Journal: Nucleic Acids Research

Article Title: PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression

doi: 10.1093/nar/gkad042

Figure Lengend Snippet: Loss of PTBP1 upregulates PHLDA3 and impairs AKT activation in crypt cells. ( A ) Volcano plot demonstrates mRNA abundance changes in the knockout crypt cells. Red dots represent significantly upregulated genes (log 2 FC > 1, FDR < 0.05), blue dots represent significantly downregulated genes (log 2 FC < 1, FDR < 0.05), and the black dots represent genes with no significant changes in expression between the knockout and control crypt cells. Phlda3 showed the highest fold change among the upregulated genes (arrow). ( B ) Real-time PCR shows significant upregulation of Phlda3 mRNA level at 20, 24, 36 and 50 h PTI. Data were normalized to Gapdh . Each symbol in the graph indicates a fold change of Phlda3 expression in individual mice. Bars show mean values. At each time point, sibling littermates were used for analysis. Data shown are representative of at least three independent experiments. ( C , D ) Immunofluorescence staining using an anti-PHLDA3 antibody shows that PHLDA3 protein expression is increased in the bottom crypt cells in the knockout mice (D). Dotted boxes show the crypt bottom region. ( E ) Double immunofluorescence staining using anti-PHLDA3 and anti-GFP antibodies shows PHLDA3 protein is localized in cells adjacent to LGR5-positive CBC stem cells (*) in wild-type mice. PHLDA3-positive cells are delineated by white dotted lines. ( F ) Double immunofluorescence staining using anti-PHLDA3 and anti-lysozyme antibodies shows that PHLDA3 protein is localized in Paneth cells in wild-type mice. Crypt cells positive for PHLDA3 and lysozyme are delineated by white dotted lines. ( G ) Western blot shows decreased AKT phosphorylation on ser473 in knockout crypt cells at 24 h PTI. Quantification of the ratio of Phospho-AKT to Pan-AKT is shown in the right panel. Mice used in the assay are littermates. PTBP1 was efficiently depleted in all three knockout mice. ( H , I ) Immunofluorescence staining with an anti-P53 antibody shows an upregulation of P53 in the knockout crypt cells at 48 h PTI as indicated by nuclear localization of P53 (inset and arrows in I). ** P < 0.01; **** P < 0.0001. WT, wild-type; KO, knockout. Scale bars, 50 μm.

Article Snippet: Primary antibodies used are mouse anti-PTBP1 (Life Technologies, 324800), mouse anti-PTBP2 antibody (ABCAM, sc-376316), rabbit anti-Pan AKT (Cell Signaling, 4685), rabbit anti-Phospho-AKT (Ser473) (Cell Signaling, 4060), mouse anti-HSC70 (Santa Cruz Biotechnology, SC-7298), and rabbit anti-GRP78 (Abcam, ab32618).

Techniques: Activation Assay, Knock-Out, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Double Immunofluorescence Staining, Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: PRMT5/FGFR3/AKT Signaling Axis Facilitates Lung Cancer Cell Metastasis

doi: 10.1177/15330338231161139

Figure Lengend Snippet: Silencing of PRMT5 impairs FGFR3/Akt signaling in lung cancer cells. (A) A549 cells were infected with lentivirus containing PRMT5-shRNAs, and the indicated protein expression levels were detected by Western blotting. Representative pictures were shown. (B) The indicated protein expression levels were quantified. * P < .05 versus Scr, n = 3. (C) The PRMT5 expression and localization were measured by immunofluorescence in PRMT5 depletion A549 cells. Representative pictures were shown (n = 3). Scale bar = 50 µm. (D) The phospho-Akt-Ser473 expression and localization were measured by immunofluorescence in PRMT5 depletion A549 cells. Representative pictures were shown (n = 3). Scale bar = 50 µm. (E) The proliferation marker PCNA expression was measured by immunofluorescence in PRMT5 depletion A549 cells (n = 3). Scale bar = 50 µm. (F) Cell viability was determined by MTS assay in PRMT5 depletion A549 cells (n = 4). * P < .05 versus Scr, n = 3.

Article Snippet: The membrane was incubated with the following antibodies: PRMT5 (cat# sc-376937; Santa Cruz Biotechnology), Symmetric Di-Methyl Arginine Motif [sdme-RG] MultiMab™ (cat# 13222; Cell Signaling Technology), phospho-Ser473-Akt (cat# 4060; Cell Signaling Technology), total Akt (cat# 4691; Cell Signaling Technology), β-catenin (cat# 8480; Cell Signaling Technology), Collagen I (cat# ab34710; Abcam), FGFR3 (cat# 4574; Cell Signaling Technology), vimentin (cat# 5741; Cell Signaling Technology), E-cadherin (cat# 14472; Cell Signaling Technology), and β-actin (cat# sc-47778, Santa Cruz Biotechnology).

Techniques: Infection, Expressing, Western Blot, Immunofluorescence, Marker, MTS Assay

Journal: Technology in Cancer Research & Treatment

Article Title: PRMT5/FGFR3/AKT Signaling Axis Facilitates Lung Cancer Cell Metastasis

doi: 10.1177/15330338231161139

Figure Lengend Snippet: Inhibiting PRMT5 suppresses FGFR3/Akt signaling in lung cancer cells. (A) A549 cells were treated with indicated PRMT5 inhibitors, and the related protein expression levels were detected by Western blotting. Representative pictures were shown. (B) The phospho-Akt-Ser473 expression and localization were measured by immunofluorescence upon GSK591 treatment. Representative pictures were shown (n = 3). Scale bar = 50 µm. (C) The proliferation marker PCNA expression was measured by immunofluorescence upon GSK591 treatment. Representative pictures were shown (n = 3). Scale bar = 50 µm. (D) PCNA-positive cells were quantified upon GSK591 treatment. One hundred cells were counted for each group. * P < .05 versus Vehicle. (E) Cell viability was determined by MTS assay upon GSK591 treatment (n = 4). * P < .05 versus Vehicle.

Article Snippet: The membrane was incubated with the following antibodies: PRMT5 (cat# sc-376937; Santa Cruz Biotechnology), Symmetric Di-Methyl Arginine Motif [sdme-RG] MultiMab™ (cat# 13222; Cell Signaling Technology), phospho-Ser473-Akt (cat# 4060; Cell Signaling Technology), total Akt (cat# 4691; Cell Signaling Technology), β-catenin (cat# 8480; Cell Signaling Technology), Collagen I (cat# ab34710; Abcam), FGFR3 (cat# 4574; Cell Signaling Technology), vimentin (cat# 5741; Cell Signaling Technology), E-cadherin (cat# 14472; Cell Signaling Technology), and β-actin (cat# sc-47778, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Immunofluorescence, Marker, MTS Assay