Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Full-length LATS2, LATS2-LATCH and LATS2-LATCH-4mut, were tested for binding to LIMD1 by co-immunoprecipitation. Lysates were prepared from HEK293A cells separately transfected with GFP-tagged LATS2, LATS2-LATCH, LATS2-LATCH-4mut, or LIMD1-V5. Lysates from cells expressing LIMD1-V5 were mixed separately with those expressing the different GFP-LATS2/LATCH constructs, and anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2/LATCH (GFP) and LIMD1 (V5). (B) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH or LATS2-LATCH-4mut. (C) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH treated with (Blebbistatin) or without (DMSO) Blebbistatin.

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Expressing, Construct, Control, Western Blot, Live Cell Imaging, Stable Transfection

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A-B) LIMD1-KO MCF10A cells stably expressing GFP tagged wild-type LIMD1 (WT) or LIMD1 strain insensitive mutants (F512A, F575A, and Y646A, see also .) were established by lentiviral transduction and imaged using fixed and live-cell imaging. (A) The indicated cell lines were stained using anti-LATS1 and anti-TRIP6 antibodies. Merged images show LATS1 (red), TRIP6 (green) and DNA (blue). (B) The indicated cell lines from part (A) were imaged live for GFP fluorescence. (C) Quantification of the LATS1 junction to cytoplasm mean intensity ratio. Error bars represent the standard deviation. ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the plot (mean ± SD; n = 5; ****P < 0.0001). (D-E) LATS1/2 were tested for their ability to bind to strain insensitive mutants of LIMD1 by co-immunoprecipitation. (D) LATS2 was tested for its ability to bind to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1 and LATS2-FLAG were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged WT and mutants of LIMD1 were combined with LATS2-FLAG lysates. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (V5) and LATS2-FLAG (LATS2). (E) LATS1 was tested for binding to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1, and LATS1-3xMyc were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged LIMD1 variants were combined with LATS1-3xMyc lysates. Anti-Myc or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (LIMD1) and LATS1-3xMyc (LATS1). (F) Quantification of relative amounts of LATS2 (normalized to wild-type LIMD1 in the IP fraction) immunoprecipitated by wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants in part (D) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; ***P = 0.001, ****P < 0.0001). (G) Quantification of relative amounts of wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants (normalized to wild-type LIMD1 in the input fraction) immunoprecipitated by LATS1-3xMyc from part (E) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; *P < 0.05, ***P = 0.001).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Stable Transfection, Expressing, Transduction, Live Cell Imaging, Staining, Fluorescence, Standard Deviation, Immunoprecipitation, Transfection, Control, Western Blot, Binding Assay

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS1 or LATS2 N-terminal regions (aa 1-635 in LATS1, aa 1-598 in LATS2) with and without (∆LATCH) the LATCH regions (aa 472-520 in LATS1, aa 418-466 in LATS2) as indicated. (B) AlphaFold2 model showing the three tandem LIM domains of LIMD1 (orange) and two regions of LATS2 (green) that are predicted to interact (LATS-LATCH and the Helical Hairpin). (C) Multiple sequence alignments of LATS1/2 from the indicated species showing the conserved LATCH region. (D-E) The requirement of the conserved LATCH sequence of LATS1/2 to bind with LIMD1 was tested by co-immunoprecipitation. (D) Full-length LATS2 (LATS2) or LATS2 with either the LATCH deleted (LATS2-∆LATCH, aa 418-466 deleted) or with the helical hairpin region deleted (LATS2-∆HH, aa 599-667 deleted) tagged to FLAG and (E) Full-length LATS1 (LATS1) or LATS1 with the LATCH deleted (LATS1-∆LATCH, aa 472-520 deleted) tagged with 3xMyc were tested for binding to LIMD1 by co-immunoprecipitation. FLAG-LATS2 variants, LATS1-3xMyc variants, and LIMD1-V5 were separately transfected in HEK293 cells. HEK293A cell lysates transfected with either FLAG-LATS2 variants (D) or LATS1-3xMyc variants (E) were combined with V5-tagged LIMD1. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2 variants (FLAG) (D) , LATS1 variants (Myc) (E) and LIMD1 (V5).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Sequencing, Immunoprecipitation, Binding Assay, Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

doi: 10.1101/2025.06.06.658274

Figure Lengend Snippet: (A) CRISPR-Cas9 dropout screens performed in CRISPR-Cas9 generated isogenic A549 cells using a sgRNA library of 90,709 sequences targeting 18,010 human genes with a coverage of 5 sgRNAs per gene. (B) Dot-plot showing essential genes in LIMD1 -/- (green) and LIMD1 +/+ cells (red) upon sgRNA abundance analysis following screen performed at an early time-point (10 days) and late time-point (17 days). (C-D) Schematic diagram revealing enzymes involved in ETC as essential vulnerabilities in LIMD1 +/+ (C) whereas enzymes involved in Sec t-RNA biosynthesis and incorporation along with GPX4 are essential in LIMD1 -/- A549 cells (D) .

Article Snippet: Proteins were transferred onto PVDF membranes (Sigma, #IPVH00010), blocked, and probed with the following primary antibodies: LIMD1 (1:2000; in-house), LIMD1 (1:1000; Cell Signaling #13245), GPX4 (1:1000; Cell Signaling #52455S), and β-actin (1:20000 dilution; Sigma #A5441).

Techniques: CRISPR, Generated

Journal: bioRxiv

Article Title: LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

doi: 10.1101/2025.06.06.658274

Figure Lengend Snippet: (A, B) Dose-response curves showing surviving fractions of isogenic A549 cells upon RSL3 (n=4) (A) and erastin (n=2) treatment (B) , seeded at 1000 cells per well with a double dose of drug for 48 h followed by 48 h treatment. (C) Surviving fractions of isogenic A549 cells, seeded at 1000 cells per well, upon double dose of treatment of 48 followed by 48 h with DMSO, fer-1 (2 μM), RSL3 (2 μM), and RSL3 (2 μM) in combination with fer-1 (2 μM) (n=3). (D) Surviving fractions of isogenic A549 cells, seeded at 1000 cells per well, upon double dose of treatment of 48 followed by 48 h with DMSO, fer-1 (2 μM), erastin (5 μM), and erastin (5 μM) in combination with fer-1 (2 μM) (n=3). (E-G) Dose-response curves showing surviving fractions of isogenic H1299 cells grown in plasmax upon RSL3 (E) , IKE (F) and FIN56 (G) treatment for 24 h, seeded at 2000 cells per well (n=3). (H) Surviving fractions of isogenic H1299 grown in plasmax, upon 24 h treatment with DMSO, fer-1 (2 μM), RSL3 (8 nM) and RSL3 (8 nM) combined with fer-1(2 μM) (n=3). (I, J) Dose-response curves showing surviving fractions of isogenic SAEC cells upon ML210 (I) and erastin (J) treatment for 24 h, seeded at 3000 cells per well (n=3). (K) Surviving fractions of isogenic SAEC cells, upon 24 h treatment with DMSO, fer-1 (2 μM), ML210 (0.3 μM) and ML210 (0.3 μM) in combination with fer-1 (2 μM) (n=3). (L) Surviving fractions of isogenic SAEC cells, upon 24 h treatment with DMSO, fer-1 (2 μM), erastin (0.3 μM) and erastin (0.3 μM) in combination with fer-1 (2 μM) (n=3). Data is shown as biological replicates ± S.E.M. Two-way ANOVA with Dunnett’s multiple comparisons test was performed in A-D, I-L , whereas two-way ANOVA with Tukey’s comparisons test was performed in E-H , comparing mean values of C25 LIMD1 -/- and C59 LIMD1 -/- groups to both C8 LIMD1 +/+ , shown by black asterisk, and C9 LIMD1 +/+ groups, shown by purple asterisk. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Proteins were transferred onto PVDF membranes (Sigma, #IPVH00010), blocked, and probed with the following primary antibodies: LIMD1 (1:2000; in-house), LIMD1 (1:1000; Cell Signaling #13245), GPX4 (1:1000; Cell Signaling #52455S), and β-actin (1:20000 dilution; Sigma #A5441).

Techniques:

Journal: bioRxiv

Article Title: LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

doi: 10.1101/2025.06.06.658274

Figure Lengend Snippet: (A) CRISPR-Cas9 dropout screens performed in CRISPR-Cas9 generated isogenic A549 cells using a sgRNA library of 90,709 sequences targeting 18,010 human genes with a coverage of 5 sgRNAs per gene. (B) Dot-plot showing essential genes in LIMD1 -/- (green) and LIMD1 +/+ cells (red) upon sgRNA abundance analysis following screen performed at an early time-point (10 days) and late time-point (17 days). (C-D) Schematic diagram revealing enzymes involved in ETC as essential vulnerabilities in LIMD1 +/+ (C) whereas enzymes involved in Sec t-RNA biosynthesis and incorporation along with GPX4 are essential in LIMD1 -/- A549 cells (D) .

Article Snippet: Proteins were transferred onto PVDF membranes (Sigma, #IPVH00010), blocked, and probed with the following primary antibodies: LIMD1 (1:2000; in-house), LIMD1 (1:1000; Cell Signaling #13245), GPX4 (1:1000; Cell Signaling #52455S), and β-actin (1:20000 dilution; Sigma #A5441).

Techniques: CRISPR, Generated

Journal: bioRxiv

Article Title: LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

doi: 10.1101/2025.06.06.658274

Figure Lengend Snippet: (A, B) Dose-response curves showing surviving fractions of isogenic A549 cells upon RSL3 (n=4) (A) and erastin (n=2) treatment (B) , seeded at 1000 cells per well with a double dose of drug for 48 h followed by 48 h treatment. (C) Surviving fractions of isogenic A549 cells, seeded at 1000 cells per well, upon double dose of treatment of 48 followed by 48 h with DMSO, fer-1 (2 μM), RSL3 (2 μM), and RSL3 (2 μM) in combination with fer-1 (2 μM) (n=3). (D) Surviving fractions of isogenic A549 cells, seeded at 1000 cells per well, upon double dose of treatment of 48 followed by 48 h with DMSO, fer-1 (2 μM), erastin (5 μM), and erastin (5 μM) in combination with fer-1 (2 μM) (n=3). (E-G) Dose-response curves showing surviving fractions of isogenic H1299 cells grown in plasmax upon RSL3 (E) , IKE (F) and FIN56 (G) treatment for 24 h, seeded at 2000 cells per well (n=3). (H) Surviving fractions of isogenic H1299 grown in plasmax, upon 24 h treatment with DMSO, fer-1 (2 μM), RSL3 (8 nM) and RSL3 (8 nM) combined with fer-1(2 μM) (n=3). (I, J) Dose-response curves showing surviving fractions of isogenic SAEC cells upon ML210 (I) and erastin (J) treatment for 24 h, seeded at 3000 cells per well (n=3). (K) Surviving fractions of isogenic SAEC cells, upon 24 h treatment with DMSO, fer-1 (2 μM), ML210 (0.3 μM) and ML210 (0.3 μM) in combination with fer-1 (2 μM) (n=3). (L) Surviving fractions of isogenic SAEC cells, upon 24 h treatment with DMSO, fer-1 (2 μM), erastin (0.3 μM) and erastin (0.3 μM) in combination with fer-1 (2 μM) (n=3). Data is shown as biological replicates ± S.E.M. Two-way ANOVA with Dunnett’s multiple comparisons test was performed in A-D, I-L , whereas two-way ANOVA with Tukey’s comparisons test was performed in E-H , comparing mean values of C25 LIMD1 -/- and C59 LIMD1 -/- groups to both C8 LIMD1 +/+ , shown by black asterisk, and C9 LIMD1 +/+ groups, shown by purple asterisk. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Proteins were transferred onto PVDF membranes (Sigma, #IPVH00010), blocked, and probed with the following primary antibodies: LIMD1 (1:2000; in-house), LIMD1 (1:1000; Cell Signaling #13245), GPX4 (1:1000; Cell Signaling #52455S), and β-actin (1:20000 dilution; Sigma #A5441).

Techniques: