concentration ( MIC ) and Minimum Bactericidal Dose ( MBD ) estimation of CP1 - WT on stationary phase cells of ( E ) S . Typhimurium , ( F ) A . baumannii , ( G ) S . aureus and ( H ) P . aeruginosa . No bactericidal action is observed for P . aeruginosa . In each of the panels and its corresponding bar graph , the black lines and black bars represent the MIC data indicated by their OD600 values , and the red lines and red bars represent the MBD data indicated by their CFU / mL values . The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO , peptides and ciprofloxacin ( Cip - postive control ; 10µg / mL ) . The strain of A . baumannii used was reported to be ciprofloxacin - resistant ( Nagarajan et al , 2018 ) as is observed here ( inset in panel F ) . In panels E - H , n = 5 biological replicates . Data are presented as mean ± SD values , averaged across all the replicates . For the bar graphs in these panels , 7 % , 13 % , 8 % and 4 % DMSO , and 80µM , 100µM , 100µM and 50µM of the peptides , were used for S . Typhimurium , A . baumannii , S . aureus and P . aeruginosa respectively . N . T - No treatment ; WT - CP1 - WT peptide ; Mut - CP1 - Mut peptide ( negative control ) . P values are calculated by Student ' s unpaired t - test ; nsP > 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 Student ' s unpaired t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Dose (MBD) estimation of CP1-WT on stationary phase cells of (E) S. Typhimurium, (F) A. baumannii, (G) S. aureus and (H) P. aeruginosa. No bactericidal action is observed for P. aeruginosa. In each of the panels and its corresponding bar graph, the black lines and black bars represent the MIC data indicated by their OD600 values, and the red lines and red bars represent the MBD data indicated by their CFU/mL values. The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO, peptides and ciprofloxacin (Cip - postive control; 10µg/mL). The strain of A. baumannii used was reported to be ciprofloxacin-resistant (Nagarajan et al, 2018) as is observed here (inset in panel F). In panels E-H, n = 5 biological replicates. Data are presented as mean ± SD values, averaged across all the replicates. For the bar graphs in these panels, 7%, 13%, 8% and 4% DMSO, and 80µM, 100µM, 100µM and 50µM of the peptides, were used for S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively. N.T- No treatment; WT - CP1-WT peptide; Mut - CP1-Mut peptide (negative control). P values are calculated by Student's unpaired t-test; nsP > 0.05; *** P < 0.001; **** P < 0.0001 Student's unpaired t-test.


A - D Transmission electron micrographs of the indicated bacterial strains ( left ) depicting the loss of membrane integrity upon treatment with the CP1 - WT peptide which is time - dependent . Arrows indicate membrane blebs , generalized membrane damage and the exclusion of cytoplasmic contents out of the cell . 1h , 2h and 3h indicate different treatment times of 1 , 2 and 3 hours respectively with the CP1 - WT and CP1 - Mut peptides as indicated in the panels . Untreated cells , used as controls , are also shown . Scale bars : 2μm . Data information : In each panel , across each fluorescent channel , the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 ( immediate addition ) and 2 hours respectively . nsP > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; Student ' s unpaired t - test . WT and Mut indicate CP1 - WT and CP1 - Mut treatments respectively .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D Transmission electron micrographs of the indicated bacterial strains (left) depicting the loss of membrane integrity upon treatment with the CP1-WT peptide which is time-dependent. Arrows indicate membrane blebs, generalized membrane damage and the exclusion of cytoplasmic contents out of the cell. 1h, 2h and 3h indicate different treatment times of 1, 2 and 3 hours respectively with the CP1-WT and CP1-Mut peptides as indicated in the panels. Untreated cells, used as controls, are also shown. Scale bars: 2μm. Data information: In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.


E - H Mean fluorescence intensities across the three fluorescent channels ( FM 4 - 64FX , FAM and DAPI fluorophores ) were estimated from confocal microscopy experiments to understand if the peptide was bound to the membrane or internalized in the cytosol of the bacteria indicated in each of the panels ( top ) . Triple staining procedure with FM 4 - 64FX ( membrane - binding dye ; red ) , FAM - conjugated peptides ( green ) and DAPI ( nuclear staining dye ; blue ) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2 . 3 software . The bacterial cells were treated with 2 % DMSO / FAM - labelled CP1 - WT / FAM - labelled CP1 - Mut for 0 ( 0hr ) or 2 hours ( 2hr ) , followed by staining with FM 4 - 64FX and DAPI consecutively . The 0th hour data represents the data obtained after immediate addition of DMSO or peptide . In each panel , green circles represent autofluorescence ( cells emitting light of wavelength corresponding to that observed for FAM fluorophore ) of DMSO - treated cells at 0 and 2 hours . Data information : In panels E - H , n = 5 biological replicates are used . In each of these panels , the mean ± SD intensity values ( averaged across all the replicates ) obtained across each fluorescent channel ( FM 4 - 64FX , FAM and DAPI ) are presented for a particular bacterial strain ( indicated on top of the panel ) . In each panel , across each fluorescent channel , the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 ( immediate addition ) and 2 hours respectively . nsP > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; Student ' s unpaired t - test . WT and Mut indicate CP1 - WT and CP1 - Mut treatments respectively .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H Mean fluorescence intensities across the three fluorescent channels (FM 4-64FX, FAM and DAPI fluorophores) were estimated from confocal microscopy experiments to understand if the peptide was bound to the membrane or internalized in the cytosol of the bacteria indicated in each of the panels (top). Triple staining procedure with FM 4-64FX (membrane-binding dye; red), FAM-conjugated peptides (green) and DAPI (nuclear staining dye; blue) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2.3 software. The bacterial cells were treated with 2% DMSO/ FAM-labelled CP1-WT/ FAM-labelled CP1-Mut for 0 (0hr) or 2 hours (2hr), followed by staining with FM 4-64FX and DAPI consecutively. The 0th hour data represents the data obtained after immediate addition of DMSO or peptide. In each panel, green circles represent autofluorescence (cells emitting light of wavelength corresponding to that observed for FAM fluorophore) of DMSO-treated cells at 0 and 2 hours. Data information: In panels E-H, n = 5 biological replicates are used. In each of these panels, the mean ± SD intensity values (averaged across all the replicates) obtained across each fluorescent channel (FM 4-64FX, FAM and DAPI) are presented for a particular bacterial strain (indicated on top of the panel). In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . A Changes in body weights relative to day 0 were noted . The mean percentage changes in body weights for each cohort are plotted across all the days . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panel A , across the entire experimental tenure of 14 days , data are presented as mean ± SD values for each cohort , averaged across both the replicates for all the five mice ( studied in the respective cohort ) . Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO - treated and the respective peptide treated cohorts ( ns P > 0 . 05 ) .

O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. A Changes in body weights relative to day 0 were noted. The mean percentage changes in body weights for each cohort are plotted across all the days. Data information: n = 2 biological replicates with 5 mice per cohort were used. In panel A, across the entire experimental tenure of 14 days, data are presented as mean ± SD values for each cohort, averaged across both the replicates for all the five mice (studied in the respective cohort). Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO-treated and the respective peptide treated cohorts (ns P > 0.05).


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . B , After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( B ) alanine transaminase ( SGPT ) , alanine aminotransferase ( SGOT ) , alkaline phosphatase ( ALP ) Relative to the values obtained for the DMSO cohort , insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic ( as seen in panel B ) Data information : biological replicates with 5 mice per cohort were used . In the panels B representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) .

O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. B, After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (B) alanine transaminase (SGPT), alanine aminotransferase (SGOT), alkaline phosphatase (ALP) Relative to the values obtained for the DMSO cohort, insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic (as seen in panel B) Data information: biological replicates with 5 mice per cohort were used. In the panels B representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . C After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( C ) creatinine and blood urea nitrogen ( BUN ) levels . Relative to the values obtained for the DMSO cohort , insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects ( as seen in panel C ) . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panels C , representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . For panels C , P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) .

O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. C After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (C) creatinine and blood urea nitrogen (BUN) levels. Relative to the values obtained for the DMSO cohort, insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects (as seen in panel C). Data information: n = 2 biological replicates with 5 mice per cohort were used. In panels C, representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. For panels C, P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . ( A ) Survival for symptoms were monitored . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panel A , percent survival was compared between untreated cohort ( N . T ) and the DMSO / peptide / ciprofloxacin - treated cohort using a two - way ANOVA test using Bonferroni correction ( ns P > 0 . 05 ; * P < 0 . 01 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. (A) Survival for symptoms were monitored. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panel A, percent survival was compared between untreated cohort (N.T) and the DMSO/peptide/ciprofloxacin-treated cohort using a two-way ANOVA test using Bonferroni correction (ns P > 0.05; * P < 0.01). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . C - E Bacterial loads in S . Typhimurium infected organs , namely , ( C ) liver , ( D ) kidney and ( E ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panels , ( C - E ) , Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. C-E Bacterial loads in S. Typhimurium infected organs, namely, (C) liver, (D) kidney and (E) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panels, (C-E), Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . F - J Histopathology analyses with representative H & E - stained liver sections at different magnifications are shown for S . Typhimurium infected liver treated with various agents . K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F - J . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels K to assign histopathological scores , data are collected from five mice in each cohort , and the representative data for only one biological replicate are presented as mean ± SD values . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively . For histopathology analyses in panels ( F - J ) except for uninfected and peptide - treated mice , pathology sections from infected or treated ( DMSO or ciprofloxacin ) mice showed aggregation of multiple inflammatory cells ( INF ) and edema ( E ) in liver parenchyma , central vein congestion ( C ) , distortion of hepatic portal vein ( HPV ) , hemorrhage ( H ) , necrosis ( N ) and pyknotic nuclei ( arrows in panels H , Scale bar : 50µm in panels ( F - J )

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-DISEASE O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-DISEASE O O O O B-DISEASE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. F-J Histopathology analyses with representative H&E-stained liver sections at different magnifications are shown for S. Typhimurium infected liver treated with various agents. K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F-J. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels K to assign histopathological scores, data are collected from five mice in each cohort, and the representative data for only one biological replicate are presented as mean ± SD values. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively. For histopathology analyses in panels (F-J) except for uninfected and peptide-treated mice, pathology sections from infected or treated (DMSO or ciprofloxacin) mice showed aggregation of multiple inflammatory cells (INF) and edema (E) in liver parenchyma, central vein congestion (C), distortion of hepatic portal vein (HPV), hemorrhage (H), necrosis (N) and pyknotic nuclei (arrows in panels H, Scale bar: 50µm in panels (F-J)


Action of the CP1 - WT peptide in treatment of A . baumannii intraperitoneal infections . L - N Bacterial loads in A . baumannii infected organs , namely , ( L ) liver , ( M ) kidney and ( N ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels ( L - N ) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O

Action of the CP1-WT peptide in treatment of A. baumannii intraperitoneal infections. L-N Bacterial loads in A. baumannii infected organs, namely, (L) liver, (M) kidney and (N) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels (L-N) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


( F ) UMAP plot showing the expression levels of EMT markers . The magenta dotted lines indicate cluster 6 ( partial EMT ) .

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(F) UMAP plot showing the expression levels of EMT markers. The magenta dotted lines indicate cluster 6 (partial EMT).


( E ) , ( F ) Heatmap of DEGs in ROR1high and ROR1low cells in the S2 - VP10 xenograft ( E ) or PDO # 1 xenograft ( F ) .

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(E), (F) Heatmap of DEGs in ROR1high and ROR1low cells in the S2-VP10 xenograft (E) or PDO#1 xenograft (F).


( A ) Kaplan - Meier disease - specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database ( n = 154 ) . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; log - rank test ( A )

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(A) Kaplan-Meier disease-specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database (n = 154) . Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; log-rank test (A)


( C ) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies ( n = 3 or n = 5 , biological replicates ) . Mouse cells expressing H - 2Kd / H - 2Dd were eliminated . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; two - sided t - test ( C )

O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies (n = 3 or n = 5, biological replicates). Mouse cells expressing H-2Kd/H-2Dd were eliminated. Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; two-sided t-test (C)


( G ) Representative images of tissue stained with H & E and AB / PAS , and for ROR1 in the PDO # 1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO # 1 xenograft . Data information : Scale bars , 100 µm ( G ) .

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(G) Representative images of tissue stained with H&E and AB/PAS, and for ROR1 in the PDO#1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO#1 xenograft. Data information: Scale bars, 100 µm (G).


( H ) The expression level of AURKB in PANC - 1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2 - VP10 and PDO # 1 xenografts ( n = 3 ) . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) The expression level of AURKB in PANC-1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2-VP10 and PDO#1 xenografts (n = 3). Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( I ) Western blot analysis of ROR1 , AURKB , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O

(I) Western blot analysis of ROR1, AURKB, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( J ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO ( vehicle ) or Aurora kinase inhibitors ( n = 3 , biological replicates ) . Tozasertib , pan - Aurora kinase inhibitor ; Barasertib , selective Aurora B kinase inhibitor . Representative images of organoids are shown . An area of 2 , 000 μm2 and more was identified as an organoid . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars 1 mm ( J ) Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Treatment of PDO#1 and S2-VP10 organoids with DMSO (vehicle) or Aurora kinase inhibitors (n = 3, biological replicates). Tozasertib, pan-Aurora kinase inhibitor; Barasertib, selective Aurora B kinase inhibitor. Representative images of organoids are shown. An area of 2,000 μm2 and more was identified as an organoid. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars 1 mm (J) Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( K ) Organoid formation assay of ROR1high or ROR1low cells derived from S2 - VP10 EGFP ( control ) or S2 - VP10 AURKB - EGFP xenografts ( n = 3 , biological replicates ) . Representative images and the number of organoids are shown . Data information : Scale bars , 1 mm ( K ) . Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Organoid formation assay of ROR1high or ROR1low cells derived from S2-VP10 EGFP (control) or S2-VP10 AURKB-EGFP xenografts (n = 3, biological replicates). Representative images and the number of organoids are shown. Data information: Scale bars, 1 mm (K). Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( L ) Western blot analysis of ROR1 , AKT , phospho - AKT ( Ser473 ) , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O

(L) Western blot analysis of ROR1, AKT, phospho-AKT (Ser473), and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( N ) Western blot analysis of ROR1 , c - Myc , phospho - RB ( Ser780 ) , E2F1 , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O

(N) Western blot analysis of ROR1, c-Myc, phospho-RB (Ser780), E2F1, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( C ) Tumor growth curve of the four groups ( control , Dox only , gemcitabine only , and Dox + gemcitabine ) ( n = 8 ) . The endpoint for each group is shown by a slash . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Tumor growth curve of the four groups (control, Dox only, gemcitabine only, and Dox + gemcitabine) (n = 8). The endpoint for each group is shown by a slash. Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05.


( A ) - ( C ) Representative images of ROR1 , mCherry , and aSMA staining in the primary tumor ( pancreas ) ( A ) and metastatic lesions in the lung ( B ) and mesenteric lymph node ( C ) after orthotopic transplantation of S2 - VP10 - mCherry cells into the pancreas of BRJ mice . Data information : Scale bars , 100

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-TISSUE O O B-ORGANISM O O O O O O O O

(A)-(C) Representative images of ROR1, mCherry, and aSMA staining in the primary tumor (pancreas) (A) and metastatic lesions in the lung (B) and mesenteric lymph node (C) after orthotopic transplantation of S2-VP10-mCherry cells into the pancreas of BRJ mice. Data information: Scale bars, 100 µm


( D ) , ( E ) Representative images of S2 - 013 orthotopic xenografts stained with H & E and for ROR1 in the pancreas ( D ) and mesenteric lymph node ( E ) . Data information : Scale bars , 100

O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O

(D), (E) Representative images of S2-013 orthotopic xenografts stained with H&E and for ROR1 in the pancreas (D) and mesenteric lymph node (E). Data information: Scale bars, 100 µm


( A ) , H3K4me1 , K3K4me3 , H3K27ac , and open chromatin profiles around the ROR1 gene by CUT & RUN and ATAC - sequencing in cultured S2 - VP10 cells ( A ) The ROR1 promoter ( blue - shaded boxes ) and enhancer elements ( green - shaded box ) of ROR1 are indicated .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O

(A), H3K4me1, K3K4me3, H3K27ac, and open chromatin profiles around the ROR1 gene by CUT&RUN and ATAC-sequencing in cultured S2-VP10 cells (A) The ROR1 promoter (blue-shaded boxes) and enhancer elements (green-shaded box) of ROR1 are indicated.


( E ) YAP occupancy at the ROR1 enhancer as determined by ChIP - qPCR . The known YAP targets , CTGF and CYR61 promoters , were tested as positive controls , and ROR1 intron 1 and AFM promoter were tested as negative controls ( n = 4 , biological replicates ) .

O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(E) YAP occupancy at the ROR1 enhancer as determined by ChIP-qPCR. The known YAP targets, CTGF and CYR61 promoters, were tested as positive controls, and ROR1 intron 1 and AFM promoter were tested as negative controls (n = 4, biological replicates).


( G ) Western blot analysis of YAP , TAZ , ROR1 , and β - actin in S2 - VP10 cells transfected with control or YAP1 / WWTR1 siRNA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O

(G) Western blot analysis of YAP, TAZ, ROR1, and β-actin in S2-VP10 cells transfected with control or YAP1/WWTR1 siRNA.


( I ) Relative expression of ROR1 and YAP target genes ( CTGF and CYR61 ) in S2 - VP10 cells treated with DMSO or verteporfin ( VP ) ( n = 3 , biological replicates ) . mRNA levels are normalized to that of RPS18 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O

(I) Relative expression of ROR1 and YAP target genes (CTGF and CYR61) in S2-VP10 cells treated with DMSO or verteporfin (VP) (n = 3, biological replicates). mRNA levels are normalized to that of RPS18.


( M ) Relative luciferase activity using the ROR1 reporter in S2 - VP10 cells treated with DMSO or BET inhibitor ( JQ1 ) ( n = 3 , biological replicates ) . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( M )

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Relative luciferase activity using the ROR1 reporter in S2-VP10 cells treated with DMSO or BET inhibitor (JQ1) (n = 3, biological replicates). Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (M)


( O ) , Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . Relative expression of ROR1 ( O ) , mRNA levels are normalized to that of RPS18 . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( O )

O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O), Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). Relative expression of ROR1 (O), mRNA levels are normalized to that of RPS18. Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (O)


( P ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . representative images of organoids , and organoid area ( P ) are shown . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars , 1 mm ( P ) . Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( P ) .

O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(P) Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). representative images of organoids, and organoid area (P) are shown. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars, 1 mm (P). Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (P).


A BAK - 6H permeabilising activity is exacerbated by cBID . BAK - 6H ( mBAK ∆ C21 - 6H ) was loaded at the indicated concentrations onto Ni - NTA liposomes ( 5 μM ) prior to the addition of cBID . The release of carboxyfluorescein was measured over time . Results representative of at least 3 independent experiments .

O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.


B BAK - 6H oligomerises on liposomes induced by cBID . Liposomes were incubated with BAK - 6H ( 150 nM ) and the indicated concentration of cBID or a BID BH3 peptide for 60 min . Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN - PAGE . In parallel , samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran , normalised to total fluorescence of detergent permeabilised liposomes . Results representative of 3 independent experiments .

O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O

B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.


C BAK - 6H oligomerisation on liposomes is blocked by BCL - XL . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( WT , 90 nM ) or cBID M97A ( 90 nM ) that has reduced affinity for BCL - XL , in the presence or absence of BCL - XL for 60 mins prior to analysis of BAK oligomerisation by BN - PAGE . Results representative of 2 independent experiments . Liposome permeabilisation was monitored with the indicated combinations of cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and BCL - XL ( 1 μM ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum .

O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


D BAK - 6H oligomerisation on liposomes involves its BH3 domain . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( 90 nM ) . Antibody recognising the BAK BH3 domain ( 4B5 ) or α1 - α2 loop ( 7D10 ) , as shown on the structure of BAK ( PDB : 2IMS ) , were added ( 2 . 4 μg / μl ) either prior to ( pre ) or after ( post ) incubation with cBID for 60 mins . BAK : Ab complex is indicated ( arrowhead ) . 7D10 which recognises the activated form of mBAK ( Iyer et al . , 2016 ) , bound to and gel - shifted BAK when added either before or after activation with cBID . In contrast , 4B5 failed to appreciably gel - shift inactive BAK ( lanes 3 and 5 ) or BAK that was already activated ( lane 6 ) , but gel - shifted BAK when present during cBID activation ( lane 4 ) . Liposome permeabilisation was assessed after 20 min incubation with cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and antibodies 4B5 ( 2 . 5 μg ) or 7D10 ( 2 . 5 μg ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum .

O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


B BAK , or BAK with cysteine introduced at the indicated positions ( on a BAK ∆ Cys background ) were stably expressed in Bax - / - Bak - / - MEFs and assess for BAK expression by immunoblotting ( inset panel ; * indicates non - specific band . ) and apoptotic activity in response to etoposide treatment .

O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O

B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.


C E24 and R169 are proximal in inactive BAK on mitochondria . Mitochondria - enriched membrane fractions from cells were incubated with oxidant ( CuPhe ) and induced intramolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


D E24 and R169 dissociate during BAK activation . Mitochondria - enriched membrane fractions from cells were incubated with cBID ( 100 nM ) prior to oxidant ( CuPhe ) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


C Bax - / - Bak - / - MEFs expressing the indicated BAK variants were treated with doxycycline ( Dox , 3 h ) , incubated for a further 24 h in the presence or absence of BH3 - mimetic compounds , and cell death was assessed by PI uptake .

O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O

C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.


D Mutation of BH4 promotes BAK activating conformation change . Cells were treated with doxycycline ( 3 h ) to induce BAK expression followed by incubation with BH3 - mimetics where indicated ( for 2 h ) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK ( G3172 ) .

O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O

D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).


D BAK wild - type , or BAK with an engineered α1 - α2 loop thrombin cleavage site were stably expressed in Bax - / - Bak - / - MEFs . Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline ( CuPhe ) to induce disulphide - linkage . Samples were run on SDS - PAGE under non - reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N - terminus upstream of the thrombin cleavage site ( aa23 - 38 ) or an antibody recognising the BH3 domain ( 4B5 ) downstream of the cleavage site . Schematic indicates the nature of the BAK protein detected by each antibody . Disulphide - linkage indicated by a hashed line . Note that the N - terminal portion of thrombin cleaved BAK could not be detected with the N - terminal antibody unless it was intramolecularly disulphide - linked . * cBID ( 100 nM ) was added to activate BAK during thrombin cleavage . Data representative of two independent experiments .

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D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.


E Cleavage of the BAK α1 - α2 loop potentiates MOMP . Membrane fractions from Bax - / - Bak - / - MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID ( 10 nM ) for the indicated times . Membrane ( P ) and supernatant ( S ) fractions were separated and immunoblotted for cytochrome c or BAK 4B5 .

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E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.


A ) H & E and immunohistochemistry reveal that β - catenin accumulates in tumors emerging in one - year - old female miR - 424 ( 322 ) / 503 - / - mice . The panel is a representative image of n = 5 independent tumors . The scale bar indicates 100 micrometers ( µM ) .

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A) H&E and immunohistochemistry reveal that β-catenin accumulates in tumors emerging in one-year-old female miR-424(322)/503-/- mice. The panel is a representative image of n=5 independent tumors. The scale bar indicates 100 micrometers (µM).


B ) Bar graphic showing that human breast cancers with deletion of the miR - 424 / 503 locus have higher protein levels of β - catenin ( RPPA ) . P - value is calculated based on one - sided Wilcoxon rank - sum test . In the box - plots in B and C the relative expression of β - catenin in the RPPA data from TCGA is shown . The yellow line indicates the median and the bars indicate the 75th percentile ( Q3 ) + 1 . 5 interquartile range ( IQR ) .

O O O O O O B-ORGANISM B-DISEASE I-DISEASE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Bar graphic showing that human breast cancers with deletion of the miR-424/503 locus have higher protein levels of β-catenin (RPPA). P-value is calculated based on one-sided Wilcoxon rank-sum test. In the box-plots in B and C the relative expression of β-catenin in the RPPA data from TCGA is shown. The yellow line indicates the median and the bars indicate the 75th percentile (Q3)+1.5 interquartile range (IQR).


C ) The bar graph shows that human breast cancers with low expression levels of miR - 424 and miR - 503 have higher protein levels of β - catenin . P - value is calculated based on one - sided Wilcoxon rank - sum test .

O O O O O O O B-ORGANISM B-DISEASE I-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

C) The bar graph shows that human breast cancers with low expression levels of miR-424 and miR-503 have higher protein levels of β-catenin. P-value is calculated based on one-sided Wilcoxon rank-sum test.


B ) The bar graphs show the number of PROCR + in 1 - year - old female WT and miR - 424 ( 322 ) / 503 KO mice during diestrus . The error bars show the standard deviation .

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B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation.


E ) The panel shows the lack of induction of bona fide WNT targets in miR - over expressing cells from panel C compared with EV control . UT indicates samples untreated with WNT3 .

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E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.


D ) Western blot of LRP6 and TBLR1 in the miR - cluster - Dox - MCF - 10A model upon upregulation of miR - cluster with 100 ng / ml of doxycycline for the specified number of days . The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β - actin . The expression level of miR - 424 and miR - 503 is indicated in the bar graph below the blots . UN indicates samples untreated with doxycycline . The error bars show the standard deviation .

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D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.


F ) Western blot of LRP6 and TBLR1 expression in WT and miR - 424 / 503 - KO basal cell - derived organoids after 14 days in culture with and without Wnt3a ( 100ng / ml ) . The arrow shows the correct TBLR1 band .

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F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.


G ) IHC staining showing that LRP6 accumulates in the epithelium of female miR - 424 ( 322 ) / 503 - / - mice ( 1 - year - old ) . The scale bar indicates 100 micrometers ( µM ) .

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G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).


A ) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR - cluster - Dox - MCF - 10A cells after the miR - 424 / 503 cluster is induced with 100ng / ml of dox for three days . A CDC25A - binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls . The error bars show the standard deviation .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.


B ) The WT - blot shows the expression of LRP6 upon upregulation of miR - 424 / 503 in miR - cluster - Dox - MCF - 10A cells ( Dox + ) when one or both of the conserved microRNA binding sites ( # 1 and # 3 ) are blocked by target protection .

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B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.


C ) The WT - blot shows the expression of LRP6 and β - catenin in cells in miR - cluster - Dox - MCF - 10A cells cultured in the presence of Dox and with and without WNT3a ligand ( 100ng / ml for 6 hours ) when the microRNA binding sites # 1 and # 3 are blocked by target protection .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.


Nurr1 ( N ) and Foxa2 ( F ) synergistically downregulate SGK1 expression in cultured glia . ( A ) Up - and downregulated genes in the microarray data for the cultured glia transduced with N + F ( vs . mock - transduced control ) . Sgk1 is marked with an arrow in the top downregulated genes .

B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N+F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes.


Effect of N and F on SGK1 expression in glial cells shown in the microarray ( B ) , RNA - seq ( C ) , Data in ( B and C ) represent values of fold change ( FC ) or read count ( RC ) with color intensities . n = 3 . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n=3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Effect of N and F on SGK1 expression in glial cells shown in the RT - PCR analyses . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

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Effect of N and F on SGK1 expression in glial cells shown in the RT-PCR analyses. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation

O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY

Downregulation of SGK1 is responsible for N+F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB

O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD


WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation ( E ) and thus blocking the release of NFκB from the NFκB - IκB inhibitory complex ( F ) .

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O

WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F).


N + F - mediated downregulation of NFκB signaling ( phosphorylation of p65 , p - p65 , G ) were alleviated by forced SGK1 expression in the VM - glial cultures .

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N+F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) were alleviated by forced SGK1 expression in the VM-glial cultures.


H ) N + F - mediated downregulation of pro - inflammatory cytokine expression ( IL - 1β , TNFα , H ) were alleviated by forced SGK1 expression in the VM - glial cultures .

O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE O B-CELL_TYPE O O

H) N+F-mediated downregulation of pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures.


( I ) SGK1 inhibition blocks glial NFκB signaling . Data are represented as mean ± SEM . n = 4 - 6 . One - way ANOVA . Significantly different at p = 0 . 002 * , 0 . 00004 * * , 0 . 00001 # , 3 . 01E - 10 # # , 3 . 78E - 06 + , 1 . 59E - 07 + + in graph I .

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(I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n=4-6. One-way ANOVA. Significantly different at p=0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I.


Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition

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Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition


Glial pro - inflammatory cytokine expression regulated by SGK1

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Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition ( J - M ) and overexpression ( N ) .

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Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J-M) and overexpression (N).


In the RNA - seq data ( GSE145490 , normalized read count ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in the VM - glia treated with the SGK1 inhibitor GSK - 650394 vs the vehicle ( DMSO ) - treated control were analyzed . A and B , Top gene ontologies ( GOs ) and KEGG pathways of the genes downregulated in VM - glia by the SGK1 inhibitor treatment . The purple bar indicates the number of genes under the designated GO term / KEGG pathway . The red bar indicates the p - value , and the negative log of the p - value ( bottom ) is plotted on the X - axis .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed. A and B, Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis.


In the RNA - seq data ( GSE145490 , normalized read count ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in the VM - glia treated with the SGK1 inhibitor GSK - 650394 vs the vehicle ( DMSO ) - treated control were analyzed .

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In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed.


C , Gene set enrichment analysis ( GESA ) for inflammatory and immune responses .

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C, Gene set enrichment analysis (GESA) for inflammatory and immune responses.


D , Scatterplots for the differentially expressed genes ( DEGs ) highlighting the inflammatory / immune genes that are most significantly downregulated in the inhibiter - treated cultures .

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D, Scatterplots for the differentially expressed genes (DEGs) highlighting the inflammatory/immune genes that are most significantly downregulated in the inhibiter-treated cultures.


E , Heatmap for selected inflammatory genes in the SGK1 inhibitor - treated and - untreated glial cultures . Data represent the RC values ( inside box ) and log2 [ SGK1 inhibitor - treated / control ] ( color intensity ) .

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E, Heatmap for selected inflammatory genes in the SGK1 inhibitor-treated and -untreated glial cultures. Data represent the RC values (inside box) and log2[SGK1 inhibitor-treated/control] (color intensity).


F , Microglia and astrocyte reactivation - specific marker expressions in the RNA - seq data .

F, Microglia and astrocyte reactivation-specific marker expressions in the RNA-seq data.


mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the RNA - seq Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + +

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the RNA-seq Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++


B , mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the q - PCR analyses in other independent VM - glial cultures treated with the inhibitor ( GSK - 650394 ) and sh - Sgk1 and si - Sgk1 ( vs . DMSO , sh - control , and si - control , respectively ) ( B ) . n = 3 ; Student ' s t - test . Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + + in graph B .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the q-PCR analyses in other independent VM-glial cultures treated with the inhibitor (GSK-650394) and sh-Sgk1 and si-Sgk1 (vs. DMSO, sh-control, and si-control, respectively) (B). n=3; Student's t-test. Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++ in graph B.


C , Immunoblot analysis to assess NLRP3 inflammasome activation . To activate the inflammasome pathway , VM - derived glial cells were treated with LPS ( 0 . 25 µg / ml , 3h ) and then ATP ( 2 . 5 mM , 30 min ) in the presence or absence of the SGK1 inhibitor GSK - 650394 . Two days later , intracellular and released levels of pro - and activated caspase - 1 and IL - 1β proteins were determined in the culture media and cell lysates , respectively .

O O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

C, Immunoblot analysis to assess NLRP3 inflammasome activation. To activate the inflammasome pathway, VM-derived glial cells were treated with LPS (0.25 µg/ml, 3h) and then ATP (2.5 mM, 30 min) in the presence or absence of the SGK1 inhibitor GSK-650394. Two days later, intracellular and released levels of pro- and activated caspase-1 and IL-1β proteins were determined in the culture media and cell lysates, respectively.