Journal: American Journal of Physiology - Renal Physiology

Article Title: Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes

doi: 10.1152/ajprenal.00097.2021

Figure Lengend Snippet: Nephrotic syndrome (NS) extracellular vesicles (EVs) stimulate significantly higher phosho-p38 (p-p38) expression compared with NS relapse plasma and relapse NS EV-depleted plasma on cultured podocytes. A: representative images of flow cytometry plots displaying p-p38 expression. Anti-p-p38 labeled with PE-Cy7 and the appropriate isotype control was used. The gating strategy after excluding cell aggregates with forward and side scatter parameters is shown. PE-Cy7-positive cells were prominently shifted with IL-13 stimulation for 30 min. Both relapse NS plasma and relapse EV stimulation caused increases in the p-p38-positive cell percentage (A) and mean fluorescent intensity (MFI; B). C: MFI ratio of p-p38 on podocytes was significantly higher with relapse EV stimulation compared with NS plasma and NS EV-depleted plasma stimulation from the same patients (n = 10). Data are presented as means ± SD. A paired Student’s t test was performed. D: Western blot imaging showed increased p-p38 expression and decreased synaptopodin expression at 1–4 h of plasma and EV stimulation. E: quantification of band intensity of p-p38 compared with α-tubulin by ImageJ analysis. Means and SDs of three independent experiments are shown. F: Quantification of band intensity of synaptopodin as fold induction (FI) to α-tubulin by ImageJ analysis. Columns represent means; error bars are defined as SDs of three independent experiments.

Article Snippet: Twenty-five micrograms of protein were loaded per well and separated by 10% or 12% SDS-PAGE, transferred to a PVDF membrane, and probed with 1:1,000 diluted primary antibodies against anti-phospho-p38 (p-p38; clone E-1, Santa Cruz Biotechnology), anti-tumor susceptibility gene 101 (TSG-101) antibody [clone EPR7130(B), Abcam, Cambridge, UK)], anti-α-tubulin (clone B-7, Santa Cruz Biotechnology), anti-synaptopodin (clone D-9, Santa Cruz Biotechnology), anti-flotillin (Cat. No. 3252, Cell Signaling Technology, Frankfurt am Main, Germany), anti-glucose-regulated protein 94 (GRP94; clone D6X2Q, Cell Signaling Technology), and anti-ALG-2-interacting protein X (ALIX; clone 3A9, Cell Signaling Technology).

Techniques: Expressing, Cell Culture, Flow Cytometry, Labeling, Western Blot, Imaging

Journal: bioRxiv

Article Title: Constitutive expression of IκBζ promotes tumor growth and immunotherapy resistance in melanoma

doi: 10.1101/2024.09.19.613946

Figure Lengend Snippet: A. Immunoblot analysis of total and phosphorylated STAT3 (Y705), STAT1 (Y701), IκBα (S32), and p65 (S536) in control or IκBζ-depleted LOX-IMVI or D4M-3A cells, and in control or IκBζ-overexpressing B16-F10 cells. B. IHC staining of phosphorylated STAT3 (Y705) in control or IκBζ-depleted D4M-3A tumors, and in control or IκBζ-overexpressing B16-F10 tumors at the experimental endpoint. Scale: 100 µM. C. Chromatin fractionation of LOX-IMVI control or NFKBIZ knockdown cells. GAPDH and H3 served as internal controls for the chromatin-unbound and chromatin-bound fraction, respectively. D. Chromatin immunoprecipitation of STAT3 and p65 in control or NFKBIZ knockdown LOX-IMVI cells. E. Gene expression of IκBζ target genes in control or IκBζ-overexpressing MV3 cells, in the presence or absence of STAT3. STAT3 was lentivirally knocked down using shRNA and control cells were generated using non-coding shRNA (sh ctrl). Gene expression was subsequently analyzed in the presence or absence of transient IκBζ overexpression, normalized to the reference gene RPL37A. F. Control or IκBζ-depleted LOX-IMVI cells were treated with 100 ng/mL IL-6 for 24 h. Relative mRNA levels were analyzed and normalized as in ( E ). G. Tumor cell viability of MV3 cells, treated with the IKK inhibitor IMD-0354 (5µM) to inhibit NF-κB or a Stattic (10 µM) to suppress STAT1 and STAT3 activity. Cell proliferation was assessed using the CellTiterGlo assay from Promega. N = 3. H. Immunoblot detection of pSTAT3 (Y705) levels in IgG- or anti-PD-1-treated control or IκBζ-overexpressing tumors at the endpoint. β-actin controlled equal protein loading. Data represent the mean of 3 biological replicates ± standard deviation (SD). Significance was calculated using a 2-tailed Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: After 20 min of sonification, chromatin was incubated with protein G-coupled Dynabeads (Thermo Fisher, Cat.10003D) and 2 µg self-made antibody against IκBζ (raised against the peptide CRKGADPSTRNLENEQ, ordered at Eurogentec), 3 µg α-STAT3 (Thermo Fisher, Cat. MA1-13042), 2 µg α-p65 (Diagenode, Cat. C15310256), or an IgG antibody as control (Abcam, ab46540), overnight at 4°C on a rotator.

Techniques: Western Blot, Control, Immunohistochemistry, Fractionation, Knockdown, Chromatin Immunoprecipitation, Expressing, shRNA, Generated, Over Expression, Activity Assay, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: IKKβ acts as a tumor suppressor in cancer-associated fibroblasts during intestinal tumorigenesis

doi: 10.1084/jem.20150576

Figure Lengend Snippet: Fibroblast-specific Ikkβ deletion promotes colon tumorigenesis. (A) Tumor incidence in Ikkβ F/F and Ikkβ ΔFib mice at the end of the AOM/DSS regimen on day 84. Data are mean from two independent experiments ± SE; n ≥ 7; ns, not significant. (B) Mean tumor size in Ikkβ F/F and Ikkβ ΔFib mice. Data are mean ± SE; n ≥ 7; ***, P < 0.0001 by Student’s t test. (C) Histogram showing size distribution of tumors in Ikkβ F/F and Ikkβ ΔFib mice. (D and E) Overview of representative sections of the Swiss rolls that were used for tumor counting; bars, 1 mm. (F–H) Immunohistochemical analysis of BrdU incorporation (F and G) and proliferation index (H) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bars, 50 µm. (I) Quantification of cleaved caspase 3 staining for the apoptotic index in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. (J) Tumor incidence in Ikkβ F/F and Ikkβ ΔFib mice at the end of the sporadic tumor regimen (six weekly AOM injections without additional DSS administration) at week 22. Data are mean from two independent experiments ± SE; n ≥ 9; ns, not significant. (K) Mean tumor size in Ikkβ F/F and Ikkβ ΔFib mice. Data are mean ± SE; n ≥ 9; **, P < 0.005 by Student’s t test. (L) Quantification of the proliferation index in Ikkβ F/F and Ikkβ ΔFib tumors. Data are mean ± SE; n ≥ 20 tumors per genotype; *, P < 0.05 by Student’s t test. (M and N) Quantification of blood vessel FCD length and area in tumors from fluorescein injected Ikkβ F/F and Ikkβ ΔFib mice by CLSM. Data are mean ± SE; n ≥ 6. ***, P < 0.0001 by Student’s t test. (O–Q) Quantitative analysis of EpCAM − CD45 − CD31 + endothelial cells (O), EpCAM − CD45 − PDGFRα + fibroblasts (P), and CD45 + leukocytes (Q) in tumors from Ikkβ F/F and Ikkβ ΔFib mice analyzed by flow cytometry. Data are mean ± SE; n ≥ 6. *, P < 0.05 by Student’s t test. (R–T) Immunohistochemical analysis of p-Stat3 Y705 (R and S) and quantification (T) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bar, 200 µm. (U–W) Immunohistochemical analyses of p-Akt S473 (U and V) and quantification (W) in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bar, 300 µm. (X–Z) Immunohistochemical analysis of β-catenin (X and Y) and quantification (Z) of nuclear expression in tumor epithelial cells from Ikkβ F/F and Ikkβ ΔFib mice. Bars, 300 µm. Data in H, I, T, W, and Z are mean ± SE; n ≥ 10 tumors of each genotype; *, P < 0.05; ***, P < 0.0001 by Student’s t test.

Article Snippet: The following primary antibodies were used: α-BrdU (MCA2060; AbDSerotec), α-cleaved caspase 3 (9661S; Cell Signaling Technology), α-p-Stat3 Y705 (9145; Cell Signaling Technology), α-pAkt S473 (3787S; Cell Signaling Technology), α-β-catenin (06–734; EMD Millipore), α-p-Met (ab5662; Abcam), α-E-cadherin (610182; BD).

Techniques: Immunohistochemical staining, BrdU Incorporation Assay, Staining, Injection, Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: IKKβ acts as a tumor suppressor in cancer-associated fibroblasts during intestinal tumorigenesis

doi: 10.1084/jem.20150576

Figure Lengend Snippet: Met inhibition prevents tumor promotion in Ikkβ ΔFib mice upon CAC challenge. (A) Schematic overview of the CAC model and mode of capmatinib application. During DSS administration, capmatinib was paused and mice received tamoxifen containing AIN-76A diet. (B and C) Tumor incidence (B) and average tumor size (C) in Ikkβ F/F and Ikkβ ΔFib mice at the end of the AOM/DSS regimen that received capmatinib or were left untreated. Data are mean ± SE; n ≥ 7; **, P < 0.005; ***, P < 0.0001 by ANOVA, followed by Bonferroni post hoc test for multiple datasets. (D) Quantification of blood vessel FCD length and area in tumors from capmatinib-treated Ikkβ F/F and Ikkβ ΔFib mice by CLSM. Data are mean ± SE; n ≥ 6; ns, not significant. (E) Quantification of immunohistochemical analysis of p-Akt S473 in tumor epithelial cells from untreated and capmatinib treated Ikkβ ΔFib mice. (F) Quantification of immunohistochemical analysis of p-Stat3 Y705 in tumor epithelial cells from untreated and capmatinib-treated Ikkβ ΔFib mice. (G) Quantification of immunohistochemical analysis of nuclear β-catenin in tumor epithelial cells from untreated and capmatinib-treated Ikkβ ΔFib mice. Data in E–G are mean ± SE; n ≥ 10 tumors of each genotype; *, P < 0.05; ***, P < 0.0001 by Student’s t test. (H) Flow cytometric analysis of lamina propia lymphocytes in Ikkβ F/F and Ikkβ ΔFib mice on day 15 of the CAC model revealed no difference in the numbers recruited CD3 + CD4 + , CD4 + FoxP3 + , and CD4 + IFNγ + when the mice received a diet containing the Met inhibitor capmatinib together with tamoxifen. Data are mean ± SE; n ≥ 7 mice per group; ns, not significant.

Article Snippet: The following primary antibodies were used: α-BrdU (MCA2060; AbDSerotec), α-cleaved caspase 3 (9661S; Cell Signaling Technology), α-p-Stat3 Y705 (9145; Cell Signaling Technology), α-pAkt S473 (3787S; Cell Signaling Technology), α-β-catenin (06–734; EMD Millipore), α-p-Met (ab5662; Abcam), α-E-cadherin (610182; BD).

Techniques: Inhibition, Immunohistochemical staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Cilia ablation reduces STAT3 activation in response to Pkd1 inactivation. (A) Representative images and (B and C) quantification of pSTAT3Y705 immunostaining in kidneys from 12-week-old control, iKif3aΔTub, iPkd1ΔTub, and iPkd1ΔTub; iKif3aΔTub mice (6 weeks after the completion of doxycycline treatment). Scale bar, 50 µm. Each dot represents one individual mouse. One-way ANOVA followed by Tukey–Kramer test, **P<0.01, ***P<0.001. (D) Representative images of STAT3, F4/80, and CD3 staining on serial sections from 12-week-old control iPkd1ΔTub (n=3). Scale bar, 100 µm.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Activation Assay, Immunostaining, Control, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Ccl2 expression by tubular cells is required for STAT3 activation in response to Pkd1 disruption. (A) Western blot of STAT3 phosphorylation in kidney lysates from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iCcl2ΔTub mice (7 weeks after the completion of doxycycline treatment). Each lane indicates one individual mouse. (B and C) Quantification of (B) Socs3 and (C) Lcn2 mRNA abundance in kidneys from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iCcl2ΔTub mice. (D) Representative images and (E and F) quantification of pSTAT3Y705 immunostaining in kidneys from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iCcl2ΔTub mice. Scale bar, 50 µm. (B, C, E, and F) Each dot represents one individual mouse. One-way ANOVA followed by Tukey–Kramer test, *P<0.05, **P<0.01, ***P<0.001. AU, arbitrary units.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Expressing, Activation Assay, Disruption, Western Blot, Phospho-proteomics, Control, Immunostaining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Tubule-specific Stat3 inactivation does not prevent early kidney growth in response to Pkd1 disruption. (A and B) Representative (A) Western blot and (B) quantification of STAT3 in kidney homogenates from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice (7 weeks after the completion of doxycycline treatment). Each lane indicates one individual mouse. Bars are mean±SEM of six mice per group. Two-tailed t test, ***P<0.0001. (C) STAT3 immunostaining of kidney sections from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Representative images of three mice per group. Scale bar, 100 µm. (D) Representative images and (E) quantification of STAT3 immunostaining in tubular cysts from 13-week-old iPkd1ΔTub and iPkd1ΔTub; iStat3ΔTub mice. (F) Quantification of Socs3 mRNA abundance in kidneys from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. (G) Representative kidneys from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Scale bar, 2 mm. (H) Kidney weight to body weight ratio (KW/BW) at 13 weeks. (I) Representative PAS-stained kidney sections from 13-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice at 13 weeks. Scale bar, 0.5 mm. Each dot represents one individual mouse. One-way ANOVA followed by Tukey–Kramer test, **P<0.01, ***P<0.001. AU, arbitrary units.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Disruption, Western Blot, Control, Two Tailed Test, Immunostaining, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Tubule-specific Stat3 inactivation does not prevent cystic kidney growth nor kidney function decline in response to Pkd1 disruption. (A) Kaplan–Meier survival curves of control, iPkd1ΔTub (n=16), and iPkd1ΔTub; iStat3ΔTub (n=20) mice. (B) Body weight (BW) of 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub animals (12 weeks after the completion of doxycycline treatment). (C) Representative kidneys from the same animals. Scale bar, 2 mm. (D) Kidney weight to body weight ratio (KW/BW) at 18 weeks. (E) Representative PAS staining of kidneys sections and (F) measurement of cysts area fraction from 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Scale bar, 0.5 mm. Mann–Whitney test, * P<0.05. Quantification of (G) Socs3, (H) Lcn2, and (I) Havcr1 mRNA abundance in kidneys from 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Measurement of (J) plasma creatinine and (K) urea from 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub animals. (B, D, F, G–K) Each dot represents one individual mouse. (B, D, G–K) One-way ANOVA followed by Tukey–Kramer test, *P<0.05, **P<0.01, ***P<0.001. AU, arbitrary units.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Disruption, Control, Staining, MANN-WHITNEY, Clinical Proteomics

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Tubule-specific Stat3 inactivation increases polycystic kidney inflammation. (A) Representative examples of interstitial inflammation scoring and distribution of renal interstitial inflammation scores in PAS-stained kidneys from 18-week-old iPkd1ΔTub and iPkd1ΔTub; iStat3ΔTub mice. Numbers in the bars indicate number of mice per score; P is the P value from Mann–Whitney test. (B) Representative images of F4/80, CD3, and CXCL10 immunostaining of kidney sections from 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Scale bar, 100 µm. (C–H) Quantification of (C) Cd3, (D) Ccr2, (E) Ccl2, (F) Il6, (G) Ccl5, and (H) Cxcl10 mRNA abundance in kidneys from 18-week-old control, iPkd1ΔTub, and iPkd1ΔTub; iStat3ΔTub mice. Each dot represents one individual mouse. One-way ANOVA followed by Tukey–Kramer test, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Staining, MANN-WHITNEY, Immunostaining, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1681/ASN.2019090959

Figure Lengend Snippet: Stat3 inactivation in tubular cells increases inflammatory cytokine transcriptional response. (A–C) Quantification of (A) Ccl5, (B) Cxcl10, and (C) Ccl2 mRNA abundance in mIMCD3 cells stably expressing shRNAs targeting STAT3 (sh1 and sh2) or a scramble control shRNA (scr) treated with LPS or vehicle for 24 hours. Bars are mean±SEM of eight independent experiments. (D) Representative Western blot and (E) quantification of three independent experiments of STAT3 phosphorylation and RELA expression in whole-cell lysates from mIMCD3 cells expressing the indicated shRNAs, 24 hours after LPS or vehicle treatment. (F) Representative Western blot and quantification of four independent experiments of (G) STAT3 and (H) RELA expression in nuclear extracts from mIMCD3 cells expressing the indicated shRNAs, 24 hours after LPS or vehicle treatment. Whole-cell lysates (WCL) served as a positive control for tubulin labeling. (A–C, E, G, and H). Dots represent independent experiments. Two-way paired ANOVA followed by Tukey–Kramer test, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: For Immunostaining We used the following antibodies: pSTAT3 Y705 (1:100, 9145; Cell Signaling Technology), STAT3 α (1:100, clone D1A5, 8768; Cell Signaling Technology), F4/80 (1:100, clone Cl:A3-1, MCA497R; Bio-Rad), CD3 (1:100, ab16669; Abcam), LY6B (1:100, ab53457; Abcam), CXCL10 (1:100, ab9807; Abcam), and KI67 (1:100, ab15580; Abcam).

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Phospho-proteomics, Positive Control, Labeling