Journal: bioRxiv

Article Title: Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

doi: 10.1101/2025.10.08.681214

Figure Lengend Snippet: A . Confocal images showing fat bodies from animals expressing an ATF4 reporter (4E-BP intron -DsRed, magenta) and GFP (cyan) driven by Dcg-GAL4 , which also drives expression of the indicated RNAi . B . Quantification of DsRed intensity from A. Each data point represents one nucleus, and black bar represents mean of data from at least 5-7 animals across two independent crosses. Error bars represent standard error. Statistical comparisons were made using Kruskal-Wallis test with Dunn’s correction. C, E. Confocal images of fat bodies expressing 4E-BP intron -DsRed (magenta) from wildtype ( w 1118 ), and transheterozygous mutants for Hbs1 ( Hbs1 1 /Hbs1 48 ), and pelo ( pelo 1 /pelo PB60 ). D, F. Quantification of DsRed intensity from C and E respectively. Each data point represents one nucleus, and black bar represents mean of data from at least 5-7 animals across two independent crosses. Error bars represent standard error of mean. P-values were calculated using Mann-Whitney test. Scale bars in A, C, E: 25μm; Nuclei are counterstained by DAPI (yellow). ****=p<0.0001, ***=p<0.001, **=p<0.01, *=p<0.05 with only significant comparisons shown here and in future figures.

Article Snippet: Antibodies: ATF4 (Cell Signaling 11815S, 1:1000 in 5% BSA PBST, HBS1L (Proteintech 10359-1-AP, 1:2000 in 5%BSA PBST), Pelo (Proteintech 0582-1-AP, 1:2000 in 5% BSA PBST), Tubulin (DSHB 12G10,1:2000 PBST), GFP (Immunology consultants 50-196-2090, 1:2000 PBST), Puromycin (Sigma MABE343, 1:2000 in 5% BSA PBST).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

doi: 10.1101/2025.10.08.681214

Figure Lengend Snippet: A. Representative western blot analyses of lysates prepared from HEK293T cells co-transfected with siRNA (Ctrl (negative control), HBS1L, Pelo) and an ATF4 5’ leader-GFP reporter where the coding sequence of ATF4 is replaced with GFP (see schematic in D). Cells were treated with 10μg/ml Tunicamycin (Tu) for 4 h to induce ER stress prior to lysis. Tubulin (Tub) serves as a loading control. B-C. Quantification of ATF4 intensity (B) and GFP intensity (C) from (A) normalized to loading control (Tub). Data represent mean of at least five independent experiments; error bars are standard error of mean. D. (Top) schematic of control ATF4 5’ leader-GFP reporter, and (bottom) a mutant reporter where all the stop codons upstream of the ATF4 start codon are deleted. Green triangles mark start codons, red squares mark stop codons. Arrows in solid black outline represent open reading frames corresponding to uORFs. Dotted black arrow (bottom) represents the new putative reading frame generated by mutating stop codons in the 5’ leader. E. Western blot analysis of mutant ATF4 5’ leader-GFP in HEK293T cells where HBS1L and Pelo are RNAi depleted. F. Quantification of GFP signal from E normalized to Tub. Data represent mean of at least five independent experiments; error bars are standard error of mean. G. Quantification of Puromycin signal normalized to total protein (as measured by BCA) in HEK293T cells where HBS1L and Pelo are RNAi depleted. Data represent mean of four independent experiments; error bars are standard error of mean. See S2B for representative blot. Statistical comparisons with Ctrl RNAi -DMSO were performed using the Wilcoxon signed rank test for and are shown as floating asterisks when significant. Comparisons with Ctrl RNAi -Tunicamycin were performed using the Friedman test with Dunn’s correction. Other comparisons were performed using the Wilcoxon matched pairs signed rank test.

Article Snippet: Antibodies: ATF4 (Cell Signaling 11815S, 1:1000 in 5% BSA PBST, HBS1L (Proteintech 10359-1-AP, 1:2000 in 5%BSA PBST), Pelo (Proteintech 0582-1-AP, 1:2000 in 5% BSA PBST), Tubulin (DSHB 12G10,1:2000 PBST), GFP (Immunology consultants 50-196-2090, 1:2000 PBST), Puromycin (Sigma MABE343, 1:2000 in 5% BSA PBST).

Techniques: Western Blot, Transfection, Negative Control, Sequencing, Lysis, Control, Mutagenesis, Generated

Journal: bioRxiv

Article Title: Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

doi: 10.1101/2025.10.08.681214

Figure Lengend Snippet: A. q-RTPCR data measuring ATF4 mRNA normalized to house-keeping gene (GAPDH) in cells transfected with siRNA targeting HBS1L or Pelo and treated with 10μg/ml Tunicamycin to induce ER stress. Data represent mean of four independent experiments; error bars are standard error of mean. Statistical comparisons with Ctrl RNAi -DMSO were performed using the Wilcoxon signed rank test for and are shown as floating asterisks when significant. Comparisons with Ctrl RNAi - Tunicamycin were performed using the Friedman test with Dunn’s correction. Other comparisons were performed using the Wilcoxon matched pairs signed rank test. B. Representative western blot for puromycin-incorporation in cells where HBS1L or Pelo is depleted. Quantification presented in 2G.

Article Snippet: Antibodies: ATF4 (Cell Signaling 11815S, 1:1000 in 5% BSA PBST, HBS1L (Proteintech 10359-1-AP, 1:2000 in 5%BSA PBST), Pelo (Proteintech 0582-1-AP, 1:2000 in 5% BSA PBST), Tubulin (DSHB 12G10,1:2000 PBST), GFP (Immunology consultants 50-196-2090, 1:2000 PBST), Puromycin (Sigma MABE343, 1:2000 in 5% BSA PBST).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot

Journal: bioRxiv

Article Title: Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

doi: 10.1101/2025.10.08.681214

Figure Lengend Snippet: A-D. Representative ERG traces from 3-5 day old wildtype ( w 1118 , A), Hbs1 mutants ( Hbs1 1 /Hbs1 48 , B), Hbs1 and pelo hemizygous mutants ( pelo 1 /+;Hbs1 48 /+ , C), and ATF4 heterozygous mutants ( crc GFSTF /+ , D). The solid line represents average of three measurements from an individual animal, and shaded error envelope represents the standard error of mean across the measurements. The three quantifiable parameters of ERGs, on-transient, off-transient, and receptor potential are marked by parentheses in (A). E-G. Quantification of on-transient (E), off-transient (F), and receptor potential (G) from (A-D). Data bars are color coded to genotypes in (A-D). Each data point is the average of three independent measurements from a single animal. Data represent the mean from at least four independent animals recorded on separate days; error bars are standard error of mean. Statistical comparisons were made using the Kruskal-Wallis test with Dunn’s correction.

Article Snippet: Antibodies: ATF4 (Cell Signaling 11815S, 1:1000 in 5% BSA PBST, HBS1L (Proteintech 10359-1-AP, 1:2000 in 5%BSA PBST), Pelo (Proteintech 0582-1-AP, 1:2000 in 5% BSA PBST), Tubulin (DSHB 12G10,1:2000 PBST), GFP (Immunology consultants 50-196-2090, 1:2000 PBST), Puromycin (Sigma MABE343, 1:2000 in 5% BSA PBST).

Techniques:

Journal: bioRxiv

Article Title: Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

doi: 10.1101/2025.10.08.681214

Figure Lengend Snippet: A. Representative ERG traces from 3-5 day old animals where Gcm-GAL4 is used to deplete pelo (middle, maroon trace), ATF4 (bottom, orange trace) or express a control LacZ RNAi (top, black trace). Data are graphed as in 3A-D. B-D. Quantification of on-transient (B), off-transient (C), and receptor potential (D) from animals in (A). Data are plotted as in 3E-G. Statistical comparisons were made using Kruskal-Wallis test with Dunn’s correction. E. Representative ERG traces from 3-5 day old animals where Gcm-GAL4 drives expression of a control UAS-LacZ transgene in wildtype animals (top, black trace), Hbs1 mutants (middle, purple trace) or of a leaderless ATF4 in Hbs1 mutants (bottom, blue trace). Data are graphed as in 3A-D. F-H. Quantification of on-transient (F), off-transient (G), and receptor potential (H) from animals in (A). Data are plotted as in 3E-G. Statistical comparisons with Gcm>LacZ were made using the Kruskal-Wallis test with Dunn’s correction and are indicated as floating asterisks when significant. Other comparisons were made using the Mann-Whitney test.

Article Snippet: Antibodies: ATF4 (Cell Signaling 11815S, 1:1000 in 5% BSA PBST, HBS1L (Proteintech 10359-1-AP, 1:2000 in 5%BSA PBST), Pelo (Proteintech 0582-1-AP, 1:2000 in 5% BSA PBST), Tubulin (DSHB 12G10,1:2000 PBST), GFP (Immunology consultants 50-196-2090, 1:2000 PBST), Puromycin (Sigma MABE343, 1:2000 in 5% BSA PBST).

Techniques: Control, Expressing, MANN-WHITNEY

Journal: Nature

Article Title: Synthetic lethality of mRNA quality control complexes in cancer

doi: 10.1038/s41586-024-08398-6

Figure Lengend Snippet: (a) Guide level analysis of 9p21 genes. NCI-H1299 parent (orange) and PELO −/− (blue) models shown. Two-tailed Student’s t -test of n = 4 sgRNA per gene, Parent vs. PELO −/−; FOCAD, P = 5.7 × 10 −3 . (b) SL-ID screen results in SKIV2L −/− and FOCAD −/− models. NCI-H1299 parental (orange) SKIV 2L −/− or FOCAD −/− (blue). Each dot represents an individual sgRNA ( n = 4) for each gene, mean of n = 3 independent biological replicates. (c) Immunoblot of the PELO/HBS1L/ABCE1 rescue complex across isogenic SKIc knockout models. (d) PELO dependency by siRNA in HAP1 or HAP1 SKIV2L −/− models. Data shown as mean ± s.e.m, n = 3 biologically independent replicates, two-tailed Student’s t-test; P = 9.9 × 10 −7 . (e) Validation of siRNA efficiency by immunoblot in (d). Data generated from a single experiment. (f) RNA quantification of SKIc levels (FOCAD, TTC37, SKIV2L, AVEN) post FOCAD degradation. Data shown as mean ± s.e.m, n = 3 biologically independent replicates, two-tailed Student’s t-test . (g) sgWDR61 in NCI-H1299 parental, PELO −/−, and SKIV2L −/− models. Confluency of sgB2M (black), sgWDR61 (blue). Data shown as mean ± s.e.m, n = 3 biologically independent replicates (h) Immunoblot validation of sgWDR61 efficiency and effects on SKIc in models from (g). Data is generated from a single independent experiment.

Article Snippet: The following primary antibodies were used to detect protein abundance in Simple Western assays: PELO (ProteinTech, 10582-1-AP), HBS1L (ProteinTech, 10359-1-AP), SKIV2L (ProteinTech, 11462-1-AP), TTC37 (Abcam, ab122421), WDR61 (Invitrogen, PA5-59052), FOCAD (Invitrogen, PA5-63051), AVEN (ProSci, 2413), ABCE1 (Abcam, ab185548), JNK (Cell Signaling, 4672), pJNK (Cell Signaling, 4668), p38 (Cell Signaling, 9212), phospho-p38 (Cell Signaling, 9211), EIF2S1 (Cell Signaling, 5324), phospho-EIF2S1 (Abcam, ab32157), IRE1α (Cell Signaling, 3294), pIRE1α (Novus Biologicals, NB100-2323), PERK (Cell Signaling, 3192), phospho-PERK (Cell Signaling, 3179), CHOP (ThermoFisher Scientific, MA1-250), JUN (Cell Signaling, 9165), p21 (Cell Signaling, 2947), PARP (Cell Signaling, 3898), HSP90 (Cell Signaling, 4877), GAPDH (Cell Signaling, 2118), HiBIT (Promega, N7200).

Techniques: Two Tailed Test, Western Blot, Knock-Out, Biomarker Discovery, Generated

Journal: Nature

Article Title: Synthetic lethality of mRNA quality control complexes in cancer

doi: 10.1038/s41586-024-08398-6

Figure Lengend Snippet: (a) Correlation of HBS1L and PELO Chronos scores across DepMap models from 23Q4 release ( n = 1078). (b) Correlation of HBS1L dependency to RNA expression data across all lineages and models. The top 1000 correlated genes are shown. Highlighted 9p21.3 genes in red. P-value calculated by two-tailed Student’s t -test. (c) Correlation of HBS1L dependency to proteomic expression data across all lineages and models. The top 1000 correlated proteins are shown. Highlighted SKIc member proteins shown in purple. P-values calculated by F -test. (d) Validation of HBS1L knockout in models assessed in Fig. . (e) Confluency over time of MDA-MB-231 cells after transfection of sgB2M or four independent sgRNA’s targeting HBS1L (e7.1, e7.2, e13.1, e17.1). Data shown as mean ± s.e.m. of n = 2 independent biological replicates. (f) Representative confluency images of MDA-MB-231 cells containing sgB2M control or one of four sgRNA’s targeting HBS1L . Image acquired on day 7. Representative image of a single replicate of n = 3 independent biological experiments. (g) Immunoblot of HBS1L and PELO from in vivo doxycycline sg HBS1L study, n = 4 tumors harvested at each time-point post dox (D7, D14, D21) for both the no dox and dox conditions. (h) Quantification of HBS1L and PELO after in vivo sg HBS1L inducible knockout, normalized to HSP90 loading control. Shown as the mean of n = 4 animals per group.

Article Snippet: The following primary antibodies were used to detect protein abundance in Simple Western assays: PELO (ProteinTech, 10582-1-AP), HBS1L (ProteinTech, 10359-1-AP), SKIV2L (ProteinTech, 11462-1-AP), TTC37 (Abcam, ab122421), WDR61 (Invitrogen, PA5-59052), FOCAD (Invitrogen, PA5-63051), AVEN (ProSci, 2413), ABCE1 (Abcam, ab185548), JNK (Cell Signaling, 4672), pJNK (Cell Signaling, 4668), p38 (Cell Signaling, 9212), phospho-p38 (Cell Signaling, 9211), EIF2S1 (Cell Signaling, 5324), phospho-EIF2S1 (Abcam, ab32157), IRE1α (Cell Signaling, 3294), pIRE1α (Novus Biologicals, NB100-2323), PERK (Cell Signaling, 3192), phospho-PERK (Cell Signaling, 3179), CHOP (ThermoFisher Scientific, MA1-250), JUN (Cell Signaling, 9165), p21 (Cell Signaling, 2947), PARP (Cell Signaling, 3898), HSP90 (Cell Signaling, 4877), GAPDH (Cell Signaling, 2118), HiBIT (Promega, N7200).

Techniques: RNA Expression, Two Tailed Test, Expressing, Biomarker Discovery, Knock-Out, Transfection, Control, Western Blot, In Vivo

Journal: Nature

Article Title: Synthetic lethality of mRNA quality control complexes in cancer

doi: 10.1038/s41586-024-08398-6

Figure Lengend Snippet: a , HBS1L knockout in a panel of cell lines. Relative viability of sgHBS1L or sgB2M cells at day 7. Data are mean ± s.e.m. of n = 3 independent biological replicates. b , Model of the PELO–HBS1L complex, adapted from Protein Data Bank ID 5LZZ . PELO is shown in blue, HBS1L is in grey. Key HBS1L residues (568–682) along the PELO interaction surface are shown in salmon. GTPase catalytic residues T325 and H348 are circled with green and pink dotted lines, respectively. c , Validation of a stable NCI-H1299 sgHBS1L model ( HBS1L KO) and HBS1L rescue constructs. d , Functional characterization of HBS1L. Data shown from n = 3 independent biological replicates. EV, empty vector, P = 1.7 × 10 −5 ; wild type, P = 0.21; Δ1–140, P = 0.13; H348A, P = 6.6 × 10 −5 ; T325A, P = 4.8 × 10 −4 ; Δ568–682, P = 2.0 × 10 −5 . e , Co-immunoprecipitation of full-length HBS1L and HBS1L(Δ568–682) and PELO. Representative image of a single experiment from two independent biological replicates. f , Immunoblot for validation of the inducible sgHBS1L model. Experiment performed once. g , In vivo HBS1L dependency. Growth of size-matched (200 mm 3 ) implanted doxycycline-inducible sgHBS1L MIAPACA2 model. Doxycycline (625 mg kg −1 ) chow was administered ad libitum. Data show mean ± s.e.m. of n = 8 mice per group. P value calculated at study end, P = 3.9 × 10 −4 . Two-tailed Student’s t -test ( a , d , g ).

Article Snippet: The following primary antibodies were used to detect protein abundance in Simple Western assays: PELO (ProteinTech, 10582-1-AP), HBS1L (ProteinTech, 10359-1-AP), SKIV2L (ProteinTech, 11462-1-AP), TTC37 (Abcam, ab122421), WDR61 (Invitrogen, PA5-59052), FOCAD (Invitrogen, PA5-63051), AVEN (ProSci, 2413), ABCE1 (Abcam, ab185548), JNK (Cell Signaling, 4672), pJNK (Cell Signaling, 4668), p38 (Cell Signaling, 9212), phospho-p38 (Cell Signaling, 9211), EIF2S1 (Cell Signaling, 5324), phospho-EIF2S1 (Abcam, ab32157), IRE1α (Cell Signaling, 3294), pIRE1α (Novus Biologicals, NB100-2323), PERK (Cell Signaling, 3192), phospho-PERK (Cell Signaling, 3179), CHOP (ThermoFisher Scientific, MA1-250), JUN (Cell Signaling, 9165), p21 (Cell Signaling, 2947), PARP (Cell Signaling, 3898), HSP90 (Cell Signaling, 4877), GAPDH (Cell Signaling, 2118), HiBIT (Promega, N7200).

Techniques: Knock-Out, Biomarker Discovery, Construct, Functional Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, In Vivo, Two Tailed Test