hsf1 coding sequences  (New England Biolabs)

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