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Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 5. Regulation of RBMY oncogenicity by its upstream kinases. (A) AlamarBlue cell viability assay evaluated the survival and proliferation ability of the indicated transfectants. Cell viability represented by the measured absorbance at 570 nm was plotted as the means ± SDs after normalization to the corresponding values at 0 hours. *P < .005 vs the GFPþHA control was determined by the Student t test. (B) 3D spheroid invasion assay of indicated transfectants. The invasiveness of spheroids grown in a 3D matrix was determined by the formation and enrichment of spindle-like protrusions (black arrows). Representative micrographs acquired before and after the formation of protrusions from spheroid bodies are shown. The mean percentage of the invaded spheroids was calculated and is shown in a bar graph. The error bars indicate the SDs of 3 wells in 2 independent experiments, and significance was evaluated by the Student t test. (C) A wound healing assay showed the migration efficacy of the indicated transfectants. A representative image of each group before (0 h) and after (48 h) cell migration is displayed. Cells expressing GFP and HA served as controls. The migration distances were measured and are reported as the means ± SDs (Student t test). The data shown are representative of 3 independent experiments. (D) Transwell invasion assay to determine the invasive potential of the indicated transfectants. The numbers of invaded cells are presented as the means ± SDs of 4 independent experiments, and significance was evaluated by the Student t test. (E) Scratch wound healing assay showed the migratory abilities of SNU-387 cells cotransfected with plasmids indicated. The migration distances were depicted as the means ± SDs (Student t test). (F) Transwell invasion assay on the indicated transfectants. The number of invaded cells was plotted as means ± SDs and evaluated by the Student t test for statistical significance. (G) Scratch wound healing and Transwell invasion assays on cells transfected twice with siCtrl/siRBMY, then once with HA/HA-PIM1 expression plasmids. Migration distance (means ± SDs) and number of invaded cells (means ± SDs) were analyzed 48 hours postseeding, and differences between groups were estimated by the Student t test.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Viability Assay, Control, Invasion Assay, Wound Healing Assay, Migration, Expressing, Transwell Invasion Assay, Transfection
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 6. Increasing cytoplasmic accumulation of RBMY by PIM1. (A) Subcellular fractionation coupled with Western blot assay on Huh-7 cells co-expressed with proteins indicated. Detection of Lamin B1 and glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) was performed as for controls of the purities of nuclear (Nuc) and cytoplasmic (Cyt) fractions, respectively. Bar plot represents the densitometry analysis of GFP-RBMY-p. The densities of GFP-RBMY-p in nuclear and cytoplasmic fractions were normalized to the intensities of their corresponding markers, Lamin B1 and GAPDH, respectively, and then divided by the total nuclear þ cytoplasmic densities of GFP-RBMY-p to obtain a percentage value. (B) Fluorescence imaging distinguished the impact of the indicated kinases on the distribution of GFP-RBMY in Huh-7 cells. The arrowheads and arrows indicate the RBMY-Nþ and RBMY-Cþ/Mix patterns, respectively. (C) Comparison of the subcellular distribution of GFP-RBMY upon co-expression with HA-PIM1, HA-MEK2, or HA-CK1ε kinases in HCC cell lines. The microscopic signals of GFP-RBMY- Mix/Cþ and Nþ were plotted as a mean percentages of the cell population (±SDs). (D) Fluorescence microscopy analysis showed the Nþ (arrowhead) and Cþ/Mix (arrow) patterns of GFP-RBMY in Huh-7 cells upon treatment of kinases inhibitors. (E) Western blot analysis exposed the expression level of GFP-RBMY in nuclear and cytoplasmic fractions of HCC cells treated with dimethyl sulfoxide (DMSO) solvent control or kinase inhibitors. Cells were incubated with the indicated inhibitors (30 mmol/ L per well) after attachment. Transfection of GFP-RBMY was performed 24 hours post-treatment. Cells then were refreshed with medium containing the indicated inhibitors and cultured for another 24 hours before subcellular fractionation. Quantitative densitometry analysis was accomplished as mentioned previously. DAPI, 40,6-diamidino-2-phenylindole; RBMY-p, phosphory- lated RBMY.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Fractionation, Western Blot, Fluorescence, Imaging, Comparison, Expressing, Microscopy, Solvent, Control, Incubation, Transfection, Cell Culture
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 7. Tumorigenic expression of PIM1 and RBMY in primary HCC tissues. (A) PIM1-specific IHC on paraffin- embedded tissue sections of primary HCC. PIM1- Cþ–expressing tissue showed PIM1-positive signal only in the cyto- plasm. PIM1-Mix pattern comprised cells express- ing both nuclear and cytoplasmic PIM1 (arrow- heads) and also PIM1-Cþ (arrows). (B) PIM1 and RBMY double-stained IFA was accomplished on pri- mary HCC tissues using anti-PIM1 and SRGY clone 1 antibodies. Yellow ar- rows, PIM1-Mix/RBMY- Nþ; white arrows, PIM1- Mix/RBMY-Mix; and white arrowheads, PIM1-Cþ/ RBMY-Cþ. (C) PIM1- and RBMY-specific IHC on serial paraffin-embedded tissue sections of primary HCC. BV, blood vessel; DAPI, 40,6-diamidino-2- phenylindole.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Expressing, Staining
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 8. PIM1-associated cytoplasmic expression of RBMY is a risk factor for poor prognosis in HCC. (A) Subcellular fractionation and Western blot were performed to analyze the levels of PIM1 and RBMY in the nuclear and cytoplasmic fractions of primary HCC tissues (N ¼ 42). Lamin A/C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the controls for the nuclear and cytoplasmic fractions, respectively, to confirm the purity and loading amount. (B and C) A significant positive correlation between PIM1 and RBMY in patients with HCC. (A) Densitometry analysis was conducted to measure the nuclear and cytoplasmic intensities of PIM1 p34 and RBMY and standardized with the intensities of Lamin A/C and GAPDH, respectively. (B) Linear correlations were established between the nuclear and cytoplasmic fractions of PIM1 p34 and RBMY. (C) The levels of nuclear and cytoplasmic RBMY were compared among cases expressing PIM1- (N ¼ 2), PIM1- Mix (N ¼ 21), and PIM1-Cþ (N ¼ 19). Data were analyzed by the Mann–Whitney U test for statistical significance. Kaplan–Meier analysis with the log-rank test was performed to estimate the (D) 5-year metastasis-free survival rate and the (E) 5-year survival rate after metastasis according to the expression patterns of PIM1 and RBMY in (D) primary tumors and (E) metastases of HCC tissues. (F) Fluorescence microscopy analysis of PLC/PRF/5 cells cotransfected with AsRed2-PIM1 and GFP-RBMY plasmids. 40,6-diamidino-2-phenylindole (DAPI) indicates nuclear staining. White arrows point to cells expressing PIM1-Mix/RBMY-Cþ. Yellow arrowheads specify cells showing PIM1-Mix/RBMY-Mix. White arrowheads show cells expressing PIM1-Cþ/RBMY- Nþ. Yellow arrows indicate cell expressing RBMY-Nþ, but the absence of PIM1. (G) Double IFA staining of primary tumors and paired metastases of HCC. White arrows, PIM1-Mix/RBMY-Cþ or PIM-Cþ/RBMY-Mix; white arrowheads, PIM1-Cþ/RBMY- Cþ. The yellow arrows show the dissociation of the PIM1-RBMY complex. A, alive; BV, blood vessel; C, cytoplasm; D, death; M, metastasis; N, nucleus.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Expressing, Fractionation, Western Blot, MANN-WHITNEY, Fluorescence, Microscopy, Staining
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 10. The PIM1–RBMY axis controls the translational activation of EMT. (A) Immunoblotting was performed to determine the levels of EMT proteins upon depletion of PIM1 and RBMY by siPIM1 and siRBMY, as well as restoration with HA-PIM1 and GFP-RBMY. The numbers under the blots of Snail1 and ZEB1 show the band densities of these proteins relative to a-tubulin and are presented as ratios with respect to the value of siCtrl, which is set as 1. (B) A schematic domain structure of RBMY is depicted. The mutation sites of NESm and NLSm are specially marked in pink characters and blue characters, respectively, showing the substitution of amino acids (a.a.) by alanine in comparison with the original sequence of RBMY-WT. Fluorescence imaging showed the distribution patterns of GFP-RBMY WT, NESm, and NLSm in Huh-7 cells. White arrow- heads, white arrows, and yellow arrows indicate Nþ, Cþ, and Mix patterns of RBMY, respectively. Immunoblotting was performed to assess the expression of EMT proteins in cells overexpressing GFP-RBMY and its mutants with/without HA- PIM1 co-expression. (C) IFA showed the distribution of Snail1 and E-cadherin in Huh-7 cells overexpressing GFP-RBMY and GFP-NLSm. The white arrows, yellow arrows, and white arrowhead indicate GFP-RBMY-Mix, GFP-RBMY-Cþ, and GFP-RBMY-Nþ cells, respectively. (D) Immunoblotting showed the expressions of GFP-RBMY-WT/mutants and EMT proteins upon SMI-4a treatment. Dimethyl sulfoxide (DMSO) served as the solvent control. (E) Immunoblotting evaluated the expres- sions of RBMY and EMT proteins in response to the knockout of PIM1. PLC/PRF/5 cells were transfected 3 times with sgPIM1 (clones 6, 4, and 3) and sgCtrl at 24-hour intervals. Part of these sgRNAs-expressed PLC/PRF/5 cells were transfected once with HA-RBMY (lower panel). All cells were harvested for measuring the levels of endogenous RBMY (upper) and HA-RBMY (lower), as well as PIM1 and EMT proteins, 24 hours after the last transfection. (F) Schematic diagram summarizing the findings of this study showing how PIM1 and RBMY are involved in the expression of EMT proteins. DAPI, 40,6-diamidino-2- phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Activation Assay, Western Blot, Mutagenesis, Comparison, Sequencing, Fluorescence, Imaging, Expressing, Solvent, Control, Knock-Out, Transfection, Clone Assay
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 11. Positive feedback stabilization exists between PIM1 and RBMY. A CHX-chase assay followed by Western blot analysis was conducted to estimate the turnover rates of (A) HA-PIM1 upon GFP-RBMY co-expression and (B) RBMY depletion by siRBMY. Cells cotransfected with HA-PIM1 and (A) either GFP control or GFP-RBMY and (B) either siRBMY or siCtrl were harvested at the times indicated after CHX (300 mg/mL) treatment. The percentage of protein remaining was plotted by quantifying the HA-PIM1 intensities before normalization to their respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals. (C) CHX-chase assay assessing the half-life of GFP-RBMY upon HA-PIM1 overexpression was performed. Cell lysates harvested at the times indicated were analyzed by immunoblotting, and the percentage of protein remaining was calculated as described earlier. (D) Western blot analysis showed the absence of endogenous RBMY and PIM1 p34 in the HCC cell line SNU-387 derived from a female patient, as compared with the male HCC line PLC/PRF/5. (E) A CHX-chase assay was performed in SNU-387 cells to determine the half-life of HA-PIM1 in the presence or absence of GFP-RBMY. (F) Immuno- blotting of PIM1 and RBMY expressions in male (N ¼ 4) and female (N ¼ 6) HCC tissue samples. Frozen tissues (20 mg) were homogenized in 400 mL RIPA lysis buffer containing 0.5% sodium dodecyl sulfate. After sonication, the protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Bolt 4%–20% Bis-Tris Plus gels, transferred onto a polyvinylidene difluoride membrane, and subsequently probed with antibodies recognizing the indicated proteins. (G) A CHX-chase assay was achieved before subcellular fractionation of cells cotransfected with HA-PIM1 and GFP-RBMY. The fractions were analyzed by Western blot. Protein degradation was estimated by quantifying the nuclear and cytoplasmic GFP- RBMY intensities before normalization to their respective markers, Lamin A/C and GAPDH. (H) CHX-chase assay determined the half-life of HA-PIM1 upon co-expression of GFP-RBMY-WT and its mutants.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Western Blot, Expressing, Control, Over Expression, Derivative Assay, Lysis, Sonication, Polyacrylamide Gel Electrophoresis, Membrane, Fractionation
![Figure 12. PIM1 fosters the mitochondrial translocation of RBMY to regulate mitochondrial integrity and dynamics. Subcytoplasmic localization of RBMY was analyzed by double-stained IFA probing for the colocalization of RBMY and organelle markers in (A) GFP-RBMY–transfected Huh-7 cells and (B) primary HCC tissue. Glucose-regulated protein of 94 kDa (GRP94), Golgi phosphoprotein 2 (GOLPH2), and COX4 are markers of endoplasmic reticulum (ER), Golgi, and mitochondria, respectively. (C and D) IFA showed the distribution of GFP-RBMY in Huh-7 cells transfected 3 times with siCtrl or siPIM1. (D) The percentage of the cell population (means ± SDs, Student t test) was determined with respect to the different subcellular distribution patterns of GFP-RBMY, which are indicated as patterns I–IV. (E) Immunoblotting showed the submitochondrial localization of PIM1 and RBMY. Mitochondria isolated from Huh-7 cells 24 hours post-transfection were subjected to pro- teinase K and/or Triton X-100 treatment and analyzed by Western blot. Mock indicates the basal levels of mitochondrial proteins. Bcl2 and COX4 represent OMM and IMM proteins, respectively. (F) Mitochondrial levels of apoptosis-related proteins (Bcl2 and Bad) upon overexpression of HA-PIM1 and GFP-RBMY. After subcellular fractionation, the isolated cytoplasmic and mitochondrial fractions were analyzed by Western blot. COX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to indicate the purity of the mitochondrial and cytoplasmic fractions, respectively. (G) Fluorescence staining of the mitochondria, cytoplasm, and nucleus of live PLC/PRF/5 cells with PIM1 or RBMY depletion using Mito-ID Green, CytoPainter Red, and Hoechst 33342, respectively. The white arrows indicate mitochondrial fusion. (H) Immunoblotting of PLC/PRF/5 cells transfected 3 times with the indicated siRNAs and once with HA-PIM1 or GFP-RBMY. A hypothetical model of the mechanism by which RBMY regulates mitochondrial dynamics is presented. (I) Western blot analyzed the Drp1 levels of the indicated cells expressing GFP-RBMY WT or mutants together with HA-PIM1 (upper) and kinase inhibitor SMI-4a (lower). Cells were treated with SMI-4a (30 mmol/L) or solvent control (dimethyl sulfoxide [DMSO]) 24 hours before transfecting with GFP-RBMY-WT or mutants. These cells were continuously supplied with SMI-4a and harvested after 24 hours post-transfection. DAPI, 40,6- diamidino-2-phenylindole.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1855/pm36191855/pm36191855__page17_image1.jpg)
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 12. PIM1 fosters the mitochondrial translocation of RBMY to regulate mitochondrial integrity and dynamics. Subcytoplasmic localization of RBMY was analyzed by double-stained IFA probing for the colocalization of RBMY and organelle markers in (A) GFP-RBMY–transfected Huh-7 cells and (B) primary HCC tissue. Glucose-regulated protein of 94 kDa (GRP94), Golgi phosphoprotein 2 (GOLPH2), and COX4 are markers of endoplasmic reticulum (ER), Golgi, and mitochondria, respectively. (C and D) IFA showed the distribution of GFP-RBMY in Huh-7 cells transfected 3 times with siCtrl or siPIM1. (D) The percentage of the cell population (means ± SDs, Student t test) was determined with respect to the different subcellular distribution patterns of GFP-RBMY, which are indicated as patterns I–IV. (E) Immunoblotting showed the submitochondrial localization of PIM1 and RBMY. Mitochondria isolated from Huh-7 cells 24 hours post-transfection were subjected to pro- teinase K and/or Triton X-100 treatment and analyzed by Western blot. Mock indicates the basal levels of mitochondrial proteins. Bcl2 and COX4 represent OMM and IMM proteins, respectively. (F) Mitochondrial levels of apoptosis-related proteins (Bcl2 and Bad) upon overexpression of HA-PIM1 and GFP-RBMY. After subcellular fractionation, the isolated cytoplasmic and mitochondrial fractions were analyzed by Western blot. COX4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to indicate the purity of the mitochondrial and cytoplasmic fractions, respectively. (G) Fluorescence staining of the mitochondria, cytoplasm, and nucleus of live PLC/PRF/5 cells with PIM1 or RBMY depletion using Mito-ID Green, CytoPainter Red, and Hoechst 33342, respectively. The white arrows indicate mitochondrial fusion. (H) Immunoblotting of PLC/PRF/5 cells transfected 3 times with the indicated siRNAs and once with HA-PIM1 or GFP-RBMY. A hypothetical model of the mechanism by which RBMY regulates mitochondrial dynamics is presented. (I) Western blot analyzed the Drp1 levels of the indicated cells expressing GFP-RBMY WT or mutants together with HA-PIM1 (upper) and kinase inhibitor SMI-4a (lower). Cells were treated with SMI-4a (30 mmol/L) or solvent control (dimethyl sulfoxide [DMSO]) 24 hours before transfecting with GFP-RBMY-WT or mutants. These cells were continuously supplied with SMI-4a and harvested after 24 hours post-transfection. DAPI, 40,6- diamidino-2-phenylindole.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Translocation Assay, Staining, Transfection, Western Blot, Isolation, Over Expression, Fractionation, Fluorescence, Expressing, Solvent, Control
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 13. The PIM1–RBMY axis increases the mitochondrial motility. Bar plots showing the (A) luminescence signal of mitochondrial ATP in Huh-7 cells and the (B) fluorescence signal of mtROS in PLC/PRF/5 cells upon transfection with the indicated plasmids. The results shown are the means ± SDs from quadruplicate samples, and a representative data from 3 separate experiments is shown. The significance of differences was analyzed by the Student t test. (C) Flow cytometry analysis of mtROS in PLC/PRF/5 cells. Stable lines expressing HA-PIM1 and/or HA-RBMY were treated with antimycin A in the absence or presence of NAC. Cells were incubated with mtROS detection reagent before staining with SYTOX blue dead cell dye. For each sample, 10,000 cells were gated. The mtROS level was estimated in PE channel, and the live/dead cells were detected using V450 channel. (D) Mitochondrial locomotion in live Huh-7 transfectants. Representative fluorescence images of nonmotile and motile cells are shown. The percentage of these cells was depicted as means ± SDs and evaluated by the Student t test. (E) Traveling distance of mitochondria from the nuclear front in Huh-7 transfectants was plotted (means ± SDs, Student t test). Representative pictures showed staining of mitochondria, cytoplasm, and nucleus in live Huh-7 cells over- expressed with proteins indicated. Orange and blue arrows indicate nonmotile and motile cells, respectively.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Transfection, Flow Cytometry, Expressing, Incubation, Staining
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 14. ROS genera- tion is crucial for PIM1/ HA-RBMY-mediated mito- chondrial motility and cellular migration/inva- sion. (A) PLC/PRF/5 cells stably expressing HA-PIM1 and/or HA-RBMY were treated with 30 mmol/L NAC and 1 PBS (mock) as sol- vent control. The distribu- tion of mitochondria of these live cells were visual- ized by staining with Mito- ID Green. Staining of 40,6- diamidino-2-phenylindole (DAPI) indicates the nu- cleus. Bar plots (means ± SDs) showed the distance of mitochondrial move- ment. The statistical signifi- cance was evaluated by the Student t test. (B) Scratch wound healing assay showed the migratory abil- ity of cells indicated in the absence and presence of NAC. The migration dis- tance was calculated and presented as means ± SDs. The Student t test was applied for the statistical analysis of the results. (C) Transwell invasion assay assessed the invasive po- tential ofcellsindicated with or without NAC treatment. The number of invaded cells was counted(means ± SDs) and the result between groups was compared by the Student t test. *P < .05, **P < .005, and ***P < .0005.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Migration, Stable Transfection, Expressing, Control, Staining, Wound Healing Assay, Transwell Invasion Assay
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 15. PIM1 and RBMY prime HCC cells for lung metastasis in a mouse model. (A) A schematic illustration of the establishment of the liver cancer lung metastasis model is shown. (B) Double-stained IFA with anti-HA and anti-human RBMY (SRGY clone 1) antibodies was performed to assess the expression of transfected HA-PIM1 and/or GFP-RBMY WT/mutants in the liver tissue sections. A representative figure from each group is shown. (C) The number of pulmonary metastatic nodules and the percentage incidence of lung metastasis in each group were plotted (means ± SEMs, Student t test ). (D) H&E staining assessed the number of pulmonary HCC metastases (black arrows). A representative image from each group is presented. (E) Double IFA staining of PIM1 and RBMY in liver tissues of HA-PIM1þGFP-NLSm mice. Positive immunoreactivity for PIM1-Mix/ RBMY-Cþ is indicated by white arrows. (F) IHC staining of the indicated proteins in serial lung sections from HA-PIM1þGFP- NLSm mice. The black arrows indicate the metastatic nodule in serial sections. BV, blood vessel; DAPI, 40,6-diamidino-2- phenylindole.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Staining, Expressing, Transfection, Immunohistochemistry
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 16. Increasing mitochondrial mass and fission is essential for PIM1 and RBMY-mediated distance metastasis of HCC. (A) Macroscopic views of livers and lungs harvested from the indicated mice. The black arrows show hepatic and pulmonary nodules. No metastatic tumors were found in the lungs of any control vector and HA-PIM1þGFP-NESm mice. (B) Immunoblotting of the protein lysates harvested from liver tissues of the indicated mice after death. A cartoon diagram illustrating that PIM1/RBMY-Cþ and PIM1/RBMY-Nþ impact different mechanisms is shown. (C) Double IFA staining of COX4 and Naþ/Kþ adenosine triphosphatase (ATPase), a plasma membrane marker, in liver tissue sections from the indicated mice. The white arrows show cells with mitochondria-surrounding nuclei. The yellow arrows indicate cells with increased mito- chondrial mass and increased mitochondrial trafficking to the leading edge. DAPI, 40,6-diamidino-2-phenylindole.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Control, Plasmid Preparation, Western Blot, Staining, Clinical Proteomics, Membrane, Marker
Journal: Cellular and molecular gastroenterology and hepatology
Article Title: PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.
doi: 10.1016/j.jcmgh.2022.09.014
Figure Lengend Snippet: Figure 17. Metastasis of cells expressing HA-PIM1 and GFP-RBMY-WT/mutants in a tail-vein injection mouse model. (A) Schematic diagram shows the steps by which a tail-vein injection metastasis mouse model was generated. (B) Western blot monitored the levels of EMT and mitochondrial proteins achieved on PLC/PRF/5 stable lines expressing proteins indicated before subjecting to tail-vein injection. (C) Number of lung metastatic masses and percentage of the metastatic occurrence in each group were counted and reported as means ± SEMs (Student t test). (D) H&E staining was performed on the lung tissues resected from mice indicated, and a representative image from each group is shown. Black arrowheads show pulmonary nodules. (E) Representative macroscopic views of lungs excised from each group at 10 weeks postinjection. Black arrowheads denote pulmonary nodules. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Article Snippet: Cultured cells plated on 8-well chamber slides (Millipore, Billerica, MA) were fixed with 4% paraformaldehyde Table 1.Detailed Information of Antibodies Used in IHC and IFA Analysis Antibody (clone/catalog number) Manufacturer Host species Dilution and incubation conditions RBMY (SRGY clone 1) Homemade Mouse 5 , 4 C, Overnight (O/N) RBMY1A1 (GTX110098) GeneTex Rabbit 150 , 4 C, O/N Her Par-1 (OCH1E5, ab75677) Abcam Mouse 1 , 4 C,
Techniques: Expressing, Injection, Generated, Western Blot, Staining