human psg5l rb1  (Addgene inc)

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: Human RB1 (RRID:Addgene_10720) was prepared using a Quikchange Mutagenesis Kit (Stratagene) and the following oligos (modified bases underlined): 5′C AAAACCCCCCGAAA G AC C GC G GCCACCGCCGCC3′ and 5′GGCGGCGGT GGC C GC G GT C TTTCGGGGGGTTTTG3′ (RRID:Addgene_229856).

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Cytotoxic effect of L12 treatment and its dependency on the RB1 pathway functionality. (A) Depiction of the structure of L12. (B) Western blotting (WB) analysis of RB1 and phosphorylated Ser 780 RB1 proteins using an anti‐RB1 (RB1) and anti‐phospho‐Ser 780 RB1 (pRB1) antibodies. Total lysates from various cell lines were probed, including the RB1‐deficient human retinoblastoma Y79, the osteosarcoma U2OS with a constitutively phosphorylated RB1 at Ser 780 (pRB1), the mammary gland epithelial cell line MCF10A, and the adenocarcinoma alveolar basal epithelial A549, both with functional RB1 pathways, as well as the RB1‐deficient small cell lung carcinoma U1690. α‐tubulin (αTubulin) served as a loading control. Cells treated with different L12 concentrations for 24 h were assessed for cell accumulation in the sub‐G1 fraction (indicating dead cells). The histograms depict mean ± SD of the percentages of cells in the sub‐G1 fractions ( N = 3; * p < .05). (C) Examination of L12's impact on the downstream gene target of TUBG, RB1 , was conducted by WB analysis using an anti‐RB1 antibody on MCF10A and A549 cell lysates. α‐tubulin and actin were utilized as loading controls. Numbers on the western blot indicate variations in RB1 expression compared to the vehicle control. To account for protein loading discrepancies, RB1's protein concentration was normalized to its ratio with the respective loading control for each treatment. The graph displays relative RB1 protein expression across MCF10A ( N = 4) and A549 ( N = 3) cell lines when treated with varying L12 concentrations, with data presented as mean ± SD. Please note that the intensity of the RB1 bands may vary between figures as a result of adjusted exposure times for western blot analysis. These adjustments were made to prevent overexposure and to accurately capture differences in RB1 expression levels across experimental conditions.

Article Snippet: The selected clone was then cotransfected with an sgRNA‐resistant RB1 gene (RRID:Addgene_10720), and coexpressing clones were obtained by isolation of Cas9‐GFP expressing clones and tested the restored RB1 expression using an anti‐RB1 antibody in Western blot analysis.

Techniques: Western Blot, Functional Assay, Control, Expressing, Protein Concentration

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: The selected clone was then cotransfected with an sgRNA‐resistant RB1 gene (RRID:Addgene_10720), and coexpressing clones were obtained by isolation of Cas9‐GFP expressing clones and tested the restored RB1 expression using an anti‐RB1 antibody in Western blot analysis.

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Variable cytotoxic effects of L12 in cells expressing TUBG1 vs. TUBG2. (A) U2OS cells, U2OS cells stably expressing Flag‐tagged TUBG2 (TUBG2‐Flag), or TUBG1 single guide (sg) RNA (sg TUBG1 , resulting in TUBG1 knockout) co‐expressing either sg‐resistant TUBG1 or a sg‐resistant TUBG2 were treated with DMSO (vehicle) or varying concentrations of L12 for 24 h. Total lysates were analyzed by western blotting (WB; N = 3). Antibodies targeting the C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) of TUBG were used to detect endogenous and recombinant TUBG proteins, with actin serving as a loading control. DNA content was measured using a nuclear counter, and histograms display cell cycle profile changes, specifically in the sub‐G1 fraction. Graphs present normalized TUBG1 and TUBG2 levels (from WB) relative to actin and mean ± SD percentages of cells in the sub‐G1 fraction ( N = 3). A schematic highlights the GTPase domain (residues 5–255) and DNA‐binding domain (DBD; residues 334–451) of the human TUBG1 (h‐ TUBG1 ) gene. Amino acid differences between TUBG1 and TUBG2 are shown, with gray and blue indicating TUBG1‐specific residues and magenta denoting TUBG2‐specific residues. (B) WB analysis of cytosolic (Cytosol) and chromatin fractions from the indicated U2OS cells. Antibodies targeting TUBG's C‐terminus (T3320, rabbit) or N‐terminus (T6557, mouse) were used to detect endogenous and recombinant TUBG proteins. T3320 preferentially detects TUBG1, while T6557 preferentially detects TUBG2, especially in fractionated samples, where T6557's specificity for TUBG1 improves. Densitometric analysis of TUBG1 and TUBG2 levels are shown, normalized to α‐tubulin (cytosolic marker) or histone (chromatin marker). An anti‐flag antibody was used to detect TUBG2–Flag. (C) WB analysis of total lysates shows RB1, TUBG1, and TUBG2 protein levels using anti‐RB, T3320, and T6557 antibodies. An α‐tubulin (αTubulin) served as a loading control. The graphs depict relative RB1, TUBG1, and TUBG2 expression across the indicated cell lines (RB1: TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 8; U2OS‐TUBG2 Flag, N = 3; TUBG1: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG1, N = 3; TUBG2: U2OS and TUBG1 ‐sgRNA‐U2OS‐TUBG2, N = 3). (D) Confocal microscopy images of U2OS‐TUBG2‐Flag cells stained with an anti‐TUBG and anti‐Flag antibodies. Images highlight the location of TUBG2‐Flag at γ‐tubules and centrosomes. Colocalization pixel maps (CPM) of magenta/red and green channels are included. White regions signify colocalization, with arrowheads and arrows indicating γ‐tubules and centrosomes, respectively. Scale bars: 10 μm. Graphs show the fluorescence intensity of T3320 (TUBG1) or anti‐Flag (TUBG2) found at γ‐tubules and centrosomes (Student's t test, **** p < .0001; γ‐tubules: N = 127; centrosomes: N = 108). Note that the issue of antibody specificity encountered in WB analysis does not affect immunofluorescence assays, as the proteins maintain a different conformation that is not influenced by SDS treatment. (E) Confocal microscopy images of TUBG1 ‐sgRNA‐U2OS‐TUBG1 and TUBG1 ‐sgRNA‐U2OS‐TUBG2 cells stained with anti‐TUBG antibodies. Graphs display the mean percentages of cells with γ‐tubules ( N = 5; Student's t test, ** p < .01) Hoechst was used for nuclear staining. Scale bars: 10 μm.

Article Snippet: The selected clone was then cotransfected with an sgRNA‐resistant RB1 gene (RRID:Addgene_10720), and coexpressing clones were obtained by isolation of Cas9‐GFP expressing clones and tested the restored RB1 expression using an anti‐RB1 antibody in Western blot analysis.

Techniques: Expressing, Stable Transfection, Knock-Out, Western Blot, Recombinant, Control, Binding Assay, Marker, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence

Journal: Cell Death Discovery

Article Title: CDK4/6 inhibition initiates cell cycle arrest by nuclear translocation of RB and induces a multistep molecular response

doi: 10.1038/s41420-024-02218-6

Figure Lengend Snippet: A T24 and RT112 cells were treated with 1µM PD for 0, 0.5, 4, 8, 12 or 24h. Protein expression was analyzed by immunoblotting. B T24 and RT112 cells were treated with 5µg/ml or 20µg/ml CHX alone or in combination with 1µM PD for 0, 4, 8, 12 or 24h. Protein expression was assessed by immunoblotting. A plus sign indicates presence, while a minus sign indicates absence. C RB signals in Fig. 3 A and B were quantified densitometrically, normalized to the respective housekeeping protein and displayed relative to 0h. Asterisks indicate a significant difference to the respective 0-hour control. D T24 and RT112 cells were treated with 1µM PD for 24h, both with and without MG-132 and epoxomicin as the indicated concentrations. Protein expression was assessed by immunoblotting. E T24 and RT112 cells were transfected with control or gankyrin siRNA oligos. After 24h, cells were treated with 1µM PD for 8 or 24h and Western blot analysis was performed using antibodies against RB, gankyrin and GAPDH. RB signals were quantified densitometrically and expressed as the ration si-gankyrin/si-ctrl at each time point after normalized to the respective housekeeping protein. F RT112 cells transfected with pCMV HA hRB-wt (RB-WT), pCMV HA hRb delta CDK (RB-ΔCDK) or pCMV HA mpRB (RB-mpRB) plasmids for 24h were then treated with 1µM PD for the indicated hours. Exogenous RB protein was detected by Western blotting. RB signals were quantified densitometrically and expressed as the ratio relative to 0h after normalized to the respective housekeeping protein. Error bars indicate SD, asterisks indicate ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The plasmids of human pCMV HA hRb-wt (RB-WT) and pCMV HA hRb delta CDK (RB-ΔCDK) were purchased from addgene [ ].

Techniques: Expressing, Western Blot, Control, Transfection