Journal: Nucleic Acids Research

Article Title: Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

doi: 10.1093/nar/gkag264

Figure Lengend Snippet: The conserved Bcl1 interacts via its WD40 β-propeller with Rpl1. ( A ) Proximity labelling assay with C-terminally miniTurbo-tagged RPL10A (HsRPL10A-miniTurbo) in HeLa cells. The RPL10A bait r-protein and selected enriched proteins are written in bold, the red dot highlights the highly enriched WDR89. ( B ) AlphaFold3 model of the Bcl1–Rpl1 complex. The seven-bladed β-propeller domain of Bcl1 is coloured in green and the C-terminal extension in light green; the position of residue Asn322 (N322) is indicated to better visualize from where the C-terminal extension emanates. ( C ) AlphaFold3 model of the WDR89–RPL10A complex (left) and its structural superposition with the AlphaFold3 model of the Bcl1–Rpl1 complex (right). ( D ) Predicted electrostatic surface potential of Bcl1 (left) and close-up view of two of the three Rpl1 sites, indicating residues predicted to form H-bonds with Bcl1, that are in contact with the negatively charged top surface of the β-propeller (right). ( E–G ) Y2H interaction assays between the full-length Rpl1 and Bcl1 proteins ( E ), between full-length Rpl1 and the C-terminally truncated Bcl1.N366 and Bcl1.N325 variants or the C-terminal extension of Bcl1 (323C) ( F ), and between the indicated Rpl1 mutant variants and either Bcl1 or Acl1 ( G ). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; K, Lys; R, Arg. ( H ) In vitro binding assay between Bcl1.N366 and Rpl1. Bcl1.N366-(His) 6 or Bcl1.N366 and Rpl1b were co-expressed in E. coli and purified by Ni–NTA affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blotting using anti-His and anti-Rpl1 antibodies (bottom). Bands corresponding to Bcl1.N366-(His) 6 and Bcl1.N366 or to Rpl1b are indicated by blue or black arrowheads.

Article Snippet: The DNA sequence coding for the Homo sapiens RPL10A protein was PCR-amplified from plasmid pADH111-HsRPL10A (pDK10427), generated by cloning the PCR-amplified RPL10A coding sequence [template pNTI194 (Addgene plasmid #84266)] into the Nde I/ Bam HI-restricted plasmid pADH111-LTV1 (pDK3331), and cloned by Gibson assembly between the Nhe I and Pst I restriction sites of the lentiviral donor vector pSKP-32, a pCW57.1-derived plasmid bearing the MND-Blasticidin resistance cassette instead of the hPGK-puromycin resistance cassette [ ], to generate plasmid pDS79 containing the RPL10A gene under the transcriptional control of a doxycycline-inducible promoter and fused at its 3′ end to sequences encoding the V5 tag, the miniTurbo (MT) biotin ligase, and the HA tag.

Techniques: Residue, Mutagenesis, In Vitro, Binding Assay, Purification, Affinity Purification, SDS Page, Staining, Western Blot

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Experimental setup for PI3K inhibitor (PI3Ki) and MEK inhibitor (MEKi) treatment and release. (B) Protein levels of pERK1/2, ERK1/2, pAKT, and AKT after HRAS G12V induction and treatment with MEKi or PI3Ki. (C) S phase percentage after HRAS G12V induction with release from PI3Ki or MEKi as determined by EdU labelling and flow cytometry. N=4. (D) Nuclear EU intensity after HRAS G12V induction and release from MEKi or PI3Ki. N=5 (MEKi N=4). (E) Average replication fork speeds after HRAS G12V induction and release from MEKi or PI3Ki. N=4. (F) Protein levels of pAKT (S473), AKT, pERK1/2 and ERK1/2 after UCL-TRO-1938 treatment of uninduced BJ-hTERT-BRAF V600E cells for 48 h. (G) Nuclear EU intensity after KRAS G12V induction +/- UCL-TRO-1938 (5 μM) for 48 h. N=4. (H) Median replication fork speeds after KRAS G12V induction +/- UCL-TRO-1938 (5 μM) for 48 h. N=3. (I) Inducible expression of oncogenic PI3K E545K , alone or with KRAS G12V or BRAF V600E , was used to increase PI3K signalling, on its own or in combination with MAPK activation. (J) Protein levels of pAKT (S473), AKT, pERK1/2 and ERK1/2 after oncogene induction or doxycycline treatment (72 h) of parental cells (BJ-hTERT). (K) Nuclear EU intensity after oncogene induction. N=4. (EL) Median replication fork speeds after oncogene induction. N=4-6. Means +/-SEM (bars) are shown with 2-way ANOVA or mixed effects analysis.

Article Snippet: A doxycycline-inducible mutant PIK3CA E545K expression vector was created by PCR subcloning of the PIK3CA E545K coding sequence (Addgene, Plasmid #73055) into a modified pTRIPZ vector (Addgene, Plasmid #206981) lacking the TurboGFP coding sequence.

Techniques: Flow Cytometry, Expressing, Activation Assay

Journal: iScience

Article Title: The microcephaly-associated protein YIPF5 differentially regulates ER export

doi: 10.1016/j.isci.2026.114791

Figure Lengend Snippet: YIPF5 regulates the secretome composition (A) Schematic representation of the experimental workflow for analyzing the glycoprotein secretome. MCF10A cells were metabolically labeled with the clickable sugar analog ManNAz, followed by a biotin-azide click reaction to tag glycoproteins ( n = 4). Secreted glycoproteins were concentrated from the culture medium, selectively purified, and identified using mass spectrometry. The image was created with BioRender.com . (B) Quantitative comparison of protein abundance in the secretome versus the total proteome of MCF10A cells. Proteins with secretion changes reflecting a similar change in total expression (“explained by proteome”) (q value ≤ 0.05) are shown in green. Proteins with increased or decreased secretion independent of total proteome changes are highlighted in red and blue, respectively. Gray (not affected) represents unchanged proteins. (C) Analysis of protein abundance changes in the secretome of YIPF5 KO versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 KO secretome are shown. Proteins with secretion changes explained by total proteome abundance are shown in green, while unaffected proteins are in gray. The vertical axis represents –log10(q value), and the horizontal axis represents log2(fold change). (D and E) Heatmap depicting proteins showing increased secretion in the YIPF5 KO secretome with a gene ontology (GO) annotation for the ER (GO: 0007029) and the Golgi apparatus (GO: 0005794) (D) or that contain a KDEL-ER retrieval sequence (E). (F) MCF10A WT and YIPF5 KO cells stably expressing the inducible ER-Ca 2+ sensor GCampER were analyzed using live fluorescence microscopy at identical settings. (G) Mean ± SD fluorescence intensity of GCampER was quantified in MCF10A WT and YIPF5 KO cells in 72 wells per cell line from n = 3 independent experiments. Unpaired t test, two-tailed p < 0.0001. (H) HeLa cells stably expressing shScramble or shRNAs against YIPF5 were transiently transfected with CNPY3-mCherry, supernatants were collected and cell lysates prepared after 24 h followed by western blot analysis using a CNPY3 antibody. A representative blot of n = 3 independent experiments is shown. For full size blot see C. (I) Quantification of CNPY3-secretion from (H). Displayed are arbitrary units normalized to the values of shScramble-expressing cells from n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test, with ∗ indicating p < 0.05. (J) Analysis of protein abundance changes in the secretome of YIPF5 KO re-expressing YIPF5 I98S mutant versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 I98S secretome are shown ( n = 4). Proteins with secretion changes explained by total proteome abundance are denoted in green, while unaffected proteins are in gray. (K) Venn Diagram of protein abundance changes in YIPF5-KO cells and YIPF5-KO re-expressing YIPF5 I98S cells. Proteins with decreased (blue) or increased (red) secretome abundance are shown. See also and , , , , and .

Article Snippet: The GCamPer coding sequence was amplified from pMOS003-lenti-CMV-GCaMPer (Addgene #65227, ) using primers GCamPer-F and GCamPer-R containing attB sites (sequences see ), gel-purified, and recombined with pDONR221 by BP ClonaseTM II (Thermo Fisher Scientific).

Techniques: Metabolic Labelling, Labeling, Purification, Mass Spectrometry, Comparison, Quantitative Proteomics, Expressing, Control, Sequencing, Stable Transfection, Fluorescence, Microscopy, Two Tailed Test, Transfection, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Divergent condensates tune transcriptional responses during stress

doi: 10.64898/2026.02.12.705659

Figure Lengend Snippet: (a) Representative pseudo-colored IF images of U2OS cells Mock treated (37 °C) or stressed (HS, 42 °C, 1 h; ACA, 10 mM, 6 h; CdC, 1 mM, 4 h; SA, 0.5 mM, 1 h; HP, 2 mM, 1 h; CC, 10 mM, 4 h; MGO 10 mM, 1 h; ATO 0.5 mM, 1 h) stained for HSF1 (green). Nuclear (Nuc.) boundary (cyan) obtained from segmentation of DAPI signal. Scale bar, 10 μm. (b) Heatmaps of mean number of HSF1 foci, HSF1 apparent partition (App. Part.), Nuc. HSF1 mean fluorescent intensity (Nuc. MFI), mean HSF1 foci area, and mean HSF1 foci eccentricity (Ecc.) per nucleus from (a). Color scale for each is also represented (n ≥ 73 cells, 1138 condensates, per condition). (c) Representative pseudo-colored IF images of U2OS cells treated with conditions from (a) and stained for HSF1 (green) and RNAPII-2P (red). Nuc. boundary (cyan) obtained from segmentation of DAPI signal. Scale bar, 10 μm. Zoom-ins are 2.7 μm × 2.7 μm. (d) Line scan metaplots of RNAPII-2P signal across segmented HSF1 condensates for each condition in (c). *=p<0.05, ns = not significant by two-sided unpaired T-test, (n ≥ 78 cells, 1958 condensates, per condition).

Article Snippet: The endogenous hAXL coding sequence was removed and replaced with a synthesized gene block (Twist Bioscience®) encoding a HaloTag, GGSx3 linker sequence and a multiple cloning site (MCS). pLVX-TetOne-Puro-Halo-GGSx3-HSF1 was created by PCR amplification of the HSF1 coding sequence from HSF1-GFPN3 (Addgene #32538) and subsequently cloned into the MCS using AgeI and NotI restriction sites. pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔDBD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ1-3, pLVX-TetOne-Puro-Halo-GGSx3-NLSx3-HSF1-ΔRD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ4 and pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔAD were created by cloning in synthesized gene blocks (Twist Bioscience®) of HSF1-ΔDBD, HSF1-ΔLZ1-3, NLS3x-HSF1-ΔRD, HSF1-ΔLZ4, HSF1-ΔAD into pLVX-TetOne-Puro-Halo-GGSx3-MCS.

Techniques: Staining

Journal: bioRxiv

Article Title: Divergent condensates tune transcriptional responses during stress

doi: 10.64898/2026.02.12.705659

Figure Lengend Snippet: (a) Representative pseudo-colored IF images of Mock treated or HS subjected U2OS cells and stained for HSF1 (green) and H3K4me3, H3K27ac, BRD4, MED12, RNAPII, or RNAPII-5P (red). Scale bar, 10 μm. Zoom-ins are 2.7 μm × 2.7 μm with 1 μm scale bar. (b) Line scan metaplots of co-IF signal across segmented HSF1 condensates from (a). Dashed red lines represent co-IF signal from spatial randomizations. *=p<0.05, **=p<0.005, ns = not significant, by two-sided unpaired T-test (n ≥ 53 cells, 1750 condensates, per condition). (c) Representative pseudo-colored IF images of U2OS or UHKO cells subjected to HS and stained for H3K27ac, BRD4, MED12, or RNAPII (red). Scale bar, 10 μm. Zoom-ins are 5 μm × 5 μm. (d) Quantification of H3K27ac, BRD4, MED12, or RNAPII App. Part. of cells from (c). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 428 cells, per condition). Line represents mean and error bars represent s.e.m. (e-f) Quantification of BRD4 or MED12 App. Part. of MCF7 (e) or HeLa (f) cells subjected to HS and siCtrl or siHSF1 siRNAs. ***=p<0.0005, by two-sided unpaired T-test (n ≥ 382 cells, per condition). Line represents mean and error bars represent s.e.m. (g) Representative pseudo-colored images of UHKO-HaH cells with or without dox. induction, subjected to HS, and stained for BRD4 or MED12 (red). Scale bar, 10 μm. Zoom-ins are 5 μm × 5 μm. (h) Quantification of BRD4 or MED12 App. Part. of cells from (g). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 895 cells, per condition). Line represents mean and error bars represent s.e.m. (i) Representative pseudo-colored fluorescence images of U2OS cells treated with HS and DMSO, A-485, or OTX-015 and stained for HSF1 (green). Scale bar, 10 μm. Zoom-ins are 7.5 μm × 7.5 μm. (j) Quantification of HSF1 MFI, App. Part., mean foci area, and mean foci Ecc. in cells from (i). **=p<0.005, ***=p<0.0005, ns = not significant, by two-sided unpaired T-test (n ≥ 290 cells). Line represents mean and error bars represent s.e.m. (k) Meta-images of MED12, H3K27ac, BRD4, or RNAPII signal (red) across segmented HSF1 condensates (green) in cells subjected to HS and further treated with DMSO, A-485, or OTX-015. Intensity scale for respective biomolecules is also represented. (n ≥ 290 cells, 9599 condensates, per condition).

Article Snippet: The endogenous hAXL coding sequence was removed and replaced with a synthesized gene block (Twist Bioscience®) encoding a HaloTag, GGSx3 linker sequence and a multiple cloning site (MCS). pLVX-TetOne-Puro-Halo-GGSx3-HSF1 was created by PCR amplification of the HSF1 coding sequence from HSF1-GFPN3 (Addgene #32538) and subsequently cloned into the MCS using AgeI and NotI restriction sites. pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔDBD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ1-3, pLVX-TetOne-Puro-Halo-GGSx3-NLSx3-HSF1-ΔRD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ4 and pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔAD were created by cloning in synthesized gene blocks (Twist Bioscience®) of HSF1-ΔDBD, HSF1-ΔLZ1-3, NLS3x-HSF1-ΔRD, HSF1-ΔLZ4, HSF1-ΔAD into pLVX-TetOne-Puro-Halo-GGSx3-MCS.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Divergent condensates tune transcriptional responses during stress

doi: 10.64898/2026.02.12.705659

Figure Lengend Snippet: (a) Representative pseudo-colored IF image of U2OS cells treated with mock and HS conditions and stained for HSF1 (green) and HSF1-pS320 (pS320, red); with normalized line scan metaplot of HSF1-pS320 signal across segmented HSF1 condensates (n ≥ 94 cells, 2766 condensates, per condition). Dashed red lines represent co-IF signal from spatial randomizations. Scale bar, 10 μm. Zoom-ins are 2.7 μm × 2.7 μm. (b) Representative pseudo-colored IF images of HS subjected U2OS cells treated with DMSO, CHX, or Doxo; with quantification of HSF1 apparent partition (App. Part.). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 900 cells per condition). Line represents mean and error bars represent s.e.m. Scale bar, 10 μm. Zoom-ins are 12.5 μm × 12.5 μm. (c) Disorder prediction from PONDR along with schematic of HSF1 domain deletion constructs. (d) Representative pseudo-colored fluorescent images of mock or HS treated FL, ΔDBD, ΔLZ1-3, ΔRD, ΔLZ4, and ΔAD cells stained with Halo-HSF1 (green). Scale bar, 10 μm. Zoom-ins are 7.5 μm × 7.5 μm. (e) Quantification of Halo-HSF1 App. Part. in from cells in (d). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 300 cells, per condition). Line represents mean and error bars represent s.e.m. (f) Representative pseudo-colored images of HS subjected FL, ΔDBD, ΔLZ1-3, ΔRD, ΔLZ4, and ΔAD cells stained with Halo-HSF1 (JF549-HL, green) and BRD4 (red); with meta-images (Av., n ≥ 69 cells, per condition). Scale bar, 10 μm. Zoom-ins and meta-images are 4.2 μm × 4.2 μm. (g) Meta-images of MED12, H3K27ac, or RNAPII signal (red) across segmented Halo-HSF1 condensates (JF549-HL, green) in FL, or ΔAD cells subjected to HS. Intensity scale for respective biomolecules is also represented. (n ≥ 67 cells, 436 condensates, per condition). (h) Representative pseudo-colored images of mock-treated ΔRD cells stained with Halo-HSF1 (JF549-HL, green) and H3K27ac, BRD4, MED12, RNAPII, RNAPII-5P, or RNAPII-2P (red); with meta-images (Av., n ≥ 644 cells, per condition). Scale bar, 10 μm. Zoom-ins and meta-images are 4.2 μm × 4.2 μm. (i) Representative pseudo-colored images of heat-shocked FL and ΔRD cells stained with Halo-HSF1 (JF549-HL, green) and RNAPII-2P (red); with meta-images (Av., n ≥ 284 cells, per condition). Scale bar, 10 μm. Zoom-ins and meta-images are 4.2 μm × 4.2 μm. (j) Model representing coordinated hub formation during HS. (k) Model representing cis elements and trans factors that drive HSF1 condensate formation and function during HS.

Article Snippet: The endogenous hAXL coding sequence was removed and replaced with a synthesized gene block (Twist Bioscience®) encoding a HaloTag, GGSx3 linker sequence and a multiple cloning site (MCS). pLVX-TetOne-Puro-Halo-GGSx3-HSF1 was created by PCR amplification of the HSF1 coding sequence from HSF1-GFPN3 (Addgene #32538) and subsequently cloned into the MCS using AgeI and NotI restriction sites. pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔDBD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ1-3, pLVX-TetOne-Puro-Halo-GGSx3-NLSx3-HSF1-ΔRD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ4 and pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔAD were created by cloning in synthesized gene blocks (Twist Bioscience®) of HSF1-ΔDBD, HSF1-ΔLZ1-3, NLS3x-HSF1-ΔRD, HSF1-ΔLZ4, HSF1-ΔAD into pLVX-TetOne-Puro-Halo-GGSx3-MCS.

Techniques: Staining, Construct

Journal: bioRxiv

Article Title: Divergent condensates tune transcriptional responses during stress

doi: 10.64898/2026.02.12.705659

Figure Lengend Snippet: (a) Meta-images of H327ac, BRD4, MED12, RNAPII, RNAPII-5P, RNAPII-2P, and HSF1-pS320 (pS320) in U2OS cells treated with mock, HS, ACA, CdC, SA, HP, CC, MGP, or ATO conditions across segmented HSF1 condensates (n ≥ 53 cells, 1094 condensates, per condition). Meta-images are 2.7 μm × 2.7 μm. (b) Representative pseudo-colored IF images of U2OS cells treated with MGO, along with DMSO, CHX, or Doxo, and stained for HSF1 (green). Nuc. Boundary (cyan) is derived from DAPI staining. Scale bar, 10 μm. Zoom ins are 5 μm × 5 μm. (c) Quantification of HSF1 App. Part. in from cells in (b). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 409 cells, per condition). Line represents mean and error bars represent s.e.m. (d) Representative pseudo-colored fluorescent images of FL, ΔDBD, and ΔLZ1-3 cells treated with MGO and stained for Halo-HSF1 (JF549-HLgreen). Nuc. Boundary (cyan) is derived from DAPI staining. Scale bar, 10 μm. Zoom ins are 5 μm × 5 μm. (e) Quantification of HSF1 App. Part. in from cells in (d). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 120 cells, per condition). Line represents mean and error bars represent s.e.m. (f) Model of transcriptionally incompetent HSF1 condensates, lacking a full complement of factors required for functional hubs, in other stresses.

Article Snippet: The endogenous hAXL coding sequence was removed and replaced with a synthesized gene block (Twist Bioscience®) encoding a HaloTag, GGSx3 linker sequence and a multiple cloning site (MCS). pLVX-TetOne-Puro-Halo-GGSx3-HSF1 was created by PCR amplification of the HSF1 coding sequence from HSF1-GFPN3 (Addgene #32538) and subsequently cloned into the MCS using AgeI and NotI restriction sites. pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔDBD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ1-3, pLVX-TetOne-Puro-Halo-GGSx3-NLSx3-HSF1-ΔRD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ4 and pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔAD were created by cloning in synthesized gene blocks (Twist Bioscience®) of HSF1-ΔDBD, HSF1-ΔLZ1-3, NLS3x-HSF1-ΔRD, HSF1-ΔLZ4, HSF1-ΔAD into pLVX-TetOne-Puro-Halo-GGSx3-MCS.

Techniques: Staining, Derivative Assay, Functional Assay

Journal: bioRxiv

Article Title: Divergent condensates tune transcriptional responses during stress

doi: 10.64898/2026.02.12.705659

Figure Lengend Snippet: (a) Schematic of intra-condensate SMT assay. (b) Representative pseudo-colored time-lapsed images of HSF1 condensates (JF646-HL, red) and single-HSF1 molecules (JF549-HL, green) from live UHKO-HaH cells subjected to HS or CC. Dotted green circles have been included to aid HSF1 molecule location. Appropriate single-HSF1 molecule trajectory are also represented. Scale bar, 60 nm. (c) Mean square displacement (MSD) vs time-lag (τιt) plots for trajectories in (b). Diffusion coefficients (D) extracted from plots are indicated. (d) Distribution of log D of HSF1 molecules in HS and other stresses (Others: CC, HP, MGO, CdC, and ACA). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 20 cells, 102 trajectories, per condition). Line represents median. (e) Representative pseudo-colored images of U2OS cells mock treated or subjected to HS, SA, or HP, and stained for HSF1 (green, by IF) and HSATIII (red, by smFISH); with meta-images (Av., n ≥ 58 cells, per condition). Nuc. Boundary (cyan) is derived from DAPI staining. Scale bar, 10 μm. Meta-images and zoom-ins are 2.7 μm × 2.7 μm. Inset represents smFISH signal, with distinct (x-fold) contrast adjustment to aid visualization. (f) Quantification of HSATIII signal at in cells from (e). ***=p<0.0005, by two-sided unpaired T-test. Line represents mean and error bars represent s.e.m. (g) Meta-images (Av.) and normalized line scan metaplots of HSATIII smFISH signal across segmented HSF1 condensates from (e). ***=p<0.0005, ns = not significant, by two-sided unpaired T-test (n ≥ 58 cells, 1750 condensates, per condition). Underscored significance denotations are relative to HS, whereas others are relative to Mock. (h) Schematic of transcription site (TS) imaging by smFISH. (i) Representative pseudo-colored smFISH images of U2OS cells mock treated or subjected to HS, SA, or HP, and stained for HSP90AA1 introns (green) or HSP90AA1 exons (red). Nuc. Boundary (cyan) is derived from DAPI staining. Scale bar, 10 μm. Zoom-ins are 2.7 μm × 2.7 μm. (j) Quantification of HSP90AA1 intron and exon signal at putative TS in cells from (h). ***=p<0.0005, by two-sided unpaired T-test (n ≥ 80 cells, per condition). Line represents mean and error bars represent s.e.m. (k) Metaplot of σ; 120 bp fragments HSF1 CUT&RUN signal at HSEs in U2OS cells that were mock treated or subjected to HS, or SA. HSEs at stress-type agnostic (left) or stress-type specific (right) loci with upregulated signal are represented. (l) Metaplot of BrU-Seq signal in U2OS cells that were mock treated or subjected to HS, or SA. HSE associated commonly (stress-type-agnostic) upregulated genes (left) or chaperones (right) are represented. (m) Representative genomic view of DNAJB1 and SERPINH1 loci with HSF1 CUT&RUN (C&R) or BrU-seq signal from U2OS cells that were mock treated or subjected to HS, or SA. (n) Proposed model of divergent functionality of HSF1 and potentially other stress-induced transcription factor condensates.

Article Snippet: The endogenous hAXL coding sequence was removed and replaced with a synthesized gene block (Twist Bioscience®) encoding a HaloTag, GGSx3 linker sequence and a multiple cloning site (MCS). pLVX-TetOne-Puro-Halo-GGSx3-HSF1 was created by PCR amplification of the HSF1 coding sequence from HSF1-GFPN3 (Addgene #32538) and subsequently cloned into the MCS using AgeI and NotI restriction sites. pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔDBD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ1-3, pLVX-TetOne-Puro-Halo-GGSx3-NLSx3-HSF1-ΔRD, pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔLZ4 and pLVX-TetOne-Puro-Halo-GGSx3-HSF1-ΔAD were created by cloning in synthesized gene blocks (Twist Bioscience®) of HSF1-ΔDBD, HSF1-ΔLZ1-3, NLS3x-HSF1-ΔRD, HSF1-ΔLZ4, HSF1-ΔAD into pLVX-TetOne-Puro-Halo-GGSx3-MCS.

Techniques: Diffusion-based Assay, Staining, Derivative Assay, Imaging