csf3r sirna  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Immunology

Article Title: The RNA m 6 A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression

doi: 10.1038/s41423-023-01115-9

Figure Lengend Snippet: ALKBH5 deficiency increases m 6 A modification of the CSF3R mRNA to promote its decay. A qRT‒PCR analysis of Csf3r mRNA level in bone marrow neutrophils isolated from Alkbh5 -deficient mice and their WT littermates 12 h after CLP ( n = 10). B qRT‒PCR analysis of CSF3R mRNA level in ALKBH5 -deficient and WT dHL-60 cells infected with E. coli (left, n = 6) or stimulated with LPS (right, n = 5). mRNA level data were normalized to Gapdh ( A ) or GAPDH ( B ) expression. C m 6 A abundance on the CSF3R mRNA in dHL-60 cells infected with E. coli as indicated by our m 6 A-seq data (GSE201060, NM_000760.4). Two independent biological replicates (Rep 1 and Rep 2). Red, m 6 A-IP; gray, input; blue box, m 6 A peaks. The y -axis shows the normalized m 6 A signal along the gene. D m 6 A-RIP-qPCR analysis of m 6 A enrichment of the CSF3R mRNA in ALKBH5 -deficient and WT dHL-60 cells infected with E. coli for 2 h ( n = 4). Different regions of human EEF1A1 mRNA were used as positive and negative controls (ctrl). The data were presented relative to those obtained with IgG. E RNA decay assay of CSF3R mRNA degradation in ALKBH5 -deficient and WT dHL-60 cells treated with actinomycin D for the indicated times ( n = 5). The data were normalized to the 18S rRNA level, and residual mRNA abundances were normalized to the abundance at t = 0 h. All data are the mean ± SEM of biologically independent samples. Two-tailed unpaired Student’s t -test ( A , B , D and E ). * P < 0.05; ** P < 0.01; **** P < 0.0001; ns not significant

Article Snippet: For CSF3R inhibition, the medium containing ATRA was replaced, and the cells were then transfected with siRNA against CSF3R with TransIT-siQUEST Transfection Reagent (Mirus) following the manufacturer’s instructions.

Techniques: Modification, Isolation, Infection, Expressing, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: The RNA m 6 A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression

doi: 10.1038/s41423-023-01115-9

Figure Lengend Snippet: Reduced binding of ALKBH5 to the CSF3R mRNA leads to decreased G-CSFR expression in bacteria-infected neutrophils. A , B FACS analyses of the protein levels of G-CSFR on the surface of WT dHL-60 cells stimulated with E. coli ( A ) or LPS ( B ) for the indicated times ( n = 6). C , D FACS analyses of total intracellular G-CSFR protein in WT dHL-60 cells stimulated with E. coli ( C ) or LPS ( D ) for the indicated times ( n = 6). E , F qRT‒PCR analysis of CSF3R mRNA level in WT dHL-60 cells stimulated with E. coli ( E ) or LPS ( F ) for the indicated times ( n = 5). G RIP-qPCR analysis of ALKBH5 binding to the CSF3R mRNA in WT dHL-60 cells without or with E. coli infection for 4 h ( n = 4). The data were presented relative to those obtained with IgG. All data are the mean ± SEM of biologically independent samples. The data are representative of 6 independent experiments with similar results ( A – D ). Two-tailed unpaired Student’s t -test ( A – G ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: For CSF3R inhibition, the medium containing ATRA was replaced, and the cells were then transfected with siRNA against CSF3R with TransIT-siQUEST Transfection Reagent (Mirus) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Bacteria, Infection, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: The RNA m 6 A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression

doi: 10.1038/s41423-023-01115-9

Figure Lengend Snippet: Roles of ALKBH5-mediated m 6 A demethylation in neutrophil production and mobilization through posttranscriptional enhancement of CSF3R mRNA stability and upregulation of G-CSFR protein expression. ALKBH5 directly binds to the CSF3R mRNA and mediates its m 6 A demethylation to increase the mRNA stability and consequent protein expression of G-CSFR, maintaining the antibacterial innate response (left). Upon ALKBH5 deletion or bacterial infection, disruption of the ALKBH5- CSF3R mRNA axis leads to an increased m 6 A level on the CSF3R mRNA, resulting in enhanced CSF3R mRNA decay and then decreased G-CSFR protein expression, which impairs neutrophil production and mobilization as well as the host innate defense (right). Me, m 6 A RNA modification

Article Snippet: For CSF3R inhibition, the medium containing ATRA was replaced, and the cells were then transfected with siRNA against CSF3R with TransIT-siQUEST Transfection Reagent (Mirus) following the manufacturer’s instructions.

Techniques: Expressing, Infection, Disruption, RNA modification

Journal: Signal Transduction and Targeted Therapy

Article Title: Colony-stimulating factor 3 as a key mediator in the progression of idiopathic pulmonary fibrosis: a novel therapeutic target

doi: 10.1038/s41392-025-02421-6

Figure Lengend Snippet: CSF3 induces pulmonary fibrosis through the CSF3R/STAT3 signaling axis. a qRT-PCR analysis of fibrosis markers (α-SMA, COL1A1, fibronectin) in human lung fibroblast cell line (HLF, left) and mouse primary lung fibroblast cells (MLF, right) treated with recombinant CSF3 (rCSF3) (200 ng/ml, 24 h). b and c Representative immunofluorescence images of α-SMA and COL1A1 ( b ), and Western blot analysis of fibrosis markers and STAT3 activation (p-STAT3) ( c ) in HLF and MLF cells treated with rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. d Hydroxyproline content assay in HLF and MLF cells treated with rCSF3. e Schematic illustration of the procedure used to induce lung fibrosis in C57BL/6 mice with recombinant mouse CSF3. BLM was administered IP on day 1, followed by IP injections of rCSF3 three times per week. f Quantification of the fibrosis area was performed using Orbit software based on Masson’s trichrome staining images. The green area represents the fibrotic region. Scale bar: 500 μm. g Hydroxyproline content in mouse lung tissues from groups treated with BLM and rmCSF3 as indicated. h–k qRT-PCR analysis of α-SMA ( h ), COL1A1 ( i ) and fibronectin ( j ) expression, and hydroxyproline content ( k ) assay in wild-type (CSF3 +/+ ) MLF cells and CSF3 knock-out (CSF3 −/− ) MLF cells treated with BLM and rCSF3. l and m qRT-PCR analysis of fibrosis markers and CSF3 expression ( l ), and representative immunofluorescence images of α-SMA and COL1A1 ( m ) in HLF cells transfected with si-CSF3R and treated with rCSF3 (200 ng/ml, 24 h). Scale bars: 200 μm. n and o Western blot analysis of α-SMA and COL1A1 ( n ), and representative immunofluorescence images of α-SMA and COL1A1 ( o ) in CSF3 −/− MLF cells treated with BLM and rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. Statistical significance was determined using ANOVA with multiple comparison or t -test

Article Snippet: The TGFβRII or CSF3R siRNA was purchased from Santa Cruz (Cat. No. sc-36657 for TGFβRII) or Genolution, Inc. for CSF3R.

Techniques: Quantitative RT-PCR, Recombinant, Immunofluorescence, Western Blot, Activation Assay, Control, Software, Staining, Expressing, Knock-Out, Transfection, Comparison