stable cell line expressing shrna  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for HER2 and cholera toxin B in MCF10A and SKBR3 cells. Scale bars represent 10 μm. (B) Lipid raft areas on the cell surface, with quantification in the bar graph on the right. (C) Flotillin1 (FLOT1), MAL, and MAL2 mRNA expression in different breast cancer cell lines as assessed by quantitative PCR (n = 3). (D) RNA-seq analysis of FLOT1 and MAL2 expression in normal breast tissue (n = 112) and HER2-positive breast tumors (n = 160) represented in The Cancer Genome Atlas database. (E) Uniform Manifold Approximation and Projection (UMAP) plots of breast cancer single-cell RNA-seq data (GEO: GSE75688, left) and co-expression pattern of HER2, FLOT1, MAL, and MAL2 in cells from cluster 2. (F) Distribution of MAL, FLOT1, MAL2, and HER2 expression level for each cell in cluster 2. (G) MAL2 ATAC-seq peak clusters in SKBR3, MCF10A, MCF7, and MDA-MB-231 cell lines. In the bar graphs, the bars represent the mean ± SEM. **p <0.01, ***p <0.001, ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for HER2 and MAL2 (top row) and MAL2 and cholera toxin B (lipid rafts) (bottom row) in SKBR3 cells. (B) Immunofluorescence staining for FLAG-tagged MAL2 or endogenous MAL2 with HER2 and actin in control and MβCD (5 mM)-treated SKBR3 cells. Scale bars represent 10 μm. (C) Proximity ligation assay (PLA) experiment for HER2 and MAL2 in control and MβCD (5 mM)-treated SKBR3 cells also stained for actin (phalloidin). Scale bars represent 10 μm. (D) MAL2 mRNA expression in control and MAL2 knockdown SKBR3 and BT474 cells as assessed by quantitative RT-PCR (n = 3). (E) Western blot analysis of HER2, phospho-HER2, EGFR, and phospho-EGFR in control and MAL2 knockdown SKBR3 and BT474 cells. (F) BrdU incorporation in MAL2KD cells relative to control SKBR3 and BT474 cells. (G) PLA experiment for HER2 and MAL2 in control and MAL2LD SKBR3 cells. Scale bars represent 10 μm. (H) Transmission electron microscopy images in control and MAL2 knockdown SKBR3 cells. (I) Immunofluorescence staining HER2 and cholera toxin B in control (top), MAL2KD-treated (middle), and MβCD-treated (5 mM) SKBR3 cells. Scale bars represent 10 μm. (J) Immunofluorescence staining for FLAG-tagged FLOT1, HER2, and phalloidin in control, MAL2KD-treated, and MβCD (5 mM)-treated SKBR3 cells. Scale bars represent 10 μm. (K) Immunofluorescence staining for HER2 and EGFR in control and MAL2KD SKBR3 cells. Scale bars represent 10 μm. (L) PLA experiment for HER2 and EGFR in control and MAL2KD SKBR3 cells also stained for actin (phalloidin). Scale bars represent 10μm. (M) Quantitation of PLA experiment by measuring fluorescent intensity of amplified PLA reactions and phalloidin. Bar graphs represent the mean ± SEM. **p <0.01, ***p <0.001, ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Immunofluorescence, Staining, Control, Proximity Ligation Assay, Expressing, Knockdown, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay, Transmission Assay, Electron Microscopy, Quantitation Assay, Amplification

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) SIM imaging of fluorescent staining for HER2, Ezrin, and phalloidin in control SKBR3 cells. Images represent different combinations of staining as noted on the figures. Bottom left: a 3D reconstruction for all three molecules. Scale bars represent 10 μm. (B) SIM imaging of fluorescent staining for HER2, HA-tagged NHERF1, and phalloidin in control SKBR3 cells. Bottom left: a 3D reconstruction for all three molecules. Scale bars represent 10 μm. (C) SIM imaging of fluorescent staining for HER2, Ezrin, and phalloidin in MAL2 knockdown SKBR3 cells. Scale bars represent 10 μm. (D) SIM imaging of fluorescent staining for HER2, HA-tagged NHERF1, and phalloidin in MAL2 knockdown SKBR3 cells. Scale bars represent 10 μm. (E) PLA for HER2 with Ezrin (left panels) or NHERF1 (right panels) in control (top row), MAL2 knockdown-treated (middle row) and MβCD-treated (bottom row) SKBR3 cells. Columns labeled “Middle” represent an optical section through the mid-portion of the cells. Columns labeled “Top“ represent an optical section taken near the apical surface of the cell. Columns labeled “w/Phalloidin” represent co-registration of the PLA signal and immunofluorescence for actin (phalloidin). Scale bars represent 10 μm (for all images). (F) Percentage of cells with positive PLA reactions located within membrane protrusions. (G) Quantitation of PLA fluorescent intensity within membrane protrusions. Bar graphs represent the mean ± SEM. ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Imaging, Staining, Control, Knockdown, Labeling, Immunofluorescence, Membrane, Quantitation Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Co-immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing control SKBR3 cells. Scale bars represent 10 μm. (B) Immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing MAL2KD_SKBR3 cells. (C) Immunofluorescence staining for Ezrin (top) or HER2 (bottom) with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with MβCD. (D) Immunofluorescence staining for Ezrin with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with wortmannin (0, 5, and 10 μM). (E) Immunofluorescence staining for HER2 with phalloidin in PH-PLCδ-GFP-expressing SKBR3 cells treated with wortmannin (0, 5, and 10 μM). (F) PLA results for HER2 and Ezrin in control and wortmannin-treated SKBR3 cells. Scale bars represent 10 μm. (G) Western blot analysis of AKT and phospho-AKT in control and MAL2 knockdown SKBR3 cells (left) and in control and MβCD-treated SKBR3 cells (right). (H–J) SIM imaging showing HER2, pAKT, and phalloidin immunofluorescence in control (H), MAL2KD (I)-treated, and MβCD-treated (J) SKBR3 cells. Scale bars represent 10 μm. (K) Immunofluorescence staining for FOXO1 in control, MAL2KD-treated, and MβCD-treated SKBR3 cells. (L) XTT cell viability assay in control, MAL2KD-treated, and MβCD-treated SKBR3 and BT474 cells. Bar graphs represent the mean ± SEM. ****p <0.0001. These results are representative of three independent experiments.

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Immunofluorescence, Staining, Expressing, Control, Western Blot, Knockdown, Imaging, Viability Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: (A) Immunofluorescence staining for cholera toxin B (lipid rafts) in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (B) Immunofluorescence staining for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (C) PLA for HER2 and MAL2 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 μm. (D–F) Quantitative results from immunoprecipitation coupled with data-independent acquisition mass spectrometry (DIA-MS) in control and trastuzumab-resistant SKBR3 cells. (D) The DIA-MS Intensity (log 2 ) of HER2 and MAL2 proteins from control and trastuzumab-resistant SKBR3 cells. (E) The DIA-MS Intensity (log 2 ) for all the peptide precursor signals of HER2 in control and resistant cells. (F) The DIA-MS peak groups visualized for quantifying HER2 (VLGSGAFGTVYK) and MAL2 (VTLPAGPDILR). Peaks above and below the middle line denote the MS2 and MS1 ion traces in DIA-MS. (G) MAL2, Ezrin, and NHERF1 mRNA expression in control and trastuzumab-resistant SKBR3 cells as assessed by quantitative RT-PCR (n = 3). (H) PLA for HER2 with Ezrin (left), NHERF1 (middle), and PMCA2 (right) in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Boxed portions are amplified at right with co-registration of PLA signal and immunofluorescence for actin (phalloidin). (I) Quantitation of PLA experiment for HER2 in combination with MAL2, Ezrin, NHERF1, or PMCA2 represented as the fluorescent intensity of amplified PLA signals associated with membrane protrusions. (J) Coimmunoprecipitation for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells. (K) PLA for HER2 and HSP90 in control and trastuzumab-resistant SKBR3 cells also stained for phalloidin. Scale bars represent 10 μm. (L) XTT cell viability assay in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 and BT474 cells. (M) Immunofluorescence staining for FOXO1 in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 cells. (N) Immunofluorescence staining for HER2 and pAKT in control, MAL2KD-treated, and MβCD-treated trastuzumab-resistant SKBR3 cells. Scale bars represent 10 μm. (O) Diagram representing the structure of MAL2- and lipid raft-enriched membrane protrusions containing multi-protein HER2 signaling complexes. These results are representative of three independent experiments.

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Immunofluorescence, Staining, Control, Immunoprecipitation, Data-independent acquisition, Mass Spectrometry, Expressing, Quantitative RT-PCR, Amplification, Quantitation Assay, Membrane, Viability Assay

Journal: Cell reports

Article Title: MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells

doi: 10.1016/j.celrep.2021.110160

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A stable cell line expressing shRNA directed against MAL2 was generated by transducing cells with commercially prepared lentiviruses containing 3 individual shRNA directed against MAL2 mRNA: MAL2 (sc-77560-V) (Santa Cruz).

Techniques: Recombinant, Real-time Polymerase Chain Reaction, RNA Sequencing, Sequencing, Microarray, Control, Software, Imaging, Light Microscopy

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: HCoV-229E and HCoV-OC43 infection in human cells. ( A ) Cell lines, including A549, H1299, MRC-5, and HCT-8 were infected with HCoV-229E (MOI 0.05) or HCoV-OC43 (MOI 0.01). Viral RNA in cell lysates was quantified by real-time PCR at 24 hpi. Data show viral RNA levels relative to GAPDH. Error bars represent SD ( n = 3). Statistical analysis was conducted with one-way ANOVA: ****, p < 0.0001. ( B ) Representative western blot images showing the expression viral N protein and GAPDH (loading control) in cell lysates at 24 hpi. Full size images are presented in . ( C ) Representative images show CPEs in the HCoV-229E or HCoV-OC43-infected H1299 cells at 48 hpi. Scale bar, 10 μm.

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Infection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: p97 inhibition by CB-5083 suppresses HCoV replication in H1299 cells. H1299 cells were infected with HCoV-229 (MOI 0.5), HCoV-OC43 (0.01), or mock-treated in the presence of CB-5083 at the indicated concentration. Cultured media containing CB-5083 was removed at 8 hpi and replaced with fresh media without CB-5083. ( A ) Cells were harvested to quantify viral RNA levels in cell lysates at 24 hpi. Data shows viral RNA levels relative to GAPDH. ( B ) Representative Western blot analysis of the expression viral N protein and GAPDH (loading control) in cells treated with CB-5083 at 24 hpi after HCoV or mock infection. Full-length blots are included in ( C ) Titer of secreted virus in culture media collected from the HCoV-infected cells at 24 hpi was quantified by determining TCID 50 in H1299 cells. ( D ) Cellular viability of infected cells was determined at 72 hpi and normalized to mock-infected and DMSO-treated controls. All conditions were performed in biological triplicate. Error bars represent SD ( n = 3). Statistical analysis was conducted with one-way ANOVA compared to infected control (0 μM of CB-5083): *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Inhibition, Infection, Concentration Assay, Cell Culture, Western Blot, Expressing, Control, Virus

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: p97 is essential for early stages of HCoV replication. H1299 cells were infected with HCoV-229E (MOI 0.5) or HCoV-OC43 (MOI 0.01). ( A ) Quantification of viral RNA in cell lysates over time after infection. Data shows RNA levels relative to GAPDH. ( B ) Representative Western blot showing viral N protein and GAPDH expression in the infected cells. Full bot images are depicted in . ( C ) H1299 cells were continuously treated with DMSO or p97 inhibitors (CB-5083 and NMS-873) at the indicated period from 30 min before infection to 8 hpi and harvested to determine viral RNA levels. All conditions were performed in biological triplicate. ( D ) Quantification of viral RNA in the cell lysates harvested at 8 hpi. Data show relative RNA levels to GAPDH. Error bars represent SD ( n = 3). Statistical analysis was conducted with one-way ANOVA compared to group 1: a , p < 0.01; b , p < 0.001; c , p < 0.0001; ns, no significance.

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Infection, Western Blot, Expressing

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: p97 knockdown suppresses HCoV replication in H1299 cells. H1299 cells with inducible control shRNA (Ctrl shRNA) or p97 shRNA were inoculated with Dox (0.5 μg/mL) for 72 h. Cells were infected with HCoV-229E (MOI 0.05) or HCoV-OC43 (MOI 0.01). After 1 h of infection, cells were washed and harvested at 0, 4, 8, or 24 hpi. Mock-infected cells were harvested at 24 hpi. ( A ) Quantification of viral RNA in cell lysates over time after infection. Data shows viral RNA levels relative to GAPDH. ( B ) Representative western blot images showing the expression of p97, viral N protein, and GAPDH (loading control) in cell lysates across the infection time course. Full-length blots can be found in . ( C ) Viral titer in culture media collected from HCoV-infected cells at 24 hpi was quantified by determining TCID 50 in H1299 cells. Error bars represent SD ( n = 3). Statistical analysis was conducted with two-way ANOVA for A and Student’s t test for C: *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Knockdown, Control, shRNA, Infection, Western Blot, Expressing

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: Cellular pathway changes in H1299 cells after HCoV-OC43 infection. Proteomic analysis of H1299 cells in response to HCoV-229E or HCoV-OC43 infection. ( A ) Volcano plots of the change in total protein levels comparing infected cells 24 hpi vs. mock infection. Up- (red) and down-regulated proteins (blue) with |log2 FC| > 0.3 and −log10 p value > 1.3 are highlighted. Detected viral proteins are labeled. ( B ) Protein abundance of all detected viral proteins across the infection time course. ( C ) Multi-list Reactome enrichment of DEPs (|log2 FC| > 0.3, p < 0.05) at each time point after HCoV-OC43 infection. Data present the top 20 statistically enriched Reactome pathway terms (–log10 p value > 1.3).

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Infection, Labeling, Quantitative Proteomics

Journal: Cells

Article Title: Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

doi: 10.3390/cells10112953

Figure Lengend Snippet: p97 depletion impacts host cell cycle regulators during HCoV infection. Proteomic analysis of H1299 cells with control shRNA (Ctrl shRNA) or p97 shRNA after HCoV-229E or HCoV-OC43 infection. ( A ) Volcano plots of the change in total protein levels comparing HCoV infected cells with control shRNA vs. p97 shRNA at 24 hpi. Up- (red) and down-regulated proteins (blue) with |log2 FC| > 0.3 and −log10 p value > 1.3 are highlighted. Detected viral proteins are labeled. ( B ) Abundance of detected viral proteins in HCoV-infected cells with control or p97 shRNA at 24 hpi. ( C ) Multi-list Reactome enrichment of DEPs (|log2 FC| > 0.3, p < 0.05) between infected cells with control or p97 shRNA at each time point after HCoV-229E or HCoV-OC43 infection. Data presents the top 20 statistically enriched Reactome pathway terms (–log10 p value > 1.3).

Article Snippet: Stable H1299 cell lines expressing doxycycline (Dox)-inducible shRNA against a control sequence or p97 were generated using the TripZ lenti-viral shRNA system (Thermo Fisher Scientific, Carlsbad, CA, USA) as described previously [ ].

Techniques: Infection, Control, shRNA, Labeling