Journal: bioRxiv

Article Title: Control of noncoding RNA production and histone levels by a 5’ tRNA fragment

doi: 10.1101/498949

Figure Lengend Snippet: A) Biotin-oligo pulldowns from murine ES cell extracts. Silver stained gel shows two replicates each for pulldowns using biotin-tRF-GG or biotin-tRF-Lys-CTT, as indicated. Arrow indicates ~50 kD band enriched in tRF-GG pulldowns. B) Domain architecture of hnRNPF and hnRNPH. C) Peptide counts for hnRNPH in control, tRF-Lys-CTT, or tRF-GG pulldowns. D) Western blots show hnRNPF/H recovery following tRF-GG or tRF-Lys-CTT pulldown. Pulldowns were washed 4 × 3 minutes with 50 mM Tris pH 8.0 supplemented with 100 mM (W1), 250 mM (W2), or 500 mM (W3) NaCl. E) Gel shift analysis of hnRNPH1 binding to tRF-GG. A synthetic oligonucleotide corresponding to tRF-Gly-GCC (GCAJULGUGGUUCAGUGGDAGAAUUCUCGC) was labeled at the 3’ end using fluorescein 5-thiosemicarbazide, then incubated at 3 nM in equilibration buffer (0.01% Igepal, 0.01 mg/ml carrier tRNA, 50 mM Tris, pH 8.0, 100 mM NaCl, 2 mM DTT) for 3 hours along with increasing concentrations of purified hnRNPH1 protein from 1.35 nM to 2000 nM. See also . F-G) Fit of gel shift binding data for tRF-Lys-CTT and tRF-Gly-GCC. Fitting the binding data yields an estimated Kd of hnRNPH1 of ~230 nM for tRF-Gly-GCC, and >1 μM for tRF-Lys-CTT. H) Fluorescence polarization data for hnRNPH1 incubations with labeled tRF-Gly-GCC. Polarization values against the protein concentrations are fit to the Hill equation using the Igor Pro software.

Article Snippet: Double hnRNPF/H knockdown was performed by transient transfection of 20 pmols of siRNAs against mouse hnRNPF and hnRNPH transcripts, respectively (sc-38273 and sc-35580, Santa Cruz Biotechnologies) in 12-well format.

Techniques: Staining, Control, Western Blot, Gel Shift, Binding Assay, Labeling, Incubation, Purification, Fluorescence, Software

Journal: bioRxiv

Article Title: Control of noncoding RNA production and histone levels by a 5’ tRNA fragment

doi: 10.1101/498949

Figure Lengend Snippet: A) Immunofluorescence images of MERVL LTR:tdTomato ES cells transfected with siRNAs against either GFP or against hnRNPF and hnRNPH. Images show anti-Coilin IF, tdTomato fluorescence, DAPI counterstaining, and all three merged, as indicated. Interestingly, in preliminary immunofluorescence experiments using our MERVL LTR reporter cell line, we observed that hnRNPF/H is undetectable in spontaneously arising tdTomato-positive cells (data not shown), suggesting that hnRNPF/H degradation could potentially be involved in spontaneous ESC transitions to the 2C-like state. B) Browser shot of Tcf3 and Eloc mRNA abundance in ES cells subjected to the indicated knockdowns. Alternative exon inclusion events are shown in red. C) Validation of hnRNPF/H-regulated alternative splicing events by semiquantitative PCR. Three alternative splicing events - hnRNPF/H-dependent skipping of cassette exons in Brd8 and Cd81 , or an hnRNPF/H-dependent detained intron in Srsf5 - identified in our hnRNPF/H RNA-Seq data are validated here by PCR. None of these alternative splicing events was recapitulated in the tRF-GG KD samples, confirming that tRF-GG only affects a subset of hnRNPF/H functions. D-E) Altered rRNA 2’-O-methylation in tRF-GG KD cells. We carried out Ribometh-seq and calculated methylation “A scores” as previously described ( ; ). (D) shows a scatterplot of methylation scores for known 18S and 28S rRNA methylation sites, with significant changes in methylation highlighted in red dots. (E) shows the change in methylation level for the known rRNA nucleotides.

Article Snippet: Double hnRNPF/H knockdown was performed by transient transfection of 20 pmols of siRNAs against mouse hnRNPF and hnRNPH transcripts, respectively (sc-38273 and sc-35580, Santa Cruz Biotechnologies) in 12-well format.

Techniques: Immunofluorescence, Transfection, Fluorescence, Biomarker Discovery, Alternative Splicing, RNA Sequencing, Methylation

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Confocal microscopic analysis of unfixed HeLa cells penetrated by Tween20 and immunostained with anti-PQBP1, antifibrillarin, and antinucleolin antibodies. Nuclei were costained with Hoechst 33342. The signals of PQBP3/NOL7 were distributed in the peripheries of nucleoli. In addition, smaller speckles were observed in the nucleoplasm and cytoplasm. ( B ) Super-resolution microscopy images of HeLa cells after fixation. The distribution pattern was similar to that observed by standard confocal microscopy. The speckle diameters were ~260 nm in the nucleoplasm and ~300 nm in the cytoplasm. Staining localized to the outer shell of the nucleolus resembled a chain or cluster of similarly sized speckles. ( C ) Super-resolution microscopy images of normal iPSC-derived neurons after fixation. The relationship of PQBP3/NOL7, nucleolin, and fibrillarin was similar to that in HeLa cells. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Super-Resolution Microscopy, Confocal Microscopy, Staining, Derivative Assay

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) PQBP3/NOL7 immunocytochemistry of HeLa cells at various cell densities. High signal intensities of PQBP3/NOL7 were observed in cells at low cell densities, while the signals were dispersed into the cytosol in cells at medium densities and eliminated in confluent cells (high density). ( B ) Relationship between percent cell margin in contact with neighboring cells and nucleolar distribution of PQBP3/NOL7. Localization of PQBP3/NOL7 was semiquantitated into three stages weighed by different values (1: robust nucleolar PQBP3/NOL7, 0.5: modest nucleolar PQBP3/NOL7, 0: no nucleolar PQBP3/NOL7). A Kendall’s rank correlation test revealed a negative relationship between % cell contact margin and PQBP3/NOL7 nucleolar distribution (tau = −0.786, p = 1.654 × 10 −62 ). ( C ) Western blot of PQBP3/NOL7 with nuclear fraction, cytoplasmic fraction and total cell extract of HeLa cells cultured at low and high cell densities. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. ( D ) Representative images of cells in the stages of “robust nucleolar PQBP3/NOL7” (upper panels) and “faint nucleolar PQBP3/NOL7” (lower panels). In cells classified as “faint nucleolar PQBP3/NOL7,” nuclear PQBP3 signals became obscure, though fibrillarin signals of nucleoli were robust, and abnormal protrusion of nuclear margin and extranuclear DNA stains were observed in Hoechst 33342 labeling (white arrows). ( E ) Electron microscopy of HeLa cells with faint nucleolar PQBP3/NOL7 (#1, #2) and robust nucleolar PQBP3/NOL7 (#3). Nuclear membrane of #1 and #2 cells became obscure and their protrusion contains chromatin. Nucleoli of #1 and #2 cells did not show normal substructures. The continuity of the protrusion and the nucleus excluded that such protrusions were micronuclei. Cells with robust nucleolar PQBP3/NOL7 (#3) showed normal structures of nucleoli and nuclear membrane. Experiments in this figure were technically replicated until the necessary N was acquired. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Immunocytochemistry, Western Blot, Cell Culture, Labeling, Electron Microscopy, Membrane

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Confocal microscopy of HeLa cells after ten passages (10G), which were penetrated with Tween20, immunostained with anti-PQBP1, and costained with Hoechst 33342. Red arrows indicate cells with dispersed nucleolar PQBP3/NOL7 staining. ( B ) Confocal microscopy images of HeLa cells after 20 passages (20G) stained as described above. Red arrows indicate cells with dispersed nucleolar PQBP3/NOL7 staining, and chromatin distribution (Hoechst 33342-stained area) shifted and deviated in the nucleus. Purple arrows indicate cells in which chromatin was nearly absent. ( C ) Enlarged image of the area indicated by dotted lines in ( B ). ( D – F ) Specific distributions of PQBP3/NOL7 during cell division. Foci of PQBP3/NOL7 localized to the centrosome (white arrow) were observed in addition to the diffuse cytoplasmic distribution. ( G ) Quantitative analyses of percentage of red arrow type or purple arrow type of cells in three different passage groups. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. In red arrow type of cells, statistical significance was found in comparison of <5G and >10G (#: p = 0.0415), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0027). In purple arrow type of cells, statistical significance was found in comparison of <5G and >10G (##: p = 0.0082), <5G and >10G (###: p < 0.0001), and >10G and >20G (###: p = 0.0005). ( H – J ) Yellow arrows indicate cells exhibiting morphological changes of cell death, in which siPQBP3 signals (red) were absent or low, and PQBP3 signals (green) were relatively high. Contrastingly, siPQBP3-transfected cells with high red signals and low green signals did not exhibit such changes or apoptotic features (white arrows).

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Confocal Microscopy, Staining, Comparison, Transfection

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) PQBP3/NOL7 immunocytochemistry of HeLa cells at less than 5 passages (<5G), more than 10 passages (>10G), and more than 20 passages (>20G). Signals were detected in β-Gal staining of >10G and >20G cells in which PQBP3/NOL7 was decreased in the nucleus and shifted to the cytoplasm. ( B ) Signal intensities of β-Gal were quantified in HeLa cells (30 cells from 3 wells) and compared among three groups. Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), <5G and >20G (###: p < 0.0001), and >10G and >20G (###: p < 0.0001). ( C ) Signal intensities of PQBP3/NOL7 in nucleus, cytoplasm, or total cell were quantified and compared among three groups (30 cells from 3 wells). (Nuc) Statistical significance was found in comparison of <5G and >10G (#: p = 0.027), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0051). (Cyt) Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), <5G and >20G (###: p < 0.0001), and >10G and >20G (###: p < 0.0001). ( D ) Schematic presentation of the method to quantify the extranuclear DNA signals stained by Hoechst 33342 (left panel). The signal intensities were compared among three groups (right graph). Statistical significance was found in comparison of <5G and >10G (###: p < 0.0001), and <5G and >20G (###: p < 0.0001). ( E ) Western blot analyses of nuclear, cytoplasmic, and total PQBP3/NOL7 in 5G, >10G, and >20G HeLa cells (left panels). Statistical comparisons of band intensities among the three groups (right graphs). (Nuclear fraction) Statistical significance was found in comparison of <5G and >10G (#: p = 0.0189), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.0015). (Cytoplasmic fraction) Statistical significance was found in comparison of <5G and >10G (#: p = 0.025), <5G and >20G (###: p < 0.0001), and >10G and >20G (##: p = 0.003). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Immunocytochemistry, Staining, Comparison, Western Blot

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) protocol of mTOR signal activation in HeLa cell culture (<5G). MHY1485, an activator of mTOR (final concentration in the medium: 10 μM) was added to the culture medium 48 h after cell seeding and cells were harvested after another 4 h. ( B ) Western blot analysis of nuclear and cytoplasmic PQBP3/NOL7 under mTOR activation. ( C ) Quantitative analyses of band intensities of western blots. Statistical analyses (Welch’s t-test) revealed decrease of nuclear PQBP3/NOL7, increase of cytoplasmic PQBP3/NOL7, and shift of PQBP3/NOL7 from nucleus to cytoplasm. ( D ) Immunocytochemistry of HeLa cells treated with MHY1485. Cytoplasmic shift of PQBP3/NOL7 and faint signals of β-GAL were detected in HeLa cells treated with MHY1485. ( E ) Quantitative analyses of cytoplasmic shift of PQBP3 (PQBP3 nuclear/cytoplasmic signal ratio), senescence (β-GAL signal intensity), and cytoplasmic shift of nuclear DNA (Hoechst nuclear/cytoplasmic signal ratio) in HeLa cells by mTOR activation with MHY1485. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Activation Assay, Cell Culture, Concentration Assay, Western Blot, Immunocytochemistry

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Western blot analysis of PQBP3/NOL7 in total extracts of HeLa cells transfected with two types of PQBP3-siRNA or scrambled-siRNA. Statistical significance was found in comparison of non TF and si-PQBP3#1 (##: p = 0.0048), non TF and si-PQBP3#2 (##: p = 0.0019), si-PQBP3#1 and si-Scramble (##: p = 0.0033), and si-PQBP3 and si-Scramble (##: p = 0.0013). ( B ) Upper panels show three signals of immunostained PQBP3, Hoechst 33342, and fluorescence-labeled siRNA. Almost all cells were transfected with PQBP3 siRNA (#1, #2), and PQBP3 signals were accordingly reduced. Representative cells from nontransfected, PQBP3 siRNA-transfected (#1, #2), and Scrambled siRNA-transfected cells (green arrow in upper panels) are shown in middle panels. The PQBP3 siRNA-transfected cell exhibited a notched nucleus (white arrow) in which an additional large bleb (asterisk) was formed. When Hoechst 33342 signals were acquired at the same signal intensity threshold, cytoplasmic genomic DNA was present in the PQBP3 siRNA-transfected cell but not in the nontransfected or Scrambled siRNA-transfected cells. ( C ) Representative Hoechst 33342 images show a normal nucleus in a nontransfected cell (left panel) and an abnormal nucleus with a protrusion (light blue arrows) in a PQBP3-siRNA-transfected cell (middle panel), and a micronucleus (white arrow) that was detected in only a single cell among Scrambled-siRNA-transfected cells (right panel). ( D ) Quantitative analyses of frequency of cells with notched nuclei (left graph) and frequency of cells with micronuclei (right graph) from 5 to 6 randomly visual fields from independent wells containing 15–50 cells. (left) Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (##: p = 0.0075), non-transfect and si-PQBP3#2 (#: p = 0.0119), si-PQBP3#1 and si-Scramble (##: p = 0.0075), and si-PQBP3#2 and si-Scramble (#: p = 0.0119). (right) Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (##: p = 0.0025), non-transfect and si-PQBP3#2 (#: p = 0.0047), si-PQBP3#1 and si-Scramble (###: p = 0.0006), and si-PQBP3#2 and si-Scramble (##: p = 0.0011). ( E ) Quantitative analysis of cytoplasmic DNA signal intensity per cell in nontransfected, PQBP3 siRNA-transfected, and Scrambled siRNA-transfected cells. The original images were corrected by canceling noise signals, and signals outside of the nucleus were measured (see Methods). Cell numbers are shown in the figure, and images were captured from three wells. Statistical significance was found in comparison of non-transfect and si-PQBP3#1 (###: p = 0.0075), non-transfect and si-PQBP3#2 (#: p = 0.0119), si-PQBP3#1 and si-Scramble (##: p = 0.0075), and si-PQBP3#2 and si-Scramble (#: p = 0.0119). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Western Blot, Transfection, Comparison, Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) SAHFs was analyzed in HeLa cells treated with hydrogen peroxide. Nuclear speckles reactive to anti-H3K9me3 antibody indicating SAHFs was observed. Such cells with SAHFs were reactive to β-Gal staining. ( B ) Positive stains for β-Gal were observed in PQBP3-siRNA-transfected but not Scrambled-siRNA-transfected HeLa cell. (PQBP3) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). (β-Gal) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). ( C ) SAHFs was analyzed in human normal iPSC-derived neurons treated with hydrogen peroxide. Nuclear speckles reactive to anti-H3K9me3 antibody was observed similarly to HeLa cells treated with hydrogen peroxide. Such human iPSC-derived neurons with SAHFs were reactive to β-Gal staining. ( D ) Positive stains for β-Gal were observed in human normal iPSC-derived neurons that were transfected PQBP3-siRNA but not by Scrambled-siRNA-transfected. (PQBP3) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). (β-Gal) Statistical significance was found in comparison of non-transfection and si-PQBP3#1 (###: p < 0.0001), and si-PQBP3#1 and si-Scramble (###: p < 0.0001). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Staining, Transfection, Comparison, Derivative Assay

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: HeLa cells were transfected with pEGFP-C1-PQBP3, pEGFP-N1-PQBP3, or pEGFP-N1 plasmids to express EGFP-PQBP3, PQBP3-EGFP, or EGFP proteins, and following Hoechst 33342 staining without fixation, EGFP signals in live cells were observed with confocal microscopy. Similar expression patterns were observed in EGFP-PQBP3 and PQBP3-EGFP fusion proteins. EGFP protein alone did not exhibit the nucleolar pattern of the PQBP3 fusion proteins. Some cell images are redisplayed from Fig. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Transfection, Staining, Confocal Microscopy, Expressing

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Senescent HeLa cells were fixed with 10% formaldehyde and stained with antifibrillarin and DAPI. Signals of EGFP-PQBP3 fusion proteins were directly detected. Most of the nontransfected cells contained cytoplasmic genomic DNA, as revealed by DAPI (purple arrows), while a small part of nontransfected cells showed normal morphology (white arrows). Transfected cells did not contain cytoplasmic genomic DNA (green arrows). ( B ) Nonfixed HeLa cells were directly observed to evaluate the effect of EGFP-PQBP3 or EGFP expression on cytoplasmic genomic DNA. In the left panels, nontransfected cells (purple arrows) but not EGFP-PQBP3-expressing cells (green arrows) contained cytoplasmic genomic DNA. In the right panels, cytoplasmic DNA was not affected in EGFP-expressing cells (white arrows) in comparison to neighboring nontransfected cells. Lower tables show quantitative analysis of 50 cells from 5 wells, each transfected with pEGFP-C1-PQBP3 or pEGFP-C1 plasmid. A strong negative correlation between EGFP-PQBP3 expression and cytoplasmic genomic DNA was statistically confirmed by Fisher’s exact test ( p = 1.087 × 10 −7 , n = 5 wells, n = 50 cells), while no relationship was detected between EGFP expression and cytoplasmic genomic DNA. Experiments in this figure were technically replicated until the necessary N was acquired. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Staining, Transfection, Expressing, Comparison, Plasmid Preparation

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Protocol for transfection of EGFP-PQBP3 or EGFP expression plasmids and induction of senescence in HeLa cells with hydrogen peroxide (H 2 O 2 ). ( B ) Immunocytochemistry of PQBP3, PSME3, and Lamin B1 in HeLa cells with or without treatment. Upper image panels show HeLa cells with or without H 2 O 2 treatment. A portion of H 2 O 2 -treated cells (white arrows) lost the Lamin B1 ring at the nuclear membrane, and PQBP3 and PSME3 were dispersed to the cytosol in these cells. The right graphs show signal densities of PQBP3 or PSME3 on the Lamin B1 ring (upper and middle graphs) and numbers of PQBP3-PSME3 colocalized dots on the Lamin B1 ring (lower graph). The signal intensities of PQBP3 and PSME3 on the Lamin B1-positive area were measured by ImageJ. ( C ) Immunoprecipitation analysis of the interactions between PQBP3, PSME3, and Lamin B1. Left panels and right panels show input and output, respectively, of immunoprecipitations. Input panels show decreased PSME3 and Lamin B1, after H 2 O 2 treatment. Output panels show suppressed interactions between Lamin B1 and PSME3 by EGFP-PQBP3 in coprecipitation. Black and gray arrows indicate each proteins. ( D ) Qunatitative analyses of Lamin B1 protein levels normalized to GAPDH. (Lamin B1/GAPDH) Statistical significance was found in comparison of (1) and (3) (#: p = 0.0452), (2) and (4) (###: p = 0.0006), and (3) and (4) (#: p = 0.0494). (PQBP3-EGFP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (###: p = 0.0005), and (3) and (4) (###: p = 0.0002). (PSME3 in EGFP-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (#: p = 0.006), and (3) and (4) (#: p = 00252). (Lamin B1 in PSME3-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (2) and (4) (##: p = 0.0012), and (3) and (4) (##: p = 0.0093). (PSME3 in Lamin B1-IP) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (2) and (4) (#: p = 0.0138), and (3) and (4) (#: p = 0.0111). ( E ) Immunoprecipitation analysis of the interactions between PQBP3, PSME3, and Lamin B1 was performed by these proteins endogenously expressed in HeLa cells. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Transfection, Expressing, Immunocytochemistry, Membrane, Immunoprecipitation, Comparison

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Sequential reprobing of the same filter with anti-Ubiquitin antibody and anti-Lamin B1 antibody revealed existence of ubiquitinated Lamin B1 (black arrow), which was increased in HeLa cells treated with hydrogen peroxide despite of the decrease of total Lamin B1. ( B ) Immunoprecipitation and detection of Lamin B1 by anti-Ubiquitin, anti-SUMO1, and anti-Lamin B1 antibodies to confirm existence of ubiquitinated and SUMOylated Lamin B1. The band detected in ( A )) was confirmed by immunoprecipitation as mono-ubiquitinated Lamin B1 (black arrow). In addition, some higher bands were shown as ubiquitinated Lamin B1 in HeLa cells treated with hydrogen peroxide (red arrow). Similarly, SUMOylated Lamin B1 were confirmed. The bands of ubiquitinated Lamin B1 were also reactive to SUMO1 (red arrow), indicating that two modifications occurred simultaneously on Lamin B1. ( C ) Protein modifications were examined in GST-Lamin B1 and cell lysates from HeLa cells with or without H 2 O 2 treatment expressing EGFP-PQBP3 or EGFP by transient transfection. Considering the molecular weight 26 kDa of GST, the band indicated with balck arrow corresponds to the band in ( A ), and it is mono-Ubiquitinated Lamin B1. The band size of blue arrow in SUMO1 blot is consistent with SUMOylated and Ubiquitinated GST-Lamin B1, and corresponds to SUMOylated and Ubiquitinated Lamin B1 detected in immunoprecipitation (( B ), red arrow). Light gray arrow-indicated band in ( B ) corresponds to that in (( C )). ( D ) PSME3 knockdown by PSME3-siRNA (KD) and PSME3 overexpression by pCMV3-Myc-PSME3 revealed a reverse relationship between PSME3 and Lamin B1. Ubiquitinated and/or SUMOylated LaminB1 was examined by western blot (upper panels) or immunoprecipitation (lower panels). Red and black arrows indicate modified and non-modified Lamin B1 as described above. Right graphs show quantitative analyses of red arrow band intensities in Ub and SUMO blots subtracted by backgrounds and corrected by Lamin B1. (PSME3) Statistical significance was found in comparison of (1) and (2) (###: p = 0.0007), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p < 0.0001). (Lamin B1) Statistical significance was found in comparison of (1) and (2) (#: p = 0.0141), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p < 0.0001) (GAPDH) Statistical significance was found in comparison of (1) and (3) (##: p = 0.0014), and (2) and (3) (###: p = 0.0006). (Ub(85kDa)) Statistical significance was found in comparison of (1) and (3) (##: p = 0.009), and (2) and (3) (#: p = 0.0157). (SUMO1(85kDa)) Statistical significance was found in comparison of (1) and (3) (###: p = 0.0004), and (2) and (3) (##: p = 0.0012). (Ub-Lamin B1) Statistical significance was found in comparison of (1) and (2) (###: p < 0.0001), (1) and (3) (###: p < 0.0001), and (2) and (3) (###: p = 0.0003). (SUMO-Lamin B1) Statistical significance was found in comparison of (1) and (2) (#: p = 0.0182), (1) and (3) (###: p = 0.0002), and (2) and (3) (#: p = 0.0238). ( E ) Inhibitors of ubiquitination (0.1 μM TAK-243) or SUMOylation (10 μM 2-D08) was added to the culture medium of HeLa cells, and the cells were transfected by pCMV3-Myc-PSME3 6 h later. Inhibition of SUMOylation suppressed Lamin B1 decrease by PSME3-OE (black arrow), and the suppressive effect was smaller in inhibition of ubiquitination. Right panels confirm the effects of TAK-243 and 2-D08, respectively, on ubiquitination and SUMOylation, in which the Lamin B1 band reactive to both anti-Ubiquitin and anti-SUMO1 antibodies in Fig. 9B is indicated (red arrows). (Lamin B1) Statistical significance was found in comparison of (1) and (4) (#: p = 0.0178), (2) and (3) (#: p = 0.0126), (2) and (4) (###: p < 0.0001), and (3) and (4) (###: p = 0.0004). (Ub) Statistical significance was found in comparison of (1) and (4) (##: p = 0.003), (2) and (3) (##: p = 0.0047), and (3) and (4) (#: p = 0.0204). (SUMO1) Statistical significance was found in comparison of (1) and (4) (###: p = 0.0002), (2) and (4) (##: p = 0.0012), and (3) and (4) (###: p = 0.0002). Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Expressing, Transfection, Molecular Weight, Knockdown, Over Expression, Western Blot, Modification, Comparison, Inhibition

Journal: The EMBO Journal

Article Title: PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation

doi: 10.1038/s44318-024-00192-4

Figure Lengend Snippet: ( A ) Colocalization of PQBP3/NOL7 and polyQ disease proteins in cell models. Left panels show single expression of a polyQ disease protein fused to DsRed (DsRed-Atxn1, DsRed-Atxn7, DsRed-Htt, and DsRed-AR) containing a normal or mutant length of polyQ sequence. A small portion of DsRed-Atxn1 formed nuclear speckles in both normal and mutant polyQ lengths, while most of DsRed-Atxn1 exhibited relatively homogeneous nucleoplasm distribution. DsRed-Atxn7 was dominantly distributed in nucleoli. In most cells, DsRed-Htt was predominantly distributed around the nucleus in the cytoplasm, while a part of mutant DsRed-Htt formed cytoplasmic inclusion bodies, as we reported previously (Tagawa et al, ). DsRed-AR, in the absence of androgen treatment, was distributed in the cytoplasm. ( B ) Expression of EGFP-PQBP3 changes the cellular distribution of polyQ disease proteins. EGFP-PQBP3 colocalized with Atxn1 on the nucleoli, while a portion of EGFP-PQBP3 formed nucleoli composed only of PQBP3. EGFP-PQBP3 colocalized with Atxn7 at fibrillarin-positive nucleleoli. EGFP-PQBP3 coexpression shifted localization of DsRed-Htt and DsRed-AR from the cytoplasm to the nucleoli. ( C ) Evaluation of senescence by DHB-mVenus in three types of induction, confluence quiescence, proliferation senescence, H2O2 senescence, and mutant Atxn1 expression. Representative nuclear and cytoplasmic images of DHB-mVenus and quantitative analysis of the percentage of senescence-phenotype cells, in which DHB-mVenus was distributed only in nucleus, among total pDHB-mVenus-transfected cells are shown. ( D ) Immunohistochemistry for PQBP3/NOL7 and Lamin B1 in cerebellar cortex of Atxn1-KI mice and littermate controls at 9 weeks of age. Nucleolar PQBP3/NOL7 signal was decreased in Purkinje cells of Atxn1-KI mice, and nuclear membrane Lamin B1 signal in Purkinje cells also decreased (white arrows). High-magnification original and enhanced images revealed notches and blebbing of nuclear membranes in Purkinje cells of Atxn1-KI mice (white arrow). On the other hand, Lamin B1 signals were unaltered in granule cells. ( E ) Immunohistochemistry for Atxn1/PSME3/Ubiquitin (left panels) and PQBP3/PSME3/Ubiquitin (right panels) in cerebellar cortexes prepared from Atxn1-KI mice and littermate controls at 9 weeks of age. In a portion of Purkinje cells of Atxn1-KI mice, Atxn1 formed nuclear inclusions with ubiquitin (white arrow), while nuclear PSME3 was decreased. PQBP3 was detected in Purkinje cell nuclei of control mice (white arrow) but decreased in Purkinje cell nuclei of Atxn1-KI mice. PSME3 was localized to the nucleus of Purkinje cells in control mice but dispersed to the cytoplasm in Atxn1-KI mice. These changes in expression and localization of PSME3, PQBP3, and Lamin B1 were homologous to those observed in H 2 O 2 -induced senescent HeLa cells (Fig. ). ( F ) Immunohistochemistry for PQBP3/Atxn1/Ubiquitin (left panels) in cerebellar cortexes prepared from Atxn1-KI mice and littermate controls at 9 weeks of age. PQBP3 puncta located at the periphery of nucleus and/or in the cytoplasm (white thin arrows) were costained with Atxn1 and Ubiquitin antibodies in abnormal Purkinje cells (yellow thick arrow), while nucleolus of relatively normal Purkinje cells (light blue thick arrow) was also found in Atxn1-KI mice at 9 weeks. Lower graphs show quantitative analyses of signal intensity of PQBP3 merged with Atxn1 in Purkinje cells, signal intensity of PQBP3 non-merged with Atxn1 in Purkinje cells, and signal intensity of PQBP3 in nucleoli of Purkinje cells. Experiments in this figure were technically replicated until the necessary N was acquired. Box plots show the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. .

Article Snippet: Hela cells (1 × 10 4 Cells in 500 μL) were transfected with 6 nM of Cy5-labeled Human NOL7-siRNA (#sc-95562, Santa Cruz, Dallas, TX, USA) or Trilencer-27 universal scrambled negative control siRNA duplex (#SR30004, OriGene, Rockville, MD, USA).

Techniques: Expressing, Mutagenesis, Sequencing, Transfection, Immunohistochemistry, Membrane, Ubiquitin Proteomics, Control

Journal: Frontiers in immunology

Article Title: Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response.

doi: 10.3389/fimmu.2023.1287539

Figure Lengend Snippet: FIGURE 3 Quantitative proteomic analysis of non-infected and Leishmania-infected macrophages. THP-1 cells were cultured in SILAC media as described in “Materials and methods”. Labeled THP-1 cells were treated with an equal mix of two Ago1 siRNAs or with control scrambled siRNA. Control and Ago1- knockdown cells were differentiated and incubated with Leishmania for 24 h. Subsequently, control and infected cells were washed extensively and mass spectrometry was performed on a mix of three lysates allowing for a quantitative comparison between non-infected, infected cells and infected in Ago1 knockdown condition (three independent experiments). Shown is the comparison between non-infected (L) and Leishmania-infected (M) cells. (A) Pie chart of the log2(fold change) distribution of Leishmania-modulated proteins. M/L normalized ratios were log2 transformed and averaged for at least 2 replicates. P-values < 0.05 from one-sample T-tests were considered significant. Proteins with a negative log2(fold change) were downregulated in Leishmania-infected cells compared to non-infected cells, whereas proteins with a positive log2(fold change) were upregulated in Leishmania- infected cells compared to non-infected cells. (B) Volcano plot comparing protein expression levels in Leishmania-infected cells with those in non- infected cells, both treated with control siRNA. Proteins significantly upregulated by Leishmania infection are shown as green dots, and those that were downregulated, as red dots. The horizontal dashed line marks the p-value cut off of 0.05 or -log10(p-value) = 1.301. The vertical dashed line represents log2(M/L) = 0. L, Light (Scrambled siRNA-treated, Non-infected); M, Medium (Scrambled siRNA-treated, Ld-infected).

Article Snippet: We have previously used HiPerFect transfection reagent (Qiagen) to silence the expression of various proteins in THP-1 cells (28) and in human monocyte-derived macrophages (24) using siRNAs from OriGene, with transfection efficiency between 70-80%, as measured by fluorescent-labeled transfection control siRNA (OriGene).

Techniques: Infection, Cell Culture, Multiplex sample analysis, Labeling, Control, Knockdown, Incubation, Mass Spectrometry, Comparison, Transformation Assay, Expressing

Journal: Frontiers in immunology

Article Title: Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response.

doi: 10.3389/fimmu.2023.1287539

Figure Lengend Snippet: FIGURE 4 Proteomic analysis of Ago1-dependent Leishmania-modulated macrophage proteins. (A) Scatter plot of log2 transformed M/L ratios (y-axis) vs. log2 transformed H/M ratios (x-axis) of proteins. Proteins that were upregulated by Leishmania infection and recovered to any degree following Ago1 knockdown are shown in green. Proteins that were downregulated by Leishmania-infection and recovered to any degree following Ago1 knockdown are shown in red. The Ago1-dependent Leishmania-modulated proteins were also classified by GO terms in three categories: (B) biological process, (C) cellular component, and (D) molecular function. P-values < 0.05 from one-sample T-tests were considered significant for both log2 transformed M/L and H/M fold changes. L, Light (Scrambled siRNA-transfected, Non-infected); M, Medium (Scrambled siRNA-transfected, Ld-infected); H, Heavy (Ago1 siRNA-transfected, Ld-infected).

Article Snippet: We have previously used HiPerFect transfection reagent (Qiagen) to silence the expression of various proteins in THP-1 cells (28) and in human monocyte-derived macrophages (24) using siRNAs from OriGene, with transfection efficiency between 70-80%, as measured by fluorescent-labeled transfection control siRNA (OriGene).

Techniques: Transformation Assay, Infection, Knockdown, Transfection

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Phemindole downregulates STIM1 Expression. (A) Immunofluorescence images showing decreased expression of STIM1 in the MDAMB-231 cells in response to Phemindole treatment. Anti STIM1 antibody was FITC tagged (green) and nuclei were stained with DAPI (blue) (left panel). Corrected Fluoroscence intensity of STIM1 protein was quantified and represented as bar diagram (Right panel). Pictures showed representative cells of each population and were representative of three independent experiments. (B) Western blot analysis depicting the changes in the expression pattern of STIM1 and Orai1. MDAMB-231 cells were treated with Phemindole and Western blotted with respective antibodies as labeled in the figure (upper-left panel). Quantification of WB were depicted as bar diagram (upper-right panel). MDAMB-231 cells were treated with 10 μM Phemindole for 24 h. Lysates were subjected to SDS–PAGE followed by Western blotting (with anti-Orai1 antibodies. β–actin was used as a loading control (middle-left panel). Band intensity was quantified and represented as a bar histogram (middle-right panel). Cells were treated with 10 μM Phemindole and were harvested at 0, 6, 12, or 24 h after Phemindole treatment. Western blotting was done. Antibodies used are labeled and β-actin was used as loading control (lower-left panel). Lower-right panel represent the normalized intensity of the bands. Blots are representative of three independent experiments and quantifications are indicated by Bar diagram. Values were normalized by intensity of loading control. (C) MDAMB-231 cells were treated with Phemindole or Tg under different conditions, lysed and proteins were subjected to immune precipitation by anti STIM1 antibody and followed by Western blotting with respective antibody (upper panel). (D) Representative Fluorescence images showed the association between STIM1 (red) and ORAI1 (green) at plasma membrane. Nuclei were stained with DAPI (blue). Enlargements of the areas indicated by the color rectangles showed overlap formation of STIM1 and Orai1 in plasma membrane due to Tg treatment (Left-middle) which is invisible in Phemindole treated set (left-lower) and not prominent in un-stimulated control set (left-upper panel). Pictures showed representative cells of each population and were representative of three independent experiments. Right panel, the Pearson’s and Manders coefficients determining the level of overlap of RITC and FITC on treatment with Control, thapsigargin ( TG ) and Phemindole has been represented graphically. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.)

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Labeling, SDS Page, Control, Fluorescence, Clinical Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Inhibitions of STIM1 mimics the effects of Phemindole and over expression of STIM1 showed opposite effect. (A) Ca 2+ imaging was performed in control, in the presence of SKF 96365 (SOCE inhibitor) and STIM1 si-RNA treated MDAMB-231 cells. Analog plots of the fluorescence ratio (340/380) of the cells were shown. Knockdown of STIM1 protein was confirmed by western blot analysis (Inset) (B) Quantification (mean ± SD) of fluorescence ratio (340/380). ∗ P < 0.05 versus control. (C) Bar diagram showed MTT assay under SKF 96365, STIM1 si-RNA, treated conditions for 24 h in MDAMB231 cells. (D) STIM1 protein levels were restored by Transfection of STIM1 clones and also other protein levels were measured in presence and absence of Phemindole in STIM1 transfected cells by western blot (left panel). Quantification of band intensity in respect to loading control was shown in bar diagram (left panel). Blots are representative of three independent experiments and quantifications are indicated by bar diagram. Values were normalized by intensity of loading control. (E) Fura-2 Ca 2+ imaging showing the results of a quantitative analysis of Ca 2+ content (or Ca 2+ release in after Tg treatment) and Ca 2+ influx during SOCE as mentioned previously. Values are the mean ± SEM for all cells in plates ∗ p < 0.05 versus control group. (F) Bar diagram represented the cell proliferation pattern of STIM1 over expressed MDAMB-231 cells in presence of Phemindole treatment. Each bar gives the mean ± SEM of three separate experiments. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Over Expression, Imaging, Control, Fluorescence, Knockdown, Western Blot, MTT Assay, Transfection, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Assessment of toxicity of Phemindole. (A) 4T1 induced tumors excised from the aforementioned sets were analyzed histopathologically using eosin haematoxylin staining and phase contrast images were captured. (B) Immuno-histochemical analysis of the excised tumors from the untreated and Phemindole treated sets using anti-STIM1 antibody (left panel). Corrected Fluorescence intensity of STIM1 protein was quantified and represented as bar diagram (right panel). (C) Histological sections of liver and kidney from the animals were stained with haematoxylin and counter-stained with eosin and microscopically analyzed for histopathologically examinations of tissue toxicity like cellular damage and vacuolization. (D) Blood was collected after sacrifice the mice from control, untreated tumor and Phemindole treated sets and subsequent Liver function test and Kidney Function Test were performed. All measurements of three groups were repeated at least three times, Values are mean ± SEM of three independent experiments in each case or representative of typical experiment. ∗∗∗ P < 0.001, (E) Schematic diagram depicting the detailed molecular mechanisms of antitumorogenic activity of Phemindole against triple negative breast cancer cells.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Staining, Fluorescence, Control, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells

doi: 10.1186/s13287-022-02976-z

Figure Lengend Snippet: Shp2 activation by LIF negatively affects naïve pluripotency. A Immunoblotting at indicative time after LIF stimulation (1000 U/ml), Erk2 and β-actin were used as loading controls. B Flow cytometry of GFP after 60 h stimulated by LIF + 2i, LIF only, LIF deprivation (LIF-) media respectively (left panel), Graphical presentation of GFP positive population (right panel) (*** p < 0.001, n = 3). C List of 27 genes that were predicted as putative negative regulators of naïve pluripotency (top), gene list involved in ‘IL-6/JAK/STAT3 Signaling Pathway’ (bottom). D Immunoblotting for indicative proteins at 10 and 30 min after LIF stimulation (1000 U/ml), α-tubulin was used as a loading control. LIF starvation for 1 h was performed before the LIF stimulation. E Immunoblotting for indicative proteins at 10 and 30 min after LIF stimulation (1000 U/ml), β-actin was used as loading control. LIF starvation for 1 h was performed before the LIF stimulation. DMSO (Mock) or iShp2 (5 μm) was treated while LIF starvation. F Graphical presentation of phosphatase activity of OG2 mESCs at 10 min after LIF stimulation with or without iShp2 (5 μm) for 1 h (** p < 0.01, n = 4). G Graphical illustration of LIF mediated signaling of naïve ESCs

Article Snippet: The primary antibodies against α-tubulin (#sc-8035), β-actin (#sc-47778), Stat3 (#sc-8019), Erk2 (#sc-154) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phospho-Shp2 (Tyr542) (#3751), phospho-Stat3 (#9145s), phospho-Erk1/2 (#9101), phospho-Mek1/2 (#9121s) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Flow Cytometry, Control, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells

doi: 10.1186/s13287-022-02976-z

Figure Lengend Snippet: Role of Shp2 in naïve pluripotency. A Graphical illustration of isogenic pair of naïve (OG2) and primed (POG2) ESCs (left), brightfield and fluorescent microscopic images of OG2 and POG2 ESCs (scale bars, 500 μm) (right panel). B Representative light microscopic images of WT and KD ESCs (scale bars, 200 μm) (left panel) and fluorescent microscopic images of WT and KD ESCs (scale bars, 500 μm) (right panel). C Fold mRNA expression of typical naïve pluripotency marker genes in WT and KD ESCs (** p < 0.01 and *** p < 0.001, n = 3). D Fluorescent microscopic images of WT and KD ESCs at indicated time under LIF + 2i and LIF only culture condition (scale bars, 500 μm). E Immunoblotting for pStat3, Stat3 and Erk2 in WT and KD ESCs at indicated time after LIF stimulation (1000 U/ml), Erk2 was used as a loading control. LIF starvation for 1 h was performed before the LIF stimulation. F Immunoblotting of OG2 mESCs with control (siNC) or Ptpn11 siRNA (siPtpn11) at indicated time after LIF stimulation (1000 U/ml), α-tubulin was used as a loading control. LIF starvation for 1 h was performed before the LIF stimulation. G The normalized enrichment score by gene set enrichment analysis (GSEA) of WT and KD ESCs for Hallmark_IL6_JAK_STAT3 signaling (left panel), KEGG_JAK_STAT3 signaling (middle panel), and ABBUD_LIF signaling up (right panel). H DEGs of WT LIF + 2i vs WT (-)2i were assigned as ‘gene sets for 2i’. The number of Up-DEGs and Down-DEGs within the ‘gene sets for 2i’ group are presented. I Light microscopic images of WT and KD 48 h after indicated culture condition (scale bars, 200 µm)

Article Snippet: The primary antibodies against α-tubulin (#sc-8035), β-actin (#sc-47778), Stat3 (#sc-8019), Erk2 (#sc-154) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phospho-Shp2 (Tyr542) (#3751), phospho-Stat3 (#9145s), phospho-Erk1/2 (#9101), phospho-Mek1/2 (#9121s) were purchased from Cell Signaling Technology.

Techniques: Expressing, Marker, Western Blot, Control

Journal: Stem Cell Research & Therapy

Article Title: Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells

doi: 10.1186/s13287-022-02976-z

Figure Lengend Snippet: Role of Shp2 in primed pluripotency. A Light microscopic images of WT and KD ESCs at indicated times under LIF + 2i or bFGF/Activin culture condition (scale bars, 500 μm) (left), Graphical presentation of cell number at indicated time after seeding of 1 × 10 4 ESCs (right). B Images of clonogenic assay of WT and KD ESCs at indicated time under LIF + 2i or bFGF/Activin culture condition (left), Graphical presentation of area ratio (A.U.) of clones at day 2 and day 4 determined by ImageJ (right). C Graphical illustration of competition assay with WT, labeled with red fluorescence [RF/GFP], and KD [GFP only] mESCs (top), Fluorescence microscopic images of WT or KD ESCs at indicated time after each culture condition, circles indicate KD ESCs [GFP only] (scale bars, 500 μm) (left), Relative fluorescence intensity of WT (red) and KD (green) ESCs from GFP images from 72 h was presented as a graph (right) (*** p < 0.001, n = 26). D Immunoblotting analysis of WT and KD ESCs grown under bFGF/Activin for Shp2, pStat3, pErk1/2, Erk2 and β-actin. β-actin was used as a loading control

Article Snippet: The primary antibodies against α-tubulin (#sc-8035), β-actin (#sc-47778), Stat3 (#sc-8019), Erk2 (#sc-154) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phospho-Shp2 (Tyr542) (#3751), phospho-Stat3 (#9145s), phospho-Erk1/2 (#9101), phospho-Mek1/2 (#9121s) were purchased from Cell Signaling Technology.

Techniques: Clonogenic Assay, Clone Assay, Competitive Binding Assay, Labeling, Fluorescence, Western Blot, Control

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: IQGAP1 binds directly to the MET receptor. ( A ). GST-IQGAP1 (IQ1), bound to glutathione-Sepharose beads, was incubated with 2 μg of the purified intracellular region of MET (residues 974–1390). Control pull-downs were carried out with GST-glutathione-Sepharose (GST). After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by SDS-PAGE. The gel was cut at ∼ 80 kDa. The top portion of the gel was stained with Coomassie blue. The lower portion was processed by Western blotting and probed with anti-MET and anti-GST antibodies. Input designates pure MET not subjected to pull-down. The positions of migration of molecular weight markers are indicated on the left. All images are from the same gel. The data shown are representative of two independent experiments. The multiple bands observed on the MET blot likely reflect partial degradation. ( B ). H1993 cells were cultured and lysed. Equal amounts of protein from cell lysates were subjected to immunoprecipitation (IP) with an anti-MET antibody. Control precipitation was carried out with mouse IgG. Samples were resolved by SDS-PAGE followed by Western blotting and probed with anti-MET and anti-IQGAP1 (IQ1) antibodies. Unfractionated cell lysate (Input) was processed in parallel. Both images are from the same membrane. ( C ). Immunoprecipitation was carried out from H1993 cell lysates as described for ( B ), except anti-IQGAP1 antibody was used. Control precipitation was carried out with non-immune rabbit serum (NIRS). Western blots were probed with anti-IQGAP1 (IQ1) and anti-MET antibodies. Both images are from the same membrane. All blots in panels ( B , C ) are representative of three independent experiments. Data presented in panels ( A–C ) were cropped from the full immunoblots shown in . ( D ). H1993 cells grown on coverslips were fixed, permeabilized, and incubated with anti-MET and anti-IQGAP1 antibodies. A proximity ligation assay (PLA) was carried out using Duolink in situ probes and detection reagents (Millipore Sigma). Cell images were acquired via confocal microscopy. Red spots indicate positive PLA signals. Actin was stained with phalloidin (green). Scale bar, 10 μm. Two representative images from two independent experiments (80 cells each) are shown. ( E ). HepG2 cells were incubated in the absence (left panels) or presence (right panels) of HGF. A PLA was performed as described for ( D ), and foci were quantified from confocal images using ImageJ. The boxplot shows the distribution of the number of PLA dots per cell observed with or without HGF (horizontal line, median; box, 25–75% percentiles; whiskers, 10–90% percentiles; n = 100). Statistical analysis was carried out using an unpaired t -test (***, p ≤ 0.001).

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Purification, Control, SDS Page, Staining, Western Blot, Migration, Molecular Weight, Cell Culture, Immunoprecipitation, Membrane, Proximity Ligation Assay, In Situ, Confocal Microscopy

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Knockdown of IQGAP1 enhances MET activation and signaling. HepG2 cells were transfected with control siRNA (siCtrl) or IQGAP1 siRNA (siIQ1). Next, 48 h after transfection, cells were starved of serum for 16 h and then incubated with 50 ng/mL HGF for 0, 5, or 10 min. Equal amounts of cell lysates were analyzed by SDS-PAGE and Western blotting. ( A ). The membrane was probed with an anti-IQGAP1 antibody. An anti-tubulin antibody was used for the loading control. ( B ). The intensity of the IQGAP1 band was quantified using Image Studio 2.0 (LI-COR Biosciences) and corrected for that of tubulin in the corresponding sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for three independent repetitions (mean ± SD, n = 9). The ratio observed for siCtrl cells was set as 1. ( C ). Antibodies specific to phosphorylated MET (pMET, Tyr 1234 /Tyr 1235 ) and total MET were used to probe the Western blot membrane. ( D ). The pMET bands were quantified and corrected for the amount of total MET in the corresponding sample. ( E ). The membrane was probed with antibodies to phosphorylated Akt (pAkt, Ser 473 ) and total Akt. ( F ). The pAkt and Akt signals were quantified and the pAkt/Akt ratio was calculated. ( G ). Phosphorylated ERK (pERK, Thr 202 /Tyr 204 ) and total ERK were detected on the Western blot membrane using specific antibodies. ( H ). The pERK bands were quantified and corrected for the amount of total ERK in the corresponding sample. Graphs D, F, and H show the means ± SD from three independent experiments. For these graphs, the ratio of phosphorylated to total protein in the absence of HGF (0 min) was set as 1 for both siCtrl and siIQ1 cells. Statistical analyses were performed using unpaired t -tests (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). All blots in this figure are representative of three independent experiments. The full blots of all three replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Activation Assay, Transfection, Control, Incubation, SDS Page, Western Blot, Membrane

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Phosphorylation of Tyr 1510 of IQGAP1 creates a docking site for SH2 domains. ( A ). A peptide array containing 124 different GST-tagged recombinant SH2 protein domains, each spotted in duplicate, was generated as detailed in Materials and Methods . GST alone was spotted on the array as the negative control. Biotinylated peptides comprising residues 1502–1518 of IQGAP1 with phosphorylated Tyr 1510 (pTyr 1510 ) were labeled with fluorescent streptavidin and incubated with the array for 16 h at 4 °C. After washing, fluorescence from the bound peptides (green dots) was detected. The red and orange circles delineate the duplicates for the SH2 domains of Abl1 and Abl2, respectively. ( B ). Similar analysis was conducted with the same IQGAP1 peptide, except Tyr 1510 was not phosphorylated. ( C ). The peptide array was probed with anti-GST antibody to show the positions and loading of the GST-SH2 domains and control GST.

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Peptide Microarray, Recombinant, Generated, Negative Control, Labeling, Incubation, Fluorescence, Control

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: The SH2 domains of Abl1 and Abl2 bind directly to tyrosine-phosphorylated IQGAP1. ( A ). Quantification of the fluorescence intensity of the IQGAP1 peptides with unphosphorylated (Tyr 1510 , pale green bars) or phosphorylated (pTyr 1510 , dark green bars) Tyr 1510 bound to the SH2 domains of Abl1 or Abl2 on the SH2 array. Binding to GST is the control. Data represent the mean fluorescence from two duplicates. a.u., arbitrary units. ( B ). H1993 cells were incubated with 100 nM crizotinib (criz, + ) or vehicle DMSO (−). After 24 h, cells were lysed and equal amounts of protein lysate were incubated with the purified GST-SH2 domains of Abl1 or Abl2 bound to glutathione-Sepharose. Control pull-downs were carried out with GST-glutathione-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by Western blotting. The membrane was probed with anti-IQGAP1 (IQ1) and anti-GST antibodies. Input designates unfractionated cell lysates. Both panels are from the same membrane. Blots are representative of two independent experiments. The full blots of the two replicates are shown in . ( C ). The IQGAP1 bands observed after pull-down were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of IQGAP1 in DMSO-treated cells was set as 1. Data are presented as the means of two independent replicates. ( D ). Purified GST-IQGAP1 (IQ1) on glutathione-Sepharose was incubated with purified active MET in the presence (+ATP) or absence (−ATP) of ATP. After washing, beads were incubated with 2 μg of purified Abl1 (left panel) or Abl2 (right panel). Control pull-downs were carried out with GST-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting. The membrane was probed with anti-IQGAP1 (IQ1), anti-phosphotyrosine (pTyr), and anti-Abl1 or anti-Abl2 antibodies. The overlap between IQGAP1 (red) and pTyr (green) signals is visible in the merged image (yellow). Input designates pure Abl1 or Abl2 not subjected to pull-down. The blots are representative of three independent experiments. The full blots of the three replicates are shown in . ( E ). The Abl1 and Abl2 bands observed after pull-down by GST-IQGAP1 were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of Abl1 and Abl2 signals observed with non-phosphorylated IQGAP1 (−ATP) was set as 1. Data are the means ± SD of three independent experiments. Statistical analyses were performed with unpaired t -tests (*, p ≤ 0.05).

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Fluorescence, Binding Assay, Control, Incubation, Purification, Western Blot, Membrane, SDS Page

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Endogenous IQGAP1 with phosphorylated Tyr 1510 binds to endogenous Abl1 and Abl2. ( A ). H1993 cells were cultured and lysed. Equal amounts of protein from cell lysates were subjected to immunoprecipitation (IP) with anti-Abl1 (left panel) or anti-Abl2 (right panel) antibodies. Control precipitations were carried out with mouse IgG. Samples were resolved by SDS-PAGE followed by Western blotting and probed with antibodies to IQGAP1 (IQ1) and Abl1 or Abl2. Unfractionated cell lysates (Input) were processed in parallel. Both images in each panel are from the same membrane. Blots are representative of three independent experiments. The presented data were cropped from the full immunoblots shown in . ( B ). H1993 cells were incubated with 100 nM crizotinib (criz, +) or vehicle DMSO (−) for 24 h. Cells were then lysed, Abl1 or Abl2 was immunoprecipitated, and samples were resolved by Western blotting as described for panel A. The amount of IQGAP1 was quantified and corrected for the amount of immunoprecipitated Abl1 or Abl2 in the same sample. Ratios were set as 1 for DMSO-treated (−) cells. Data are the means ± SD of three independent repetitions. Statistical analyses were performed with unpaired t -tests (***, p ≤ 0.001). All blots shown in this figure are representative of three independent experiments. The full blots of the three replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Immunoprecipitation, Control, SDS Page, Western Blot, Membrane, Incubation

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: IQGAP1 modulates Abl kinase activity. HepG2 cells were transfected with scrambled siRNA (siCtrl) or siIQGAP1 (siIQ1) RNA. After 48 h, cells were starved for 16 h before being incubated with 50 ng/mL HGF for 0, 5, or 10 min. ( A ). Cells were lysed and equal amounts of protein lysates were analyzed by Western blotting using anti-IQGAP1 and anti-tubulin antibodies. ( B ). IQGAP1 signal intensity was quantified with Image Studio 2.0 (LI-COR Biosciences) and corrected for the amount of tubulin in the corresponding sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for four independent repetitions (mean ± SD, n = 12). The ratio in siCtrl cells was set as 1. ( C ). Western blots of the same HepG2 cell lysates as those used for panel A were probed for CrkL phosphorylated on Tyr 207 (pCrkL) and total CrkL. ( D ). The pCrkL band at the 0 min HGF timepoint (basal phosphorylation) was quantified and corrected for the amount of total CrkL in the corresponding sample. siCtrl cells were set at 1 (mean ± SD, n = 4). ( E ). The pCrkL/CrkL ratio was calculated for the 0 and 10 min HGF timepoints. The ratio for 0 min HGF was set at 1 for both siCtrl and siIQ1 cells to visualize the effect of HGF stimulation on pCrkL (mean ± SD, n = 4). ( F ). The CrkL band was quantified and corrected for the amount of tubulin in the same sample. Data are expressed as the IQGAP1/tubulin ratio from the three HGF timepoints for four independent repetitions (mean ± SD, n = 12). All blots shown in this figure are representative of four independent experiments. Statistical analyses were carried out using unpaired t -tests (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). The full blots of the four replicates are shown in .

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activity Assay, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Model of IQGAP1 in receptor tyrosine kinase signaling. ( A ). Schematic of IQGAP1 that highlights known post-translational modifications. The five domains are CHD (calponin-homology domain), WW, IQ, GRD (GAP-related domain), and RGCT (RasGAP_C-terminus). Post-translational modifications that have been characterized on IQGAP1 are shown below the modified amino acid. The identified domain to which receptor tyrosine kinases bind is depicted above IQGAP1. Phosphorylation of IQGAP1 on Tyr 1510 , leading to the recruitment of SH2 domains, is also shown. ( B ). Model depicting the identified modes of action of IQGAP1 in receptor tyrosine kinase signaling. Upper panel: constitutive scaffolding by IQGAP1. IQGAP1 binds constitutively to both the growth factor receptor and a downstream signaling protein. (i) Ligand binding activates the receptor. (ii) Scaffolding by IQGAP1 facilitates activation of the signaling protein by the activated receptor, initiating downstream signaling. (iii) Signal transduction from the activated receptor to the effector protein does not occur in the absence of scaffolding by IQGAP1. The mode of action depicted here is for the EGF receptor (EGFR) and B-Raf kinase in activation of the MAPK cascade [ , ]. Lower panel: phosphotyrosine-dependent scaffolding by IQGAP1. (i) Phosphorylation of tyrosine on IQGAP1 by an activated receptor tyrosine kinase (ii) initiates recruitment of selected SH2-containing proteins. The mechanism illustrated here is for the MET receptor tyrosine kinase and the SH2-containing proteins Abl1 and Abl2. In this example, IQGAP1 (iii) impairs MET activation and signaling and (iv) decreases HGF-stimulated signaling of Abl to the adaptor protein CrkL. Therefore, IQGAP1 functions as a rheostat regulating the flux of signaling between activated MET receptors and SH2-containing signaling proteins. (v) Recruitment of SH2-containing proteins does not occur in the absence of receptor tyrosine kinase-catalyzed phosphorylation of IQGAP1. Abbreviations: EGF, epidermal growth factor; EGFR, EGF receptor; HER2, human epidermal growth factor receptor 2; HGF, hepatocyte growth factor; IR, insulin receptor; P, phosphate; pS, serine phosphorylation; pY, tyrosine phosphorylation; SUMO, SUMOylation; Ub, ubiquitination.

Article Snippet: IQGAP1 siRNA (sc-35700), control siRNA (sc-37007), and normal rabbit and mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Modification, Phospho-proteomics, Scaffolding, Ligand Binding Assay, Activation Assay, Transduction, Ubiquitin Proteomics

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication

doi: 10.3389/fcimb.2024.1399761

Figure Lengend Snippet: siRNA depletion of SNX27, Vps35, SNX1 and SNX1 + 2 prevents pericentriolar accumulation of Rab10 in the early phase of MCMV infection. (A) Balb3T3 fibroblasts were transfected with scrambled siRNA or siRNA for SNX27, Vps35, SNX1, and SNX1 + 2. After 48 h, the expression of depleted proteins was detected by Western blot using β-actin as a loading control. (B) siRNA-treated cells (48 h after transfection) were infected with Δm138-MCMV (MOI of 10), and at 16 hpi the expression of depleted proteins was analyzed by Western blot. The pIE1 expression served as a marker for infection, and β-actin was used as a loading control. (C-F) Infected cells (16 hpi) transfected with either control siRNA or siRNA for SNX27, Vps35, SNX1 and SNX1 + 2 were stained with antibodies for either SNX27 (C) or Vps35 (D) or SNX1 (E) or SNX1 + 2 (F) to visualize these proteins (green fluorescence) combined with antibodies for Rab10 (red fluorescence) and pIE1 (blue). The stained cells were analyzed by confocal imaging, and the percentage of cells expressing pericentriolar Rab10 accumulation was determined by epifluorescence image analysis. The data show individual results from five independent experiments. Ctrl., control level (mean ± SD) from 18 independent experiments. Mean values are shown as red bars, and statistical significance was determined using a two-tailed Student t-test (*** p < 0.001). Bars, 10 μm.

Article Snippet: Small interfering (si)RNA sequences were purchased as follows: non-targeting negative siRNA (1022076) and Mm_Snx27_7 sequence (SI04939543) were from Qiagen (Hilden, Germany); siRNA for SNX1 (sc-41346), siRNA for SNX2 (sc-41350) and siRNA for Vps35 (sc-63219) were from Santa Cruz Biotechnology Inc. (Dallas, USA).

Techniques: Infection, Transfection, Expressing, Western Blot, Control, Marker, Staining, Fluorescence, Imaging, Two Tailed Test

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication

doi: 10.3389/fcimb.2024.1399761

Figure Lengend Snippet: Depletion of SNX27 inhibits progression through the productive infection. Balb3T3 fibroblasts were transfected with scrambled (Scr.) or SNX27 siRNAs. After 48 hours, cells were infected with Δm138-MCMV (MOI of 10) and analyzed at 6, 16, 24, and 40 hpi by immunofluorescence and Western blot. (A) Schematic presentation of the viral replication cycle program. The available antibody reagents against seven MCMV-encoded proteins can distinguish immediate early ( ie ), three temporal classes of early ( e I , e II , and e III ), and two temporal classes of late ( l I and l II ) gene expression. The kinetics of the temporal classes of transcription are shown by dotted lines, and protein expression by full lines using different colour codes. The temporal effect of MCMV infection on cellular DNA replication (cdr) and the timing of viral DNA replication (vdr) are shown in the light-blue area. (B) Percentage of cells expressing viral proteins at 6, 16, and 40 hpi. Triple-stained (viral protein, SNX27, and DAPI) immunofluorescence samples were captured by confocal imaging ( Supplementary Figures S9, S10A ) and quantitatively analyzed using epifluorescence microscopy. Open circles represent data from three independent experiments, and red bars show the average value. Ctrl., control level (mean ± SD) determined in independent experiments (n=25 for pIE1, n=22 for pE1, n=12 for pM57, and n=8 for pM74). (C) Quantification of ie, e I , e II , and e III viral protein expression by Western blot. Viral proteins pIE1, pE1, pm06, and pM57 were analyzed simultaneously (at the same membrane) with SNX27 and actin ( Supplementary Figure S7 ). The signals were quantified, viral protein expression in SNX27-depleted cells normalized to actin expression, and the result was shown as a relative to control (scr-siRNA-treated cells). Results are presented as fold changes compared to scr-siRNA in the respective kinetics (means ± SD) by empty circles, and averages by red bars. (D) EdU labeling of viral DNA replication. Cells transfected with Scr. and SNX27 siRNA were infected and labeled 16-24 hpi with 10 μM EdU, followed by staining with antibodies against SNX27 and pIE1, and visualization of EdU-labeled DNA with the click reaction. Shown is the percentage of EdU-positive cells, whereas the triple-labeled images are shown in Supplementary Figure S10B . The empty circles show the values in an independent experiment, and the red bars show the mean value. Ctrl., control level (mean ± SD) determined in 15 independent experiments. (E) Quantification of l I and l II viral protein expression (pM55, pM74, and pm116) was performed by Western blot as described in (C) . The representative images of Western blots are shown in Supplementary Figure S7 . Two forms of pM55 (55 and 130 kDa) were analyzed. (F) Effect of depletion of Vps35 and SNX1+2 on the expression of the late protein pM74. Shown are the representative Western blots of pM74, Vps35, and SNX1+2 expression in Vps35- and SNX1+2-depleted cells at 40 hpi, respectively. The asterisk represents remnants of Vps35 detection overlaying the lower band of pIE1. The pM74 signals were quantified, normalized to actin, and shown as a fold change compared to the scr-siRNA (empty circles). The average values are shown as red bars. (G) Depletion of SNX27 reduces virus production. Single-step growth kinetics in non-transfected (NT), scr-siRNA- or SNX27 siRNA-transfected Balb3T3 cells after infection with Δm138-MCMV (MOI of 10). Supernatants and cells were harvested at indicated times post-infection, and frozen at -80°C. After two rounds of freeze/thaw, the virus was quantitated by the plaque assay on MEFs. Data represents log 10 infectious units/mL of sample, and error bars indicate standard errors of the means of six biological replicates. Statistical significance was determined using a two-tailed Student t-test (B-F) or one-way ANOVA analysis (G) . The colour of the asterisks (G) denotes the statistical difference of NT (green) and Scr. (blue) from SNX27. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Small interfering (si)RNA sequences were purchased as follows: non-targeting negative siRNA (1022076) and Mm_Snx27_7 sequence (SI04939543) were from Qiagen (Hilden, Germany); siRNA for SNX1 (sc-41346), siRNA for SNX2 (sc-41350) and siRNA for Vps35 (sc-63219) were from Santa Cruz Biotechnology Inc. (Dallas, USA).

Techniques: Infection, Transfection, Immunofluorescence, Western Blot, Gene Expression, Expressing, Staining, Imaging, Epifluorescence Microscopy, Control, Membrane, Labeling, Virus, Plaque Assay, Two Tailed Test