Journal: PLoS ONE

Article Title: Inhibitory effect of ginsenoside Rg3 on cancer stemness and mesenchymal transition in breast cancer via regulation of myeloid-derived suppressor cells

doi: 10.1371/journal.pone.0240533

Figure Lengend Snippet: (A) Tumor volume curves comparing the Rg3 treatment groups and the control group. (B) Spleen weight was measured on the day of sacrifice. (C) The percentage of MDSCs among spleen cells was analyzed on the day of sacrifice. (D) Western blot analysis of the expression levels of pSTAT3, ALDH1, OCT4, KLF4, and Vimentin in tumor tissues. β-Actin was used as a control. (E) NOTCH-associated gene expression in tumor tissues was analyzed by real-time PCR. (F) G-CSF and VEGF concentrations were detected with ELISA. (G) Representative immunohistochemical images showing pSTAT3 expression in tumor tissues. Original magnification, 200× (scale bar, 100 μm). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 indicate significant differences compared with the respective control groups.

Article Snippet: The membranes were blocked with 5% skim milk in TBST at room temperature (RT) for 1 h and incubated with antibodies against pSTAT3 (cat. #9145, Cell Signaling Technology, NY, USA), ALDH1 (cat. #50385, Santa Cruz Biotechnology, CA, USA), GLUT1 (cat. #7903, Santa Cruz Biotechnology), β-catenin (cat. #610154, BD Biosciences), NOTCH1 (cat. #6014, Santa Cruz Biotechnology), OCT4 (cat. #611203, BD Biosciences), KLF4 (cat. #TA324722, OriGene, MD, USA), Vimentin (cat. #550513, BD Biosciences), NOTCH2 (cat. #5732, Cell Signaling Technology), and β-Actin (cat. #47778, Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: Podocin-Cre Klf4fl/fl and Podocin-Cre Klf4+/+ mice were treated with nephrotoxic serum (NTS) or VEH for 7 and 14 days. (A) Representative images of periodic acid-Schiff (PAS) staining (×20) (n = 5 in each group at 7 days and n = 3 in each group at 14 days). Arrowheads show crescents. Pound signs mark tubular casts and dilation. Asterisks show sclerotic glomeruli. (B) Albuminuria (urine albumin/creatinine) and (C) serum creatinine at 7 and 14 days after treatment (n = 5 at 7 days, n = 3 at 14 days; *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal-Wallis test with Dunn’s post hoc test). Scale bars: 100 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Staining

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: Podocin-Cre Klf4fl/fl and Podocin-Cre Klf4+/+ mice were treated with nephrotoxic serum (NTS) or VEH for 7 days. (A) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo; middle panel) and WT1 (lower panel) (×20). The glomerular region was selected and intensity of staining (OD) is shown as a relative fold change to untreated Podocin-Cre Klf4+/+ mice for (B) Nephrin (n = 5 in each group) and (C) Synpo (n = 3–4 in each group). (D) Number of WT1+ cells per glomerular cross-sectional area (n = 5 in each group). *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal-Wallis test with Dunn’s post hoc test. Scale bars: 40 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Immunostaining, Staining

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: Podocin-Cre Klf4fl/fl and Podocin-Cre Klf4+/+ mice were treated with nephrotoxic serum (NTS) or VEH for 7 days. (A) Representative images of phospho-STAT3 immunostaining. Arrowheads show nuclear phospho-STAT3 staining (×20). Scale bars: 20 µm. (B) Percent of glomeruli with nuclear phospho-STAT3 staining (n = 5 in each group, 30 glomeruli per mouse; **P < 0.01; Kruskal-Wallis test with Dunn’s post hoc test). (C) Icam-1, (D) Il-6, (E) Socs3, and (F) Stat3 mRNA expression in glomeruli (n = 4–5 in each group; *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal-Wallis test with Dunn’s post hoc test). (G) Immunostaining for Ki67 was performed, and the number of Ki67+ cells per glomeruli were determined (n = 5 in each group, 30 glomeruli per mouse; **P < 0.01; Kruskal-Wallis test with Dunn’s post hoc test). (H and I) Representative images of Claudin-1 immunostaining (×20) with quantification of percent area stained (n = 5 in each group, 30 glomeruli per mouse; *P < 0.05, **P < 0.01; Kruskal-Wallis test with Dunn’s post hoc test). Scale bars: 40 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Immunostaining, Staining, Expressing

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Representative images of periodic acid-Schiff (PAS) staining for Podocin-Cre Klf4+/+ (FVB/N) mice (i: ×10 and ii: ×40) and Podocin-Cre Klf4fl/fl (FVB/N) mice (iii: ×10 and iv–viii: ×40). Representative images show (iv–v) podocyte detachment (arrowhead, inset 120×), (vi) extracapillary proliferation (pound sign), (vi) glomerular epithelial cell mitosis (arrow, inset 120×), and (vii–viii) parietal epithelial cytoplasmic extensions (asterisk). Scale bars: 50 µm (10× images), 20 µm (40× images). (B) Electron microscopy was performed to assess ultrastructural changes in podocyte morphology (×6,800; ×13,000; and ×49,000; scale bars: 3 µm, 750 nm, and 600 nm, respectively). Representative images show normal upright foot processes (arrow), podocyte effacement and denuded basement membrane (arrowhead), parietal epithelial cytoplasmic extensions (asterisk), and enlarged mitochondria with disrupted cristae (pound sign). (C) Albuminuria (urine albumin/creatinine) (n = 10 in Podocin-Cre Klf4+/+ and 17 in Podocin-Cre Klf4fl/fl; **P < 0.01; Mann-Whitney U test) and (D) serum creatinine at 12 weeks of age (n = 7 in Podocin-Cre Klf4+/+ and 10 in Podocin-Cre Klf4fl/fl; **P < 0.01; Mann-Whitney U test). (E) Survival curves for Podocin-Cre Klf4+/+ and Podocin-Cre Klf4fl/fl mice are shown till age 52 weeks (n = 30–40 in each group; P = 0.002; log-rank [Mantel-Cox] test).

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Staining, Electron Microscopy, Membrane, MANN-WHITNEY

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Representative images of immunostaining for Nephrin (upper panel), Synaptopodin (Synpo) (middle panel), and WT1 (lower panel) in Podocin-Cre Klf4fl/fl (FVB/N) and Podocin-Cre Klf4+/+ (FVB/N) mice (×20). (B) The glomerular region was selected, and intensity (OD) was measured and quantified as a relative fold change to untreated Podocin-Cre Klf4+/+ (FVB/N) mice for Nephrin (upper panel) and Synpo (middle panel) (n = 5 in each group, 30 glomeruli per mouse). The number of WT1+ cells per glomerular cross-sectional area (lower panel) (n = 4–5 in each group, 30 glomeruli per mouse). *P < 0.05; **P < 0.01; Mann-Whitney U test. (C) Nephrin, Synaptopodin, and Wt1 mRNA expression levels in glomeruli (n = 4 in each group; *P < 0.05; Mann-Whitney U test). (D) Representative images of p57 immunostaining (top panel). Quantification of percent p57+ cells per glomeruli (bottom panel) (n = 5 in each group; **P < 0.01; Mann-Whitney U test). Scale bars: 40 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Immunostaining, MANN-WHITNEY, Expressing

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Representative images of immunostaining for phospho-STAT3 in Podocin-Cre Klf4fl/fl (FVB/N) and Podocin-Cre Klf4+/+ (FVB/N) mice (left panel) (×20). Percent of glomeruli with nuclear phospho-STAT3 staining (right panel) (n = 5–9 in each group, 30 glomeruli per mouse; **P < 0.01; Mann-Whitney U test). Representative images for phospho-STAT3 and Nestin (B) and phospho-STAT3 and Claudin-1 (C) coimmunostaining in Podocin-Cre Klf4fl/fl mice (×20). Arrowheads demonstrate coexpression of nuclear phospho-STAT3 with Nestin and Claudin-1. (D) Representative images for Claudin-1 and Ki67 immunostaining (×20) (n = 5 per group). Arrowheads demonstrate coexpression of nuclear Ki67 with Claudin-1. (E) Socs3, Il-6, Icam-1, and Stat3 mRNA expression levels in glomeruli (n = 4–5 in each group; *P < 0.05, **P < 0.01; Mann-Whitney U test). (F) Podocin-Cre Klf4fl/fl (7 weeks) with dipstick-positive proteinuria were randomized to receive S3I-201 (10 mg/kg) or DMSO for 3 weeks. Albuminuria (urine albumin/creatinine) at 7 weeks (pretreatment) and at 11 weeks of age (after treatment) (n = 4–5 in each group, *P < 0.05, **P < 0.01, Kruskal-Wallis test with Dunn’s post hoc test). Scale bars: 40 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Immunostaining, Staining, MANN-WHITNEY, Expressing

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: Control (Scr-shRNA) and KLF4 knockdown (KLF4-shRNA21 and KLF4-shRNA22) human podocytes were transferred from 33°C (permissive) to 37°C (nonpermissive) to induce differentiation. (A) Cell survival (counting) at 3, 7, 10, and 14 days in 37°C (n = 6; ***P < 0.001 as compared with Scr-shRNA, #P < 0.05, ##P < 0.01, and ###P < 0.001 as compared with KLF4-shRNA21; 2-way ANOVA with Tukey’s multiple comparisons post hoc test). (B) Cell viability (MTS assay) at day 3, 7, and 10 in 37°C (n = 6–8; ***P < 0.001; Mann-Whitney U test). (C) Cell cycle analysis at day 3 in 37°C. Percentages of human podocytes in sub-G0, G0/G1, S, and G2/M phases in left panel (n = 3; *P < 0.05, ***P < 0.001; Mann-Whitney U test). Histograms generated with ModFit software to evaluate the relative cell count in each cell cycle stage. (D) CCNA2, CCNB1, CCNE1, CCND1, and p57 mRNA expression are shown relative to Scr-shRNA podocytes at day 3 in 37°C (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney U test). (E) Representative images of Phalloidin and Hoechst staining in surviving Scr-shRNA and KLF4-shRNA22 podocytes at 5 days in 37°C to evaluate for actin stress fiber formation and apoptotic bodies (n = 3, ×20). Inset shows higher magnification (×60). Arrowheads show apoptotic bodies. (F) Annexin V/propidium iodide staining in combination with FACS at 3 days in 37°C (n = 3; *P < 0.05; Mann-Whitney U test). Scale bars: 100 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Control, shRNA, Knockdown, MTS Assay, MANN-WHITNEY, Cell Cycle Assay, Generated, Software, Cell Counting, Expressing, Staining

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Representative images of Western blot for phospho-STAT3, total STAT3, and GAPDH in WT human podocytes at 33°C and after 14 days of differentiation at 37°C (n = 3, left panel). Quantification of phospho-STAT3 expression by densitometry (right panel) (n = 3; *P < 0.05; Mann-Whitney U test). (B) IL-6 mRNA expression relative to Scr-shRNA podocytes at day 3 in 37°C (n = 3; *P < 0.05; Mann-Whitney U test). (C) Representative images of phospho-STAT3 and Hoechst immunostaining (n = 3, ×20). Arrowheads show nuclear phospho-STAT3 staining. Inset shown for apoptotic bodies and phospho-STAT3 staining (×60). (D) KLF4-shRNA22 podocytes were treated with S3I-201 (12.5 μm) or DMSO for 48 hours under nonpermissive conditions. Cell viability (MTS assay) as a fold change in absorbency (n = 10–12; ***P < 0.001; Mann-Whitney U test). (E) IL-6 levels in supernatant from KLF4-shRNA22 and Scr-shRNA podocytes at day 3 in 37°C (n = 3; **P < 0.01; Mann-Whitney U test). (F) Glomeruli were isolated from WT mice and cultured for 4 days before treatment with supernatant from Scr-shRNA and KLF4-shRNA22 podocytes for 24 hours. Representative images of Claudin-1 and phospho-STAT3 immunostaining to assess STAT3 activation in PECs (n = 3, ×20). (G) Differentiated mouse PECs treated for 24 hours with supernatant from Scr-shRNA and KLF4-shRNA22 and either S3I-201 or DMSO. Cell proliferation (MTS assay) as a fold change in absorbency (n = 10–12; *P < 0.05, ***P < 0.001; Mann-Whitney U test). Scale bars: 50 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Western Blot, Expressing, MANN-WHITNEY, shRNA, Immunostaining, Staining, MTS Assay, Isolation, Cell Culture, Activation Assay

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Representative Western blot confirms stable overexpression for KLF4 (LentiORF-KLF4) and control vector (LentiORF-RFP) (n = 3). (B) Cell cycle analysis of LentiORF-KLF4 and LentiORF-RFP human podocytes under permissive conditions (n = 3, **P < 0.01; Mann-Whitney U test). (C) Differentiated LentiORF-KLF4 and LentiORF-RFP human podocytes were treated with leukemia inhibitory factor (LIF) (100 ng/ml for 15 minutes) to activate STAT3 signaling, with vehicle (VEH) as control and IL-6 mRNA expression measured (n = 3; **P < 0.01; Mann-Whitney U test). (D) Representative blot of co-IP with initial IP of KLF4 with anti-KLF4 antibody and immunoblotted for phospho-STAT3 (n = 3). IgG isotype serves as control for IP, and GAPDH serves as the loading control. (E) KLF4-shRNA22 podocytes were transfected with LentiORF-KLF4 or LentiORF-RFP, and MTS assay was performed at day 3, 7, and 10 in 37°C (n = 6–10, **P < 0.01 Kruskal-Wallis test with Dunn’s post hoc test).

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Western Blot, Over Expression, Control, Plasmid Preparation, Cell Cycle Assay, MANN-WHITNEY, Expressing, Co-Immunoprecipitation Assay, Transfection, MTS Assay

Journal: JCI Insight

Article Title: Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

doi: 10.1172/jci.insight.98214

Figure Lengend Snippet: (A) Previously reported gene expression from microdissected glomeruli (38) were used to analyze KLF4 mRNA expression from patients with RPGN as compared with normal controls (*P < 0.05; unpaired 2-tailed t test). (B) Immunofluorescence for KLF4 and phospho-STAT3 was performed in control (n = 4) and RPGN (n = 8) specimens. Six to 12 glomeruli per biopsy were selected, and the percentage of KLF4+ and phospho-STAT3+ cells were determined by measuring the number of KLF4+ and phospho-STAT3+ cells relative to total glomerular cells (Hoechst+) (*P < 0.05 as compared with control; Mann-Whitney U test). (C) The representative images in each group are shown (×20). (D) %KLF4+ and %phospho-STAT3+ in each glomerulus were plotted for healthy donor (n = 36) and RPGN biopsies (n = 59) and analyzed using hierarchal clustering (Ward’s method). With significance set at P < 0.05, cluster 4 is significantly different from clusters 1 and 2 , cluster 5 is significantly different from clusters 1 and 2, and cluster 3 is different from cluster 2; the remaining clusters are not significantly different. Scale bars: 50 µm.

Article Snippet: For IP studies, cells were lysed with radioimmunoprecipitation assay buffer with protease inhibitors and immunoprecipitated with antibodies against rabbit anti-KLF4 antibody (PM057, MBL).

Techniques: Gene Expression, Expressing, Immunofluorescence, Control, MANN-WHITNEY